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Evolution of pathologic T-cell subsets in patients with atopic dermatitis from infancy to adulthood
Journal Pre-proof Evolution of pathologic T-cell subsets in atopic dermatitis from infancy to adulthood Tali Czarnowicki, MD MSc, Helen He, BSc, Talia Canter, BS, Joseph Han, BS, Rachel Lefferdink, MD, Taylor Erickson, BS, Stephanie Rangel, PhD, Naoya Kameyama, PhD, Hyun Je Kim, MD PhD, Ana B. Pavel, PhD, Yeriel Estrada, BS, James G. Krueger, MD PhD, Amy S. Paller, MD, Emma Guttman-Yassky, MD PhD PII:
S0091-6749(19)31319-3
DOI:
https://doi.org/10.1016/j.jaci.2019.09.031
Reference:
YMAI 14212
To appear in:
Journal of Allergy and Clinical Immunology
Received Date: 21 April 2019 Revised Date:
23 August 2019
Accepted Date: 12 September 2019
Please cite this article as: Czarnowicki T, He H, Canter T, Han J, Lefferdink R, Erickson T, Rangel S, Kameyama N, Kim HJ, Pavel AB, Estrada Y, Krueger JG, Paller AS, Guttman-Yassky E, Evolution of pathologic T-cell subsets in atopic dermatitis from infancy to adulthood, Journal of Allergy and Clinical Immunology (2019), doi: https://doi.org/10.1016/j.jaci.2019.09.031. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 American Academy of Allergy, Asthma & Immunology
Evolution of polar T-cell subsets in healthy controls and atopic dermatitis patients from infancy to adulthood
Normal Cytokine Subset Development
AD Cytokine Subset Development
IFN-γ
(CLA+>CLACD4+>CD8+)
IL-13
(CLA+>CLACD4+>CD8+)
IL-9 IL-17 (CLA-)
IL-22 Tregs
(CLA+)
Blood Phenotype
0-5y/o
6-11y/o
12-17y/o
>18y/o
0-5y/o
6-11y/o
12-17y/o
>18y/o
Abbreviations: AD, Atopic dermatitis; CLA, Cutaneous lymphocyte antigen; IFN-γ, Interferon γ; IL, Interleukin; Tregs, T-regulatory cells
1 1
Evolution of pathologic T-cell subsets in atopic dermatitis from infancy to
2
adulthood
3 4
Tali Czarnowicki, MD MSc,1,3 Helen He, BSc,1 Talia Canter, BS,2 Joseph Han,
5
BS,1 Rachel Lefferdink, MD,2 Taylor Erickson, BS,2 Stephanie Rangel, PhD,2
6
Naoya Kameyama, PhD,1 Hyun Je Kim, MD PhD,1 Ana B Pavel, PhD,1 Yeriel
7
Estrada, BS,1 James G Krueger, MD PhD,3 Amy S Paller, MD,*2 Emma Guttman-
8
Yassky, MD PhD*1
9
*These authors contributed equally to the manuscript
10 11
1
12
Medicine at Mount Sinai, New York, NY, USA
13
2
14
School of Medicine, IL, USA
15
3
16
NY, USA
Department of Dermatology and the Immunology Institute, Icahn School of
Departments of Dermatology and Pediatrics, Northwestern University Feinberg
Laboratory for Investigative Dermatology, The Rockefeller University, New York,
17 18
Corresponding Author:
19
Emma Guttman-Yassky MD PhD
20
Department of Dermatology, Icahn School of Medicine at Mount Sinai Medical
21
Center, 5 E. 98th Street, New York, NY 10029
22
Email: [email protected]
23
Telephone: 212-241-9728/3288; Fax: 212-876-8961
2 24
Funding: This study was funded by a grant from the LEO Foundation. The study
25
was also supported by the Northwestern Skin Disease Research Center (NIAMS
26
P30 AR057216) and the Northwestern University Clinical And Translational
27
Sciences (NUCATS) Institute (UL1TR001422). JGK has received research
28
support (grants paid to his institution) and/or personal fees from Pfizer, Amgen,
29
Janssen, Lilly, Merck, Novartis, Kadmon, Dermira, Boehringer, Innovaderm,
30
Kyowa, BMS, Serono, BiogenIdec, Delenex, AbbVie, Sanofi, Baxter, Paraxel,
31
Xenoport, and Kineta. ASP is an investigator for Galderma, Incyte, Leo, Pfizer,
32
and Regeneron (grants paid to Northwestern Univ.) and has received honoraria
33
related to AD consulting from AbbVie, Asana, Boehringer, Dermavant, Dermira,
34
Eli Lilly, Forte, Galderma, Leo, Matrisys, Menlo Therapeutics,
35
Morphosys/Galapagos, Novartis, Pfizer, Regeneron, and Sanofi-Genzyme. EGY
36
is an employee of Mount Sinai and has received research funds (grants paid to
37
the institution) from Abbvie, Asana Biosciences, Celgene, Dermavant, DS
38
Biopharma, Galderma, Glenmark, Innovaderm, Janssen Biotech, Leo Pharma,
39
Novan, Novartis, Pfizer, Ralexar, Regeneron, and Union Therapeutics. EGY is
40
also a consultant for Abbvie, Allergan, Amgen, Asana Biosciences, Celgene,
41
Concert, DBV Technologies, Dermira, DS Biopharma, Eli Lilly, EMD Serono,
42
Escalier, Flx Bio, Galderma, Glenmark, Kyowa Kirin, Leo Pharma, Mitsubishi
43
Tanabe, Novartis, Pfizer, Regeneron, Sanofi, and Union Therapeutics.
44
Disclosures: Other authors have declared that they have no conflict of interest.
45
Word Count: 4,645
3 46
Keywords: Atopic dermatitis, endotypes, T-cell, CLA, IL-13, IL-22, IFN-γ, ICOS,
47
HLA-DR
48
4 49
Abstract
50
Background: The circulating immune phenotype was defined in adults and
51
young children with early atopic dermatitis/AD, but chronologic changes in blood
52
of AD infants and children through adolescence have not been explored.
53
Objective: To compare immune activation and cytokine polarization in blood of
54
0-5y/o (n=39), 6-11y/o (n=26), 12-17y/o (n=21) and ≥18y/o (n=43) patients with
55
AD vs. age-matched controls.
56
Methods: Flow cytometry was used to measure interferon-γ, interleukin (IL)-9,
57
IL-13, IL-17, and IL-22 cytokines in CD4+/CD8+ T-cells, with ICOS and HLA-DR
58
defining mid- and long-term T-cell activation, respectively, within skin-
59
homing/CLA+ vs. systemic/CLA- T-cells. Unsupervised clustering differentiated
60
patients based on their blood biomarker frequencies.
61
Results: While CLA+ Th1 frequencies were significantly lower in infants with AD
62
vs. all older patients (P<0.01), CLA+ Th2 T-cells were similarly expanded across
63
all AD age groups compared with controls (P<0.05). After infancy, CLA- Th2
64
frequencies were increased in AD in all age groups, suggesting systemic immune
65
activation with disease chronicity. IL-22 frequencies serially increased from
66
normal in infants to highly significant levels in adolescents and adults compared
67
to respective controls (P<0.01). Unsupervised clustering aligned the AD profiles
68
along an age-related spectrum from infancy to adulthood (ICOS, IL-22 etc.).
69
Conclusions: The adult AD phenotype is achieved only in adulthood. Unique
70
cytokine signatures characterizing individual pediatric age endotypes may require
71
age-specific therapies. Future longitudinal studies, comparing the profile of
5 72
cleared vs. persistent pediatric AD, may define age-specific changes that predict
73
AD clearance.
74 75
Capsule Summary: Decreased Th1/Th2 ratio characterizes the atopic dermatitis
76
(AD) phenotype across all age groups, while chronic activation, IL-9, IL-22, and
77
Treg changes characterize older patients. Even the adolescent AD profile differs
78
from adult AD.
79 80
Clinical Implications: Diverse immune signatures in different pediatric and adult
81
atopic dermatitis age groups argue for age-specific, rather than uniform,
82
therapeutic interventions.
83 84 85 86 87 88 89 90 91 92 93 94
6 95
Abbreviations
96
AD: Atopic dermatitis
97
ADQ: Atopic dermatitis quickscore
98
CD69: Cluster of Differentiation 69
99
CLA: Cutaneous Lymphocyte Antigen
100
EASI: Eczema area and severity index
101
HLA-DR: human leukocyte antigen DR
102
IFN-γ: Interferon gamma
103
IL: Interleukin
104
ICOS: Inducible co-stimulator molecule
105
ILC: Innate lymphoid cell
106
MHC: Major histocompatibility complex
107
PBMC: Peripheral Blood Mononuclear Cell
108
SCORAD: SCORing of Atopic Dermatitis
109
Tc1: Type 1 Cytotoxic T-cell
110
Tc17: Type 17 cytotoxic T-cell
111
Tc2: Type 2 cytotoxic T-cell
112
Tc22: Type 22 cytotoxic T-cell
113
TEWL: Transepidermal water loss
114
Tc: T cytotoxic
115
Th: T helper
116
Y/o: Years old
117
7 118
Introduction
119
Infancy, childhood and adolescence are critical periods for immune system
120
maturation.1 Early abnormal immune development may cause immune-related
121
disorders. Indeed, 85% of atopic dermatitis (AD) cases present before 5 years of
122
age.2 While young adults have a different AD phenotype from elderly patients,3
123
immune changes in AD between early childhood and adulthood are unknown.4
124
Blood studies that dissect developmental changes from infancy through
125
adulthood are limited and primarily focused on T-helper (Th)1/Th2 subsets.
126
Some studies with healthy controls showed expansion of Th1/Th2/T-cytotoxic
127
(Tc)1 subsets with age,5, 6 while others reported no changes in Th2/Tc2 over
128
time.7 Immature interferon (IFN)-γ response8 and low Th1/Tc1 frequencies9-11
129
were seen in early stages of normal development, and abnormal Th1/Th2 ratios
130
in cord blood and infants who develop AD were described.12-14 However, few
131
studies compared pediatric and adult AD populations6, 15-17 and none directly
132
compared consecutive age groups of AD with age-matched controls, which is
133
critical in understanding normal vs. pathological development of acquired
134
immunity.
135
The therapeutic arsenal available for 0-12 year old (y/o) AD patients is
136
limited to topical agents and/or broad systemic immune suppressants.4, 18 Active
137
development of targeted therapeutics is ongoing for adults and adolescents with
138
AD and will eventually move to children, further necessitating the elucidation of
139
pediatric endotypes at successive pre-adult age groups to introduce safe,
140
effective, age-tailored targeted approaches.19
8 141
We compared T-cell memory subset activation and polarized CD4/CD8
142
subset frequencies within the skin-homing/cutaneous lymphocyte antigen/CLA+
143
and systemic/CLA- compartments, in the blood of infants and toddlers (0-5y/o),
144
young children (6-11y/o), adolescents (12-17y/o) and adults (≥18y/o) with
145
moderate-to-severe AD. To differentiate pathologic from physiologic immune
146
maturation, age-matched healthy controls were included. We found that
147
decreased Th1/Th2 ratios were shared across all AD ages, but unique
148
fingerprinting characterizes individual AD age groups, differentiating them from
149
their age-matched peers. Our intracellular and T-cell blood biomarker data
150
grouped the AD cohort, but not the controls, into three unique age phenotypes
151
aligned along a spectrum.
152 153
Methods
154
Patient characteristics and blood samples
155
Blood was obtained (with signed Institutional Review Board-approved informed
156
consent from parents and patients 12 years and older) from 39 infants and
157
toddlers (0-5y/o; mean 23 months), 26 children (6-11y/o; mean 7.5yrs), 21
158
adolescents (12-17y/o; mean 14.9yrs) and 43 adults (≥18y/o; mean 43yrs) with
159
moderate-to-severe AD, as well as healthy age-matched controls (24-30 in each
160
group; demographic and laboratory data in Table E1). Sensitivity analyses on
161
patient subsets who were matched for all demographic parameters yielded
162
similar results to analysis including all subjects (Table E2A-B).
9 163
Disease severity was scored using SCORing of AD (SCORAD) for adults and
164
SCORAD, Eczema Area and Severity Index (EASI), and Atopic Dermatitis
165
Quickscore (ADQ) in those less than 18y/o. ADQ is a parent-administered tool,
166
which assesses involvement and pruritus of 7 body parts and highly correlates
167
with SCORAD.20 In subjects <18y/o, eosinophil counts and skin-barrier
168
assessment through transepidermal water loss (TEWL) of lesional (LS) and non-
169
lesional (NL) arm skin (AquaFlux Model AF200; Biox, London, England)21 were
170
measured when feasible. No patients received systemic immunosuppressive
171
treatment within 4 weeks prior to this study, thus the data present baseline
172
immune phenotyping of the studied populations. Concomitant allergic
173
manifestations were recorded, and individuals were classified as either having
174
general allergies (allergic rhinitis/asthma/allergic conjunctivitis/environmental),
175
food allergies (FA), both, and neither. Controls had no personal history of AD.
176
Only 1 child and 5 adolescent controls had history of non-cutaneous atopic
177
manifestations, however sensitivity analysis excluding these subjects did not alter
178
the results (Table E2C).
179
Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
180
PBMCs were isolated from whole blood by Ficoll-Paque Plus (GE Healthcare,
181
Sweden). Briefly, the blood was laid under Ficoll gradient; after spinning, PBMCs
182
were collected at the interface between the plasma and the Ficoll gradient (see
183
Methods in the Online Repository/OR).
184
Stimulation of blood cell populations for cytokine responses
10 185
Ex-vivo cell activation is required to detect cytokine production, as less than 1%
186
of non-stimulated cells produce cytokines. Whole blood was incubated with PMA
187
(25ng/ml) plus ionomycin (2µg/ml) in the presence of brefeldin A (10µg/ml) for 4
188
hours at 37ºC to induce cytokine responses. After stimulation, red blood cells
189
were lysed with FACS lysing solution to obtain leukocytes (see Methods in the
190
OR).
191
Cell surface staining and intracellular staining on PBMCs and stimulated
192
and non-stimulated CD4/CD8 T-cells
193
Peripheral blood mononuclear cells/PBMCs were stained with fluorochrome-
194
labeled Abs to cell surface markers (CD3, CD8, CD4, CD45RO, CCR7, ICOS,
195
HLA-DR, CLA, CCR4, CD25, CD127). Stimulated and non-stimulated blood cells
196
were also stained for cell surface markers [CD3, CD4, and CLA (CD8+ T-cells
197
were gated thorough CD3+CD4- T subpopulation] and permeabilized with
198
FACS/perm to stain for cytokines including Interleukin (IL)-13, IL-22, IL-9, IFN-γ
199
and IL-17 (see Methods in the OR).
200
Statistical analysis
201
Statistical analyses were performed with R software (www.R-projets.org). Means
202
and medians were compared using the Welch t-test and Wilcoxon-Mann-Whitney
203
test, respectively. Unsupervised hierarchical clustering of variables (T-cell subset
204
frequencies, age, and clinical scores) was performed using the R package
205
“hclust,” with a McQuitty agglomeration algorithm and Spearman coefficient as a
206
similarity metric and presented as a heat map and a dendrogram. Individual
207
scatter plots were constructed that display Spearman coefficients, 95%
11 208
confidence intervals, and p-values for AD and healthy samples. We performed k-
209
means unsupervised clustering across principal components of the frequencies
210
of all AD and separately among all healthy control subsets. We found that 3
211
clusters separated AD patients, but not controls, along a chronologic age
212
spectrum. ANOVA, in conjunction with the Tukey test, was used to find markers
213
that differentiated any two clusters. P-values and fold-changes (FCH) were
214
designated as ***(p<0.001), **(p<0.01), *(p<0.05), and +(p<0.1).
215
We also performed a power calculation based on our previous flow
216
cytometry study on moderate-to-severe adult AD patients.22 We determined that
217
39 AD patients would have greater than 90% power (at a significance level of
218
0.05) to detect differences versus controls in various inflammatory cell subsets
219
(CD4+IL-22+CLA+, CD4+IL-13+CLA-, CD8+IL-17+CLA+, CD8+ Tcm HLA-
220
DR+CLA+).
221 222
Results
223
Flow cytometry was used to measure frequencies of IFN-γ, IL-9, IL-13, IL-17A
224
and IL-22-polarized T-cells, defining Th1/Tc1, Th9/Tc9, Th2/Tc2, Th17/Tc17 and
225
Th22/Tc22 subsets in CD4+/CD8+ T-cells, respectively. Cell surface staining was
226
used to assess expression of mid (inducible co-stimulator molecule/ICOS) and
227
late (HLA-DR) activation markers in central (Tcm/CCR7+CD45RO+) and effector
228
(Tem/CCR7-CD45RO+) memory T-cells in skin homing/cutaneous/CLA+ and
229
systemic/CLA- compartments.
12 230
Patients and controls were divided into 4 consecutive age groups (infants
231
and toddlers 0-5y/o, children 6-11y/o, adolescents 12-17y/o and adults ≥18y/o).
232
To display both healthy vs. pathologic developmental changes and immune
233
abnormalities within each age group vs. controls, we present two types of
234
comparison plots; both contain similar data but focus on either AD patients vs.
235
controls for each age group or AD vs. control across all ages. To better represent
236
the effect of values distribution, the comparison plots presented below contain
237
both the mean (in black) and the median (in red) +/- standard error, and their
238
respective p-values. Results discuss the mean values, while median percentages
239
for main comparisons discussed are presented in Table E3.
240
Skin-homing memory T-cell expansion and ICOS activation feature early
241
AD
242
Tcm and Tem T-cells are main components of the adaptive immune system,
243
harboring distinct homing capacities.23 While both express the skin homing
244
marker CLA, only Tcm cells retain CCR7 positivity, which enables them to
245
migrate into lymph nodes and function as an immunologic reserve.24 After gating
246
on CD3+ viable T-cells using flow cytometry, CD3+CD4+ and CD3+CD8+ were
247
defined and analyzed separately. CCR7 and CD45RO were used to differentiate
248
memory subsets within the CD4 and CD8 populations. CCR7+CD45RO- defined
249
naïve cells, CCR7+CD45RO+ central memory T-cells (Tcm), CCR7-CD45RO+
250
effector memory T-cells (Tem) and CCR7-CD45RO- Effector/Temra/terminally
251
differentiated T-cells (Fig. E1A-C). We then further defined activated ICOS/HLA-
13 252
DR activated Tcm/Tem memory subset, using CLA to segregate skin-homing
253
(CLA+) versus systemic subsets (CLA-).
254
Normal development was characterized by a slight drop of CLA+ CD4+Tem (but
255
not Tcm) cells between infancy and childhood (infants 26.7% vs. children 19%,
256
P=0.01; Figs. 1A-B, E1D-E), but this decrease was significantly more evident in
257
AD (P<0.05; Figs. 1A-B, E1D-E). CD4+CLA+ Tem/Tcm cells were significantly
258
higher in AD infants (Tcm: 20.4% vs. 13.4%, P=0.006; Tem: 38% vs. 26.7%,
259
P=0.002; Fig. E1D-E) and children (Tcm: 18.8% vs. 11.3%, P=0.01; Tem: 27.7%
260
vs. 19%, P=0.03; Fig. E1D-E) than controls, a difference that diminished with
261
increasing age. T-cell memory subset fluctuations with age are presented in
262
Figure E2. Due to increased proportions of effector and naïve cells, CD8+
263
Tcm/Tem subsets dropped between infancy and childhood exclusively in controls
264
(Tcm: infants 12.8% vs. children 5.6%, P=0.008; Tem: infants 15.4% vs. children
265
5%, P<0.001; Fig. 1C-D), leading to higher frequencies in children with AD
266
(P<0.01), but otherwise frequencies were overall similar between AD and
267
controls over time (Fig. E1F-G). Significant CD4+/CD8+ effector cells increase
268
with age characterized AD vs. controls, who showed decrease of this subset
269
during the years (Fig. E2J, L).
270
CD4+ Tcm/Tem ICOS mid-activation continuously increased in both
271
controls and AD, however it decreased significantly between adolescence and
272
adulthood, exclusively in controls (Tcm CLA+: 24% vs. 9%, P=0.003; CLA-: 9.2%
273
vs. 3.7%, P=0.001; Fig. 1E-H). Both skin-homing/CLA+ and systemic/CLA- ICOS-
274
activated CD4+ T-cell frequencies were significantly increased in infants and
14 275
adults with AD vs. respective controls (P<0.05; Fig. 1E-H). Skin-homing CD8+
276
Tcm/Tem ICOS activation increased gradually, most notably in AD patients (Fig
277
1I-L), with frequency differences uniquely seen in adults with AD vs. controls
278
(Tcm: 18.4% vs. 12.5%, P=0.01; Tem: 26% vs. 13.4%, P<0.001; Fig. 1I-L).
279
The HLA-DR antigen, indicating chronic activation,25 had similar (or even
280
lower) expression in infants with and without AD (P>0.1; Fig. E1H-K), but started
281
to rise in AD children (Tcm+CD4+CLA+ AD: 15.8% vs. 8.4%, P=0.02; CLA-: 7.2%
282
vs. 2.8%, P=0.06; Fig. E1H-I), reaching consistently high levels across
283
CD4+/CD8+/CLA+/CLA-/Tcm/Tem cells in adults with AD compared to healthy
284
individuals (P<0.05; CD8+ Tcm/Tem data not shown; Fig. E1H-K).
285
Decreased Th1/Th2 ratio characterizes AD across ages
286
Since T-cell activation leads to cytokine polarization we next studied different
287
polar T-cell subsets. Representative flow cytometry plots and gating strategy are
288
presented in Figure E3. Congruent with past publications,9, 10 IFN-γ levels
289
increased with age in both control and AD subjects, with the lowest frequencies
290
seen in infants (Fig. 2A-D). Nevertheless, in AD IFN-γ did not reach control
291
levels, particularly within the skin homing compartment (CD4+CLA+: infants
292
control 12% vs. AD 7.7%, P=0.04; children control 19.5% vs. AD 13.6%, P=0.05;
293
adolescents control 18% vs. AD 13%, P=0.07) until adulthood (P=0.2; Figs. 2A-
294
D, E4A-D). Even in adulthood, levels trended towards lower frequencies.
295
Interestingly, the only population that showed lower systemic/CLA- CD4+/CD8+
296
IFN-γ was the 5-12y/o age group (CD4+: control 20% vs. AD 14%, P=0.04; CD8+:
297
control 39% vs. AD 28%, P=0.05; Fig. E4B, D).
15 298
In controls, IL-13+CD4+CLA+ was lowest in infants, reaching a plateau in
299
childhood. Conversely, in AD patients, levels were similarly elevated across all
300
ages (P>0.1; Fig. 2E). Systemic/CLA- Th2 was overall low in controls (Fig. 2F).
301
Levels were significantly higher in AD than controls in children (2.8% vs. 0.6%,
302
P=0.005; Fig. E4F), adolescents (1.5% vs. 0.7%, P=0.006; Fig. E4F) and adults
303
(1.3% vs. 0.7%, P=0.005; Fig. E4F). While Tc2 cells were slightly higher in
304
children with vs. without AD, differences in CD8+ subsets were more prominent in
305
adulthood (Figs. 2G-H, E4G-H). Reflecting the Th1 and Th2 imbalances
306
characterizing AD, the Th1/Th2 ratio was decreased in AD than controls in both
307
CLA+/CLA- subsets across the ages. Tc1/Tc2 was significantly lower in children
308
and adults with AD vs. controls (Figs. 2I-L, E4I-L).
309
Both healthy controls and AD patients showed Th9 increases over time,
310
peaking in adolescence and dropping in adulthood (Figs. 3A-B, E5A-B). No Th9
311
differences were observed between AD and controls, besides 5-12y/o children
312
with AD that showed significantly increased CLA- levels (0.76% vs. 0.3%,
313
P=0.04; Fig. E5B). This AD age group also showed increased CLA+/CLA- Tc9
314
levels (Figs. 3C-D, E5C-D). CLA+ Th17 cells were generally stable and similarly
315
abundant across ages among control and AD (Figs. 3E, E5E), contrary to CLA-
316
Th17 cells which showed developmental expansion in both (Figs. 3F, E5F).
317
Contrary to adolescents with AD that had lower CLA+ Tc17 than controls (1% vs.
318
4.7%, P=0.04), adults had significantly higher frequencies (2.1% vs. 1.4%,
319
P=0.05; Figs. 3G-H, E5G-H). Systemic/CLA- CD4+/CD8+ IL-22+ similarly
320
increased with age in both controls and AD (Fig. 3J, 3L, E5J, E5L), while skin-
16 321
homing Th22/Tc22 cells increased with age, primarily in AD (Figs. 3I, 3K, E5I,
322
E5K). Starting in childhood, skin-homing Th22/Tc22 were significantly higher in
323
AD vs. controls, and incrementally increased with age (Th22: children 6.7% vs.
324
4%, P=0.07; adolescents 7.9% vs. 3.6%, P=0.001; adults 8.2% vs. 4.4%,
325
P<0.0001; Figs. 3I, 3K, E5I, E5K). Polar T-cell subset development in controls
326
and AD is summarized in Table E6 and illustrated in the graphical abstract.
327
The unsupervised hierarchical clustering heatmap in Figure 4 summarizes
328
the above, displaying all polarized T-cell subsets for control and AD across age
329
groups (red: positive/increase; blue: negative/decrease). Fold-changes (FCHs) of
330
the mean frequencies of AD vs. controls for each age group are presented (stars
331
and plus signs display significance). The green cluster includes subsets that
332
were relatively low and stable among controls, but incrementally increased with
333
age in AD. Most of these subsets were significantly increased in adults with AD
334
vs. controls (FCH>1.57, P<0.05), while younger AD patients showed lower or no
335
significance. The pink box shows increased IL-9 frequencies in childhood, which
336
decline in adulthood, particularly in AD. The yellow cluster shows markers that
337
increased in both controls and AD, therefore minimizing the differences between
338
groups.
339
T-cell activation, clinical measures and IFN-γ are associated with age and
340
AD chronicity
341
We also evaluated how clinical characteristics, including AD severity (SCORAD,
342
EASI), patient age, disease duration, eosinophil counts, pruritus and TEWL relate
343
to different polar T-cell subsets. Unsupervised hierarchical clustering of all T-cell
17 344
subset frequencies, clinical scores, AD duration and age was performed using
345
Spearman correlations as a similarity metric, as displayed in the correlation
346
heatmap and dendrogram in Figure 5 (red: positive correlation; blue: negative
347
correlation, stars and plus signs display significance). Congruent with recent AD
348
data,22, 26 which showed positive correlations between IL-13- and IL-22- and
349
between IL-17- and IL-22-producing T-cells,22, 26, 27 these cytokines largely
350
grouped together (turquoise rectangle). IFN-γ-producing T-cells clustered
351
together with AD duration and patient age (yellow rectangles). A tight cluster
352
gathering multiple clinical measures (green rectangles) was located adjacent to
353
IL-9/IL-13-producing T-cells (purple rectangles). Significant correlations depicted
354
in this heatmap are listed in Table E4, with selected individual scatter plots
355
presented in Figures 6-7 and E6-E8.
356
SCORAD and EASI were positively correlated (r=0.74, P<0.001; Fig. 6A).
357
SCORAD also correlated with skin-homing CD4+ cells (r=0.24, P=0.0016; Fig.
358
6B), pruritus (r=0.54, P<0.0001, Fig. 6C) and eosinophils (r=0.25, P=0.001; Fig.
359
6D). To evaluate normal vs. pathologic development with age, we
360
comprehensively assessed age correlations in control and AD subjects,
361
presenting both on the same scatter plots for clarity, when applicable. Age
362
correlated positively with severity by SCORAD (r=0.25, P=0.0016; Fig. 6E) and
363
EASI (r=0.34, P<0.001; Fig. 6F), and with all other clinical measures, including
364
pruritus (Fig. 6G), eosinophils (Fig. 6H), and lesional TEWL (Fig. 6I-J).
365 366
While skin homing Tcm/Tem cells correlated negatively with age (red=AD, blue=controls; Fig 6K-L), the proportion of ICOS and HLA-DR-activated skin-
18 367
homing T-cells increased exclusively in AD (Fig. E6A-D). AD duration, which
368
highly correlated with patients’ age (r=0.98, P<0.0001; Fig. E6E), also
369
demonstrated significant positive correlations with EASI and pruritus (Fig. E6F-
370
H). Among the cytokine subsets (Fig. E6I-L), the most significant correlation was
371
noted between AD duration and IFN-γ-producing cells (r=0.58, P<0.0001; Fig.
372
E6L).
373
Overall IFN-γ increased with age in both AD and controls (Fig. E7A-D),
374
however, although the Th1/Th2 (but not the Tc1/Tc2) ratio, and particularly its
375
skin-homing component, increased with age in both groups, it remained
376
significantly decreased in AD across all ages vs. controls (Fig. 7A-D).
377
Conversely, differences between AD and controls were observed in skin-homing
378
Th2/Tc2 throughout development, with controls never reaching AD levels (Fig.
379
7E-H). CLA+ Th22/Tc22 showed significantly higher developmental increases in
380
AD (Fig. 7I, K), while systemic subsets generally overlapped (Fig. 7J, L). Similar
381
negative trends of IL-9+ (Fig. E7E-H), and expansion of systemic Th17 cells were
382
seen in controls and AD subjects (Fig. E7I-L).
383
Positive correlations between SCORAD and IL-13, IL-9 and IL-22-
384
producing cells were recorded (P<0.02; Fig. E8A-C). EASI correlated with Th9,
385
Th22 and pruritus (P<0.03; Fig. E8D-F), while pruritus was associated with Th22
386
and eosinophil counts (P<0.032; Fig. E8G-H). TEWL correlated with CD4+CLA+
387
cells, only in non-lesional skin (Fig. E8I-J). Th22 positively correlated with TEWL
388
in lesional tissues (Fig. E8K-L), and AD severity correlated with TEWL in non-
389
lesional skin (Fig. E8M-P).
19 390
Because Foxp3 staining requires cell permeabilization, surface markers
391
were used for Treg identification. 90% of CD25+CD127-CCR4+ cells co-express
392
Foxp3, therefore CD25+CD127-CCR4+ phenotype defined Tregs.28 In analyzing
393
T-regulatory cells (Tregs), both total Treg (CD25+CD127-) and skin-homing
394
(CCR4+CLA+ fraction) Treg frequencies were captured. The only AD age group
395
that showed significant increases of both total (Fig. E9A) and skin-homing (Fig
396
E9B) Tregs vs. control was 5-12y/o (P<0.01). Developmentally, AD showed
397
similar total Treg trends as controls, whereas discrepancies were more evident in
398
skin-homing subsets (Fig. E9C-D).
399
Cytokine polarization and T-cell activation differentiate AD patients into
400
separate age clusters along a spectrum
401
We integrated the T-cell and cytokine biomarkers to differentiate the entire AD
402
cohort based on their blood phenotype. The principal components of all
403
biomarker data for all subjects were analyzed using unsupervised k-means
404
clustering, separately for AD patients and controls. As illustrated in Figure 8, in
405
the AD cohort, the frequencies of different markers defined three meaningful
406
clusters, aligning along a spectrum. While infants (pink ellipse) clustered on the
407
far left, and adults (green ellipse) on the right, children and adolescents (blue
408
ellipse) generally clustered together, between the infants and adults. The
409
markers that best distinguished between each set of clusters appear in the boxes
410
between the two cohorts and are summarized in Table E5. Th1/Th2 ratio, CD8+
411
activation and IFN-γ-producing T-cells differentiated adults from younger groups.
412
Tregs, T-cell activation, and different cytokine subsets were able to differentiate
20 413
distinctive AD age groups. Applying the same model to the control population did
414
not distinguish between age groups (Fig. 8).
415 416
Discussion
417
This is the first comprehensive study that compares systemic immune profiles of
418
different AD age groups (0-5y/o, 6-11y/o, 12-17y/o and ≥18y/o), with appropriate
419
comparisons to controls. Since circulating CLA+ T-cells have been suggested as
420
peripheral biomarkers in AD,29 we utilized CLA to segregate between skin-
421
homing and systemic immune changes that accompany healthy and AD
422
development. Our data suggest unique endotypes in infants and toddlers,
423
children, adolescents and adults with AD, possibly advocating for personalized,
424
endotype-driven approaches rather than “one-size-fits-all” therapeutic strategies.
425
In addition, the results start to define age-specific differences between pediatric
426
AD patients and age-matched controls as a complement to our recent data
427
showing differences between elderly vs. young AD adult endotypes.3 Our data
428
suggest that even adolescents have a different profile from adults, possibly
429
contributing to the somewhat lower response to the recently approved IL-4Rα
430
antagonist, dupilumab, in adolescents vs. adults.30, 31
431
Immune responses at birth are immature10 with decreases of naïve T-cells
432
in healthy children with increasing age, paralleled by increases in Tcm/Tem
433
cells.32 Skin-homing Tcm/Tem cells are higher in infants and children with AD
434
compared to controls and decrease with age, exclusively in AD. Elevated skin-
435
homing memory cells cluster AD infants separately from other age groups,
21 436
suggesting early relevance. Excessive naïve-to-memory switch, which causes an
437
influx of memory cells into the circulation in infancy and early childhood, may play
438
a role in AD initiation, while exclusive decreases in the AD population over time
439
may suggest increased migration of CLA+ Tcm/Tem cells to skin.
440
ICOS is involved in multiple adaptive immune responses, including
441
induction of various T-cell polarizations,33-35 Treg regulation,36 antibody
442
responses (including IgE switching),37 and innate lymphoid cell (ILC)2-mediated
443
cytokine production.38, 39 ICOS has also been implicated in the pathogenesis of
444
asthma.40, 41 We have previously shown that Tcm/Tem ICOS activation is higher
445
in AD vs. control children.42 Subsequent data showed acclerated systemic/CLA-
446
ICOS T-cell activation in 0-3y/o AD infants.43 Our present data support ICOS
447
expansion in both healthy and AD development, particularly in CD4+ T-cells.
448
However, positive correlations with age were exclusively seen in AD, and
449
specifically in skin-homing memory subsets. Also, infants and adults with AD
450
have higher CD4+CLA+/CLA- ICOS activation than controls, a difference that is
451
also significant for CD8+ subsets in children. The low CD8+ frequencies in infants,
452
which increase with time,10 may explain the earlier activation of CD4+ subsets.
453
Early involvement of ICOS in AD indicates its possible role in disease induction
454
via either stimulation of T-cell subset polarization, Treg modulation, IgE induction,
455
or ILC2s activation. Furthermore, targeting ICOS/ICOS-ligand in an asthma
456
mouse model altered T-cell differentiation and ameliorated asthma.44 Since AD
457
often precedes asthma,45 CLA- ICOS expansion in AD infants may also be
458
associated with non-cutaneous atopic manifestations. These data suggest the
22 459
potential for ICOS targeting as a preventive or therapeutic approach for AD and
460
atopic associations, though this is speculative and further studies are needed.
461
The chronic T-cell activation MHC class II antigen, HLA-DR,46 increases
462
with age in healthy individuals.47, 48 HLA-DR activation has been shown in chronic
463
adult AD.42 While it is not elevated in AD infants, its activation increases with age.
464
At birth, immune responses are Th2-biased, with low Th1/IFN-γ in both
465
healthy newborns8, 9, 49, 50 and those who eventually develop AD.13 Our data
466
shows that Th1 frequencies increase with age in both AD and control infants.
467
However, and congruent with recent blood data,51 AD subjects display
468
significantly lower IFN-γ frequencies than controls, particularly among CD4+CLA+
469
subsets. Conversely, CLA+Th2 cells are similarly increased across all AD age
470
groups and are significantly higher than controls, even in infants. Systemic/CLA-
471
Th2 cells are significantly higher in AD starting in childhood, which implies
472
systemic Th2 activation with greater chronicity. Consistent with the less dominant
473
role of CD8 suggested in children with early AD42 and low CD8 abundance in
474
infants,10 Tc2 elevations are mostly prominent in adult AD. CLA+ Th1/Th2 ratio
475
correlation with age that is consistently decreased in AD compared to control,
476
and the decreased CLA+/CLA- Th1/Th2 ratio among all AD age groups best
477
reflect the immune imbalance that accompanies AD from birth to adulthood.
478
Circulating Th9 cells have skin-homing predilection.52, 53 IL-9 is elevated in
479
both skin and serum of children with AD, and was also correlated with disease
480
severity.54, 55 While the mechanism by which IL-9 contributes to AD is unknown,
481
IL-9 is linked with mast cell, eosinophil and ILC function and has been thought to
23 482
play a role in AD,56 as well as in other atopic disorders.57-60 Accordingly, IL-9
483
subsets cluster closest to all AD clinical measures
484
(eosinophils/pruritus/SCORAD/EASI/TEWL). Both normal and AD development
485
are characterized by IL-9 increases with age, which drop in adulthood. We show
486
considerably elevated systemic/CLA- T-cells exclusively among the 5-12y/o AD
487
group vs. controls, consistent with early allergy development in childhood with a
488
subsequent decline in adulthood.61, 62 CLA- systemic activation and cytokine
489
polarization with age support the systemic nature of AD and need for systemic
490
therapeutic approaches in moderate-to-severe disease, as well as the
491
association of comorbidities and increased inflammatory and cardiovascular-
492
associated proteins in more chronic AD.63-66
493
Black et al.67 showed strong Th17 skewing early in life. While similar
494
CLA+Th17 frequencies are seen in infants and older individuals regardless of AD,
495
systemic/CLA- Th17 cells are relatively low in infants of both groups and mature
496
with time. IL-17 signatures characterize pediatric AD skin,55 but these changes
497
are not mirrored in the circulation. This discrepancy may be due to increased skin
498
residence of Th17 cells, or by reciprocal peripheral regulation of Th17 and Th2
499
axes.68, 69
500
Skin-homing Th22 is elevated in children, adolescents and adults with AD
501
vs. controls. Congruent with past studies showing that IL-22 is correlated with AD
502
severity,70 we present positive correlations between IL-22-producing CD4+ cells,
503
SCORAD, EASI and pruritus. CD8+ T-cells are major producers of IL-22 in adult
504
AD skin.70 Lower proportions of CD8+ vs. CD4+ in early childhood47, 71 and the
24 505
fact that IL-22 marks AD chronicity42 may explain why skin-homing Th22/Tc22
506
cell frequencies are highest in adolescents and adults. Elevated IL-22 levels,
507
shown to negatively regulate IFN-γ,72 may also contribute to the low Th1
508
frequencies in AD. Systemic/CLA- Th22/Tc22 subset development was similar in
509
AD and controls, potentially suggesting a greater pathogenic relevance of IL-22
510
in AD skin73 and the impaired skin barrier.74 Studies have shown positive
511
correlations and cellular co-production of IL-13 and IL-2222, 75 and of IL-17 and IL-
512
22.27, 76 Indeed, these markers cluster together in the correlation heatmap. IFN-γ
513
clusters with patient age and disease duration, differentiating infants from
514
children and adolescents, and adolescents from AD adults. The association of
515
IFN-γ with disease chronicity agrees with previous chronic adult AD data,
516
suggesting that IFN-γ possibly plays a role in inflammatory disorders chronicity,
517
rather than in disease intiation.26, 42, 77 Expansion of CD8+ memory cells,
518
increases in CLA+IL-22-producing cells, and intensification of ICOS+/HLA-DR+
519
activation with maturity may account for the positive correlations between age,
520
disease duration and severity measures. Increasing severity with age was not
521
seen in a recent study that followed AD infants only between 0-11 months.78
522
TEWL increases with age in AD and correlates with IL-22 (which also increases
523
with age), likely again reflecting the contribution of IL-22 to the barrier impairment
524
in AD.
525
Besides maintaining immune tolerance,79 Tregs influence activation of
526
effector cells.80, 81 Only 5-12y/o children with AD show significantly higher total
527
and skin homing Tregs than controls. This age group is characterized by other
25 528
unique features, including increased skin-homing Tcm/Tem cells, decreased
529
systemic IFN-γ, and increased systemic IL-9 frequencies. Additionally, skin-
530
homing IFN-γ is almost doubled from infancy to childhood AD, although still
531
below age-matched controls. Multiple immunological changes occurring during
532
these years may be involved in AD clearance, or development of non-cutaneous
533
manifestations, commonly occurring during these years.82, 83
534
Profiling AD across ages is imperative for targeted therapeutic
535
development. Unlike psoriasis, in which targeted treatments lead to remarkable
536
responses in most patients, AD responses to targeted therapeutics are much
537
lower.84 This disparity may be attributed to the multi-cytokine activation in AD,
538
despite the shared Th2 activation, vs. the Th17-centered responses in
539
psoriasis.84 Additionally, AD has a highly varied endotype repertoire, with
540
different immune polarizations.3, 85, 86 Despite common features, particularly
541
elevated Th2 expression, AD is endotypically different across ages, and
542
treatments should rather be tailored to the unique age endotype. While one could
543
hypothesize that the immune changes merely reflect developmental phenomena
544
that are age-related, the lack of clear clustering in controls implicates AD as the
545
driver of the distinct, progressive age-related endotypic characteristics rather
546
than age alone. The clustering model, based on the flow cytometry biomarkers
547
splits the entire cohort into 3 separate age clusters only in AD patients.
548
Expectedly, IFN-γ, IL-22, HLA-DR increased chronologically, distinguishing older
549
subjects from infants with AD. Skin-homing Tcm/Tem cells and systemic ICOS
550
activation, both higher in AD infants, separate infants from other groups. This
26 551
model demonstrates that a limited group of blood biomarkers can distinguish
552
among various AD endotypes based on patient age, with a spectrum of age-
553
dependent vs. only ‘infantile’ or ‘adult’ phenotypes. Because our data show that
554
the ‘adult’ or ‘stable’ AD phenotype is only achieved in adulthood, it may be
555
possible to intervene before adulthood and prevent the establishment of the adult
556
AD phenotype.
557
This study has a few limitations. Despite inclusion of different age groups,
558
this study was not longitudinal and thus did not follow the same cohort with time.
559
Additionally, studies in blood do not allow one to measure T-cell subsets in skin,
560
such as tissue-resident memory/Trm T-cells. Furthermore, the study
561
characterized polyclonal T-cell responses, and not antigen-specific responses
562
induced by culprit triggers. Finally, the pathogenicity of immune axes presented
563
here cannot be further dissected without future targeted therapeutic studies to
564
check mechanism.
565
AD was initially considered an early-onset pediatric disease with 75%
566
"outgrowing" their disease by 10y/o.2, 83, 87, 88 More recent studies have
567
established AD as a disorder that often persists into adulthood.89, 90 Comparing
568
the profile of cleared vs. persistent pediatric AD, ideally through longitudinal
569
studies, will better define age-specific characteristics that predict AD clearance.
570 571 572 573
27 574
References
575
1.
576
Simon AK, Hollander GA, McMichael A. Evolution of the immune system in humans from infancy to old age. Proc Biol Sci 2015; 282:20143085.
577
2.
Bieber T. Atopic dermatitis. Ann Dermatol 2010; 22:125-37.
578
3.
Zhou L, Leonard A, Pavel AB, Malik K, Raja A, Glickman J, et al. Age-
579
specific changes in the molecular phenotype of patients with moderate-to-
580
severe atopic dermatitis. J Allergy Clin Immunol 2019; 144:144-156.
581
4.
Davidson WF, Leung DYM, Beck LA, Berin CM, Boguniewicz M, Busse
582
WW, et al. Report from the National Institute of Allergy and Infectious
583
Diseases Workshop on "Atopic Dermatitis and the Atopic March:
584
Mechanisms and Interventions". J Allergy Clin Immunol 2019; 143:894-
585
913.
586
5.
Sandmand M, Bruunsgaard H, Kemp K, Andersen-Ranberg K, Pedersen
587
AN, Skinhoj P, et al. Is ageing associated with a shift in the balance
588
between Type 1 and Type 2 cytokines in humans? Clin Exp Immunol
589
2002; 127:107-14.
590
6.
Kawamoto N, Kaneko H, Takemura M, Seishima M, Sakurai S, Fukao T,
591
et al. Age-related changes in intracellular cytokine profiles and Th2
592
dominance in allergic children. Pediatr Allergy Immunol 2006; 17:125-33.
593
7.
Chipeta J, Komada Y, Zhang XL, Deguchi T, Sugiyama K, Azuma E, et al.
594
CD4+ and CD8+ cell cytokine profiles in neonates, older children, and
595
adults: increasing T helper type 1 and T cytotoxic type 1 cell populations
596
with age. Cell Immunol 1998; 183:149-56.
28 597
8.
598 599
secretion in newborns. Biol Neonate 2004; 85:55-60. 9.
600 601
Kotiranta-Ainamo A, Rautonen J, Rautonen N. Imbalanced cytokine
Marodi L. Down-regulation of Th1 responses in human neonates. Clin Exp Immunol 2002; 128:1-2.
10.
Gasparoni A, Ciardelli L, Avanzini A, Castellazzi AM, Carini R, Rondini G,
602
et al. Age-related changes in intracellular TH1/TH2 cytokine production,
603
immunoproliferative T lymphocyte response and natural killer cell activity
604
in newborns, children and adults. Biol Neonate 2003; 84:297-303.
605
11.
606 607
Debock I, Flamand V. Unbalanced Neonatal CD4(+) T-Cell Immunity. Front Immunol 2014; 5:393.
12.
van der Velden VH, Laan MP, Baert MR, de Waal Malefyt R, Neijens HJ,
608
Savelkoul HF. Selective development of a strong Th2 cytokine profile in
609
high-risk children who develop atopy: risk factors and regulatory role of
610
IFN-gamma, IL-4 and IL-10. Clin Exp Allergy 2001; 31:997-1006.
611
13.
Herberth G, Heinrich J, Roder S, Figl A, Weiss M, Diez U, et al. Reduced
612
IFN-gamma- and enhanced IL-4-producing CD4+ cord blood T cells are
613
associated with a higher risk for atopic dermatitis during the first 2 yr of
614
life. Pediatr Allergy Immunol 2010; 21:5-13.
615
14.
616 617
Tang ML, Kemp AS, Thorburn J, Hill DJ. Reduced interferon-gamma secretion in neonates and subsequent atopy. Lancet 1994; 344:983-5.
15.
Kaminishi K, Soma Y, Kawa Y, Mizoguchi M. Flow cytometric analysis of
618
IL-4, IL-13 and IFN-gamma expression in peripheral blood mononuclear
619
cells and detection of circulating IL-13 in patients with atopic dermatitis
29 620
provide evidence for the involvement of type 2 cytokines in the disease. J
621
Dermatol Sci 2002; 29:19-25.
622
16.
Katsunuma T, Kawahara H, Yuki K, Akasawa A, Saito H. Impaired
623
interferon-gamma production in a subset population of severe atopic
624
dermatitis. Int Arch Allergy Immunol 2004; 134:240-7.
625
17.
Jung T, Lack G, Schauer U, Uberuck W, Renz H, Gelfand EW, et al.
626
Decreased frequency of interferon-gamma- and interleukin-2-producing
627
cells in patients with atopic diseases measured at the single cell level. J
628
Allergy Clin Immunol 1995; 96:515-27.
629
18.
Glines KR, Stiff KM, Freeze M, Cline A, Strowd LC, Feldman SR. An
630
update on the topical and oral therapy options for treating pediatric atopic
631
dermatitis. Expert Opin Pharmacother 2019:1-9.
632
19.
Siegfried EC, Igelman S, Jaworsk JC, Antaya RJ, Cordoro KM, Eichenfield
633
LF, et al. Use of dupilimab in pediatric atopic dermatitis: Access, dosing,
634
and implications for managing severe atopic dermatitis. Pediatr Dermatol
635
2019; 36:172-6.
636
20.
Carel K, Bratton DL, Miyazawa N, Gyorkos E, Kelsay K, Bender B, et al.
637
The Atopic Dermatitis Quickscore (ADQ): validation of a new parent-
638
administered atopic dermatitis scoring tool. Ann Allergy Asthma Immunol
639
2008; 101:500-7.
640 641
21.
Laudanska H, Reduta T, Szmitkowska D. Evaluation of skin barrier function in allergic contact dermatitis and atopic dermatitis using method
30 642
of the continuous TEWL measurement. Rocz Akad Med Bialymst 2003;
643
48:123-7.
644
22.
Czarnowicki T, Gonzalez J, Shemer A, Malajian D, Xu H, Zheng X, et al.
645
Severe atopic dermatitis is characterized by selective expansion of
646
circulating TH2/TC2 and TH22/TC22, but not TH17/TC17, cells within the
647
skin-homing T-cell population. J Allergy Clin Immunol 2015; 136:104-15.
648
23.
Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector
649
memory T cell subsets: function, generation, and maintenance. Annu Rev
650
Immunol 2004; 22:745-63.
651
24.
Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets of
652
memory T lymphocytes with distinct homing potentials and effector
653
functions. Nature 1999; 401:708-12.
654
25.
Ferenczi K, Burack L, Pope M, Krueger JG, Austin LM. CD69, HLA-DR
655
and the IL-2R identify persistently activated T cells in psoriasis vulgaris
656
lesional skin: Blood and skin comparisons by flow cytometry. J Autoimmun
657
2000; 14:63-78.
658
26.
Czarnowicki T, He HY, Wen HC, Hashim PW, Nia JK, Malik K, et al.
659
Alopecia areata is characterized by expansion of circulating
660
Th2/Tc2/Th22, within the skin-homing and systemic T-cell populations.
661
Allergy 2018; 73:713-23.
662 663
27.
Czarnowicki T, He H, Leonard A, Kim HJ, Kameyama N, Pavel AB, et al. Blood endotyping distinguishes the profile of vitiligo from that of other
31 664
inflammatory and autoimmune skin diseases. J Allergy Clin Immunol
665
2019; 143:2095-2107.
666
28.
Gorski K TJ, Gavin M, Cottrell S, Cox C, Vincent M, Ferbas J. CCR4
667
improves phenotypic identification of T-regulatory cells; Validation and
668
implementation of clinical test. Journal Immunol 2010; 184:26.
669
29.
Ferran M, Romeu ER, Rincon C, Sagrista M, Arnau AMG, Celada A, et al.
670
Circulating CLA+ T lymphocytes as peripheral cell biomarkers in T-cell-
671
mediated skin diseases. Exp Dermatol 2013; 22:439-42.
672
30.
Simpson E PA, Siegfried EC, Boguniewicz M, Pariser D, Blauvelt A,
673
Hultsch T, Staudinger H, Zhang R, Kamal MA, Davis JD, Ruddy M,
674
Graham NMH, Bansal A. Dupilumab Efficacy and Safety in Adolescents
675
with Moderate-to-Severe Atopic Dermatitis: Results from a Multicenter,
676
Randomized, Placebo-Controlled, Double-Blind, Parallel-Group, Phase 3
677
Study. Paris, France 2018; 09/15.
678
31.
Simpson EL, Bieber T, Guttman-Yassky E, Beck LA, Blauvelt A, Cork MJ,
679
et al. Two Phase 3 Trials of Dupilumab versus Placebo in Atopic
680
Dermatitis. N Engl J Med 2016; 375:2335-48.
681
32.
Saule P, Trauet J, Dutriez V, Lekeux V, Dessaint JP, Labalette M.
682
Accumulation of memory T cells from childhood to old age: central and
683
effector memory cells in CD4(+) versus effector memory and terminally
684
differentiated memory cells in CD8(+) compartment. Mech Ageing Dev
685
2006; 127:274-81.
32 686
33.
Vieira PL, Wassink L, Smith LM, Nam S, Kingsbury GA, Gutierrez-Ramos
687
JC, et al. ICOS-mediated signaling regulates cytokine production by
688
human T cells and provides a unique signal to selectively control the
689
clonal expansion of Th2 helper cells. Eur J Immunol 2004; 34:1282-90.
690
34.
Paulos CM, Carpenito C, Plesa G, Suhoski MM, Varela-Rohena A,
691
Golovina TN, et al. The Inducible Costimulator (ICOS) Is Critical for the
692
Development of Human T(H)17 Cells. Sci Trans Med 2010; 2.
693
35.
Wikenheiser DJ, Ghosh D, Kennedy B, Stumhofer JS. The Costimulatory
694
Molecule ICOS Regulates Host Th1 and Follicular Th Cell Differentiation
695
in Response to Plasmodium chabaudi chabaudi AS Infection. J Immunol
696
2016; 196:778-91.
697
36.
Vocanson M, Rozieres A, Hennino A, Poyet G, Gaillard V, Renaudineau
698
S, et al. Inducible costimulator (ICOS) is a marker for highly suppressive
699
antigen-specific T cells sharing features of TH17/TH1 and regulatory T
700
cells. J Allergy Clin Immunol 2010; 126:280-9.
701
37.
Kobayashi T, Iijima K, Dent AL, Kita H. Follicular helper T cells mediate
702
IgE antibody response to airborne allergens. J Allergy Clin Immunol 2017;
703
139:300-13.
704
38.
705 706
Wikenheiser DJ, Stumhofer JS. ICOS Co-Stimulation: Friend or Foe? Front Immunol 2016; 7:304.
39.
Simpson TR, Quezada SA, Allison JP. Regulation of CD4 T cell activation
707
and effector function by inducible costimulator (ICOS). Curr Opin Immunol
708
2010; 22:326-32.
33 709
40.
Coyle AJ, Gutierrez-Ramos JC. The role of ICOS and other costimulatory
710
molecules in allergy and asthma. Springer Semin Immunopathol 2004;
711
25:349-59.
712
41.
713 714
Maazi H, Akbari O. ICOS regulates ILC2s in asthma. Oncotarget 2015; 6:24584-5.
42.
Czarnowicki T, Esaki H, Gonzalez J, Malajian D, Shemer A, Noda S, et al.
715
Early pediatric atopic dermatitis shows only a cutaneous lymphocyte
716
antigen (CLA)(+) TH2/TH1 cell imbalance, whereas adults acquire CLA(+)
717
TH22/TC22 cell subsets. J Allergy Clin Immunol 2015; 136:941-51.
718
43.
Esaki H, Czarnowicki T, Gonzalez J, Oliva M, Talasila S, Haugh I, et al.
719
Accelerated T-cell activation and differentiation of polar subsets
720
characterizes early atopic dermatitis development. J Allergy Clin Immunol
721
2016; 138:1473-7.
722
44.
Uwadiae FI, Pyle CJ, Walker SA, Lloyd CM, Harker JA. Targeting the
723
ICOS/ICOS-L pathway in a mouse model of established allergic asthma
724
disrupts T follicular helper cell responses and ameliorates disease. Allergy
725
2019; 74:650-62.
726
45.
Czarnowicki T, Krueger JG, Guttman-Yassky E. Novel concepts of
727
prevention and treatment of atopic dermatitis through barrier and immune
728
manipulations with implications for the atopic march. J Allergy Clin
729
Immunol 2017; 139:1723-34.
730 731
46.
Ferenczi K, Burack L, Pope M, Krueger JG, Austin LM. CD69, HLA-DR and the IL-2R identify persistently activated T cells in psoriasis vulgaris
34 732
lesional skin: blood and skin comparisons by flow cytometry. J Autoimmun
733
2000; 14:63-78.
734
47.
Hulstaert F, Hannet I, Deneys V, Munhyeshuli V, Reichert T, De Bruyere
735
M, et al. Age-related changes in human blood lymphocyte subpopulations.
736
II. Varying kinetics of percentage and absolute count measurements. Clin
737
Immunol Immunopathol 1994; 70:152-8.
738
48.
Erkeller-Yuksel FM, Deneys V, Yuksel B, Hannet I, Hulstaert F, Hamilton
739
C, et al. Age-related changes in human blood lymphocyte subpopulations.
740
J Pediatr 1992; 120:216-22.
741
49.
Protonotariou E, Malamitsi-Puchner A, Rizos D, Papagianni B, Moira E,
742
Sarandakou A, et al. Age-related differentiations of Th1/Th2 cytokines in
743
newborn infants. Mediators Inflamm 2004; 13:89-92.
744
50.
745 746
Adkins B. Development of neonatal Th1/Th2 function. Int Rev Immunol 2000; 19:157-71.
51.
Brunner PM, Israel A, Leonard A, Pavel AB, Kim HJ, Zhang N, et al.
747
Distinct transcriptomic profiles of early-onset atopic dermatitis in blood and
748
skin of pediatric patients. Ann Allergy Asthma Immunol 2019; 122:318-30.
749
52.
Schlapbach C, Gehad A, Yang C, Watanabe R, Guenova E, Teague JE,
750
et al. Human TH9 cells are skin-tropic and have autocrine and paracrine
751
proinflammatory capacity. Sci Transl Med 2014; 6:219ra8.
752
53.
Wong MT, Ong DE, Lim FS, Teng KW, McGovern N, Narayanan S, et al.
753
A High-Dimensional Atlas of Human T Cell Diversity Reveals Tissue-
754
Specific Trafficking and Cytokine Signatures. Immunity 2016; 45:442-56.
35 755
54.
Ciprandi G, De Amici M, Giunta V, Marseglia A, Marseglia G. Serum
756
interleukin-9 levels are associated with clinical severity in children with
757
atopic dermatitis. Pediatr Dermatol 2013; 30:222-5.
758
55.
Esaki H, Brunner PM, Renert-Yuval Y, Czarnowicki T, Huynh T, Tran G, et
759
al. Early-onset pediatric atopic dermatitis is TH2 but also TH17 polarized
760
in skin. J Allergy Clin Immunol 2016; 138:1639-51.
761
56.
Ma L, Xue HB, Guan XH, Shu CM, Zhang JH, Yu J. Possible pathogenic
762
role of T helper type 9 cells and interleukin (IL)-9 in atopic dermatitis. Clin
763
Exp Immunol 2014; 175:25-31.
764
57.
765 766
Immunopathol 2017; 39:47-54. 58.
767 768
Clark RA, Schlapbach C. TH9 cells in skin disorders. Semin
Fujisawa T, Katsumata H, Kato Y. House dust mite extract induces interleukin-9 expression in human eosinophils. Allergol Int 2008; 57:141-6.
59.
Brough HA, Cousins DJ, Munteanu A, Wong YF, Sudra A, Makinson K, et
769
al. IL-9 is a key component of memory TH cell peanut-specific responses
770
from children with peanut allergy. J Allergy Clin Immunol 2014; 134:1329-
771
38.
772
60.
Xie J, Lotoski LC, Chooniedass R, Su RC, Simons FE, Liem J, et al.
773
Elevated antigen-driven IL-9 responses are prominent in peanut allergic
774
humans. PLoS One 2012; 7:e45377.
775 776
61.
Walton S, Nayagam AT, Keczkes K. Age and sex incidence of allergic contact dermatitis. Contact Dermatitis 1986; 15:136-9.
36 777
62.
Statescu L, Branisteanu D, Dobre C, Solovastru LG, Vasilca A, Petrescu
778
Z, et al. Contact dermatitis - epidemiological study. Maedica (Buchar)
779
2011; 6:277-81.
780
63.
Thijs JL, Strickland I, Bruijnzeel-Koomen C, Nierkens S, Giovannone B,
781
Knol EF, et al. Serum biomarker profiles suggest that atopic dermatitis is a
782
systemic disease. J Allergy Clin Immunol 2018; 141:1523-6.
783
64.
Vekaria AS, Brunner PM, Aleisa AI, Bonomo L, Lebwohl MG, Israel A, et
784
al. Moderate-to-severe atopic dermatitis patients show increases in serum
785
C-reactive protein levels, correlating with skin disease activity. F1000Res
786
2017; 6:1712.
787
65.
Brunner PM, Suarez-Farinas M, He H, Malik K, Wen HC, Gonzalez J, et
788
al. The atopic dermatitis blood signature is characterized by increases in
789
inflammatory and cardiovascular risk proteins. Sci Rep 2017; 7:8707.
790
66.
Brunner PM, Silverberg JI, Guttman-Yassky E, Paller AS, Kabashima K,
791
Amagai M, et al. Increasing Comorbidities Suggest that Atopic Dermatitis
792
Is a Systemic Disorder. J Invest Dermatol 2017; 137:18-25.
793
67.
Black A, Bhaumik S, Kirkman RL, Weaver CT, Randolph DA.
794
Developmental regulation of Th17-cell capacity in human neonates. Eur J
795
Immunol 2012; 42:311-9.
796
68.
Lynch JP, Ferreira MA, Phipps S. Th2/Th17 reciprocal regulation: twists
797
and turns in the complexity of asthma phenotypes. Ann Transl Med 2016;
798
4:S59.
37 799
69.
Choy DF, Hart KM, Borthwick LA, Shikotra A, Nagarkar DR, Siddiqui S, et
800
al. TH2 and TH17 inflammatory pathways are reciprocally regulated in
801
asthma. Sci Transl Med 2015; 7:301ra129.
802
70.
Nograles KE, Zaba LC, Shemer A, Fuentes-Duculan J, Cardinale I,
803
Kikuchi T, et al. IL-22-producing "T22" T cells account for upregulated IL-
804
22 in atopic dermatitis despite reduced IL-17-producing TH17 T cells. J
805
Allergy Clin Immunol 2009; 123:1244-52.
806
71.
Weerkamp F, de Haas EF, Naber BA, Comans-Bitter WM, Bogers AJ, van
807
Dongen JJ, et al. Age-related changes in the cellular composition of the
808
thymus in children. J Allergy Clin Immunol 2005; 115:834-40.
809
72.
Pennino D, Bhavsar PK, Effner R, Avitabile S, Venn P, Quaranta M, et al.
810
IL-22 suppresses IFN-gamma-mediated lung inflammation in asthmatic
811
patients. J Allergy Clin Immunol 2013; 131:562-70.
812
73.
Lou H, Lu J, Choi EB, Oh MH, Jeong M, Barmettler S, et al. Expression of
813
IL-22 in the Skin Causes Th2-Biased Immunity, Epidermal Barrier
814
Dysfunction, and Pruritus via Stimulating Epithelial Th2 Cytokines and the
815
GRP Pathway. J Immunol 2017; 198:2543-55.
816
74.
817 818
Fujita H. The role of IL-22 and Th22 cells in human skin diseases. J Dermatol Sci 2013; 72:3-8.
75.
Teraki Y, Sakurai A, Izaki S. IL-13/IL-22-coproducing T cells, a novel
819
subset, are increased in atopic dermatitis. J Allergy Clin Immunol 2013;
820
132:971-4.
38 821
76.
Liang SC, Tan XY, Luxenberg DP, Karim R, Dunussi-Joannopoulos K,
822
Collins M, et al. Interleukin (IL)-22 and IL-17 are coexpressed by Th17
823
cells and cooperatively enhance expression of antimicrobial peptides. J
824
Exp Med 2006; 203:2271-9.
825
77.
Gittler JK, Shemer A, Suarez-Farinas M, Fuentes-Duculan J, Gulewicz KJ,
826
Wang CQ, et al. Progressive activation of T(H)2/T(H)22 cytokines and
827
selective epidermal proteins characterizes acute and chronic atopic
828
dermatitis. J Allergy Clin Immunol 2012; 130:1344-54.
829
78.
Hulshof L, Overbeek SA, Wyllie AL, Chu M, Bogaert D, de Jager W, et al.
830
Exploring Immune Development in Infants With Moderate to Severe Atopic
831
Dermatitis. Front Immunol 2018; 9:630.
832
79.
833 834
Long SA, Buckner JH. CD4+FOXP3+ T regulatory cells in human autoimmunity: more than a numbers game. J Immunol 2011; 187:2061-6.
80.
McHugh RS, Shevach EM. The role of suppressor T cells in regulation of
835
immune responses. Journal of Allergy and Clinical Immunology 2002;
836
110:693-702.
837
81.
Hall BM. T Cells: Soldiers and Spies--The Surveillance and Control of
838
Effector T Cells by Regulatory T Cells. Clin J Am Soc Nephrol 2015;
839
10:2050-64.
840
82.
Burr ML, Dunstan FD, Hand S, Ingram JR, Jones KP. The natural history
841
of eczema from birth to adult life: a cohort study. Br J Dermatol 2013;
842
168:1339-42.
39 843
83.
Carlsten C, Dimich-Ward H, Ferguson A, Watson W, Rousseau R,
844
Dybuncio A, et al. Atopic dermatitis in a high-risk cohort: natural history,
845
associated allergic outcomes, and risk factors. Ann Allergy Asthma
846
Immunol 2013; 110:24-8.
847
84.
Guttman-Yassky E, Krueger JG. Atopic dermatitis and psoriasis: two
848
different immune diseases or one spectrum? Curr Opin Immunol 2017;
849
48:68-73.
850
85.
Czarnowicki T, He H, Krueger JG, Guttman-Yassky E. Atopic dermatitis
851
endotypes and implications for targeted therapeutics. J Allergy Clin
852
Immunol 2019; 143:1-11.
853
86.
Leung DY, Guttman-Yassky E. Deciphering the complexities of atopic
854
dermatitis: shifting paradigms in treatment approaches. J Allergy Clin
855
Immunol 2014; 134:769-79.
856
87.
Guttman-Yassky E, Nograles KE, Krueger JG. Contrasting pathogenesis
857
of atopic dermatitis and psoriasis--part I: clinical and pathologic concepts.
858
J Allergy Clin Immunol 2011; 127:1110-8.
859
88.
Burr ML, Dunstan FD, Hand S, Ingram JR, Jones KP. The natural history
860
of eczema from birth to adult life: a cohort study. Br J Dermatol 2013;
861
168:1339-42.
862
89.
863 864 865
Margolis JS, Abuabara K, Bilker W, Hoffstad O, Margolis DJ. Persistence of mild to moderate atopic dermatitis. JAMA Dermatol 2014; 150:593-600.
90.
Abuabara K, Margolis DJ. Do children really outgrow their eczema, or is there more than one eczema? J Allergy Clin Immunol 2013; 132:1139-40.
40 866
Figure legends
867
Figure 1. A-D) The frequency of CLA+ Tem (CD45RO+CCR7-) and Tcm
868
(CD45RO+CCR7+) in CD4+/CD8+ T cells and (E-L) ICOS+ activation in CLA+/-
869
CD4+/CD8+ Tcm/Tem cells in healthy controls and AD patients across ages
870
(ICOS+CLA+CD45RO+CCR7+/-). Bar plots represent means (black)/medians
871
(red) ± SEMs. AD, Atopic dermatitis; CLA, Cutaneous lymphocyte antigen; SEM,
872
Standard error of the mean/median; Tcm, central memory T-cells; Tem, effector
873
memory T-cells. P-values are designated as ***<0.001, **<0.01, *<0.05, and
874
+
875
Figure 2. A-D) IFN-γ+, (E-H) IL-13+, and (I-L) IFN-γ+/IL-13+ cytokine frequencies
876
in CLA+, CLA-, CD4+ and CD8+ T-cells in healthy controls and AD patients across
877
ages. The ratio between the % of IFN-γ+CLA+CD4+ (or CD8+) T cells and the %
878
of their IL-13+ counterpart was calculated for each sample but was not multiplied
879
by 100 and is therefore unitless. Bar plots represent means (black)/medians (red)
880
± SEMs. AD, Atopic dermatitis; CLA, Cutaneous lymphocyte antigen; SEM,
881
Standard error of the mean/median. P-values are designated as ***<0.001,
882
**<0.01, *<0.05, and +<0.1.
883
Figure 3. A-D) IL-9+ and (E-H) IL-17+ and (I-L) IL-22+ frequencies in CLA+, CLA-,
884
CD4+ and CD8+ T-cells in healthy controls and AD patients across ages. Bar
885
plots represent means (black)/medians (red) ± SEMs. AD, Atopic dermatitis;
886
CLA, Cutaneous lymphocyte antigen; SEM, Standard error of the mean/median.
887
P-values are designated as ***<0.001, **<0.01, *<0.05, and +<0.1.
<0.1.
41 888
Figure 4. Unsupervised hierarchical clustering heatmap displaying polarized T-
889
cell subsets for control and AD across age groups (red: positive/increase; blue:
890
negative/decrease). Fold-changes (FCHs) of the mean frequencies of AD vs.
891
controls for each age group are listed on the right (stars and plus signs display
892
significance). The green cluster includes subsets that were relatively low and
893
stable among controls, but incrementally increased with age in AD. The pink box
894
shows increased IL-9 frequencies in childhood, which decline in adulthood,
895
particularly in AD. The yellow cluster shows markers that increased in both
896
controls and AD.
897
Figure 5. Unsupervised hierarchical clustering of polarized cytokine subset
898
frequencies (%) with AD clinical measures using Spearman correlation as a
899
similarity metric. CD4+/CD8+ IL-13+, IL-17+ and IL-22+ subsets clustered together
900
(turquoise box). IFN-γ+-producing T-cell subsets grouped together with age and
901
disease duration (yellow box). IL-9+ and some IL-13+ and IL-22+ T-cells (purple
902
box) clustered adjacent to AD clinical measures (green box). The heatmap
903
shows the positive (red) or negative (blue) correlations of all parameters, with
904
color intensity reflecting the strength of the correlation (-1 to +1). Dendrograms
905
are shown as a tree, representing the distance between variables. CLA,
906
Cutaneous lymphocyte antigen. P-values are designated as ***<0.01, **<0.01,
907
*<0.05, and +<0.1.
908
Figure 6. Spearman correlation scatter plots (linear regression
909
[red=AD/blue=control line] with their CI [in gray]) for (A-E) SCORAD and (F-L)
910
age versus clinical measures and Tem/Tcm subset frequencies (%). Dots colors
42 911
in plots A-D, F designate different AD patient ages from infancy to adulthood, as
912
shown in Figure 1. CI, Confidence interval; CLA, Cutaneous lymphocyte antigen;
913
EASI, Eczema area and severity index; LS, Lesional; NL, Non-lesional;
914
SCORAD, SCORing of Atopic Dermatitis; Tcm, Central memory T-cells; Tem,
915
Effector memory T-cells; TEWL, Transepidermal water loss.
916
Figure 7. Spearman correlation scatter plots (linear regression
917
[red=AD/blue=control line] with their CI [in gray]) for CD4+/CD8+, CLA+/CLA- (A-
918
D) IFN-γ+/IL-13+ producing T-cell ratio, (E-H) IL-13+, and (I-L) IL-22+ producing T-
919
cell frequencies (%) versus age in AD patients (red dots/line) and controls (blue
920
dots/line). CI, Confidence interval; CLA, Cutaneous lymphocyte antigen.
921
Figure 8. Unsupervised clustering of AD patients and healthy controls across all
922
principal components of the blood flow cytometry marker frequencies (%) by k-
923
means. In AD, the frequencies of different markers defined three meaningful age-
924
clusters, aligning along a spectrum. While infants (pink ellipse) clustered on the
925
far left, and adults (green ellipse) on the right, children and adolescents (blue
926
ellipse) clustered together between the other age cohorts. The markers that best
927
distinguished between clusters appear in the boxes between two cohorts (colors
928
of markers parallel the colors of the relative age group). Arrows designate
929
elevated frequencies of a given marker among the specific age group. In healthy
930
controls, clusters did not clearly align patients along an age spectrum. CLA,
931
Cutaneous lymphocyte antigen; ICOS, Inducible co-stimulator molecule; Tregs,
932
T-regulatory cells.
933
Figure 1
A
100 100
**
** **
75
Frequency (%)
B
CLA CD4 Tcm cells
CD4 CM CLA+
150
** **
75
** **
100
50
50
25
0
40
+*
40 40
0
lts du ents
A
sc
e ol
0
0
+
+ +
+*
0
en
lts
sc
le
du
do
A
A
n re ld s nt
hi
fa
ts
AD
ICOS CLA CD8 Tem cells CD8 EM ICOS+/CLA
30 30
**
** **
C
s
L
In
lts
nt
ce
**
**
es
*** + *** *
100
du
**
ol
fa
ts
Normal
CLA CD8 Tem cells
CD8 EM ICOS+/CLA+
A
d n A re ld hi C ts n
In
lts
AD
100
20
du
n
**
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
en
**
K ICOS
sc
le
s
+
A
do
lts
nt
ce
es
A re ld hi s nt fa
C
In
ol
du
fa
+*
A
d n A re ld hi s nt
C
lts
ts
+
In
en
sc
le
+ *
40
30
Normal
CLA CD8 Tcm cells
CD8 CM ICOS+/CLA
30
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
AD
40 *** ***
**
30
du
do
A
J ICOS
A
hi
fa
s
n re ld s nt
C
In
lts
nt
ce
es
du
**
ol
* **
A
d n A re ld hi C ts n
fa
lts
ts
+
n d A re ld hi C ts n fa In s lt du
90
0
In
en
sc
le
* * +
+ +
+*
** **
50
Normal
+ *
CLA CD4 Tem cells
CD4 EM ICOS+/CLA
90
50
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
du
do
A
A
hi
fa
s
n re ld s nt
C
In
lts
nt
ce
es
fa
ol
du
A
d n A re ld hi s nt
C
In
CD8 CM ICOS+/CLA+
120
AD
30
AD
ICOS CLA CD8 Tcm cells
ts en
*
** *** *** **
120
60
0
Normal
sc
*** ***
H ICOS
60
20 0
e ol
CLA CD4 Tem cells
CD4 EM ICOS+/CLA+
100
*** **
+
A
Normal
20
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
d n A re ld hi C ts n fa In
lts du
ts en
sc
e ol
ts en
sc
e ol
G ICOS
A
n d A re ld hi C ts n fa In s lt du
A
d n A re ld hi C ts n fa In
lts du
ts en
sc
e ol
*** ***
**
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
AD
100
**
+
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
+*
+*
**
20
A
n d A re ld hi C ts n fa In s lt du
** ** ** ***
40
0
100
+
Normal
** **
**
40
25
100
**
** ** + * *** ***
** **
*** ***
20
0
CLA CD4 Tcm cells
60
* **
** ***
AD
CD4 CM ICOS+/CLA
60
25 0
ts en
**
sc
**
** **
e ol
75
A
75
** *
**
80
60
20
d n A re ld hi C ts n fa In
*** ***
+
50
lts du
100
50
ts en
sc
sc
le
e ol
100
F ICOS 80
*** ***
**
CLA CD8 Tem cells
CD8 EM CLA+
60
20
Normal
CLA CD4 Tcm cells
** **
0
AD
CD4 CM ICOS+/CLA+
** **
40
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
A
n do A re ld hi C ts n fa In s lt du ents
A
d n A re ld hi C ts n fa In
E ICOS Frequency (%)
60
**
D
CLA CD8 Tcm cells
CD8 CM CLA+
60
*** *** ** *
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
Normal
I
** ** *
50
25 0
Frequency (%)
**
100
50
0
+ *
** **
**
**
C
CLA CD4 Tem cells
CD4 EM CLA+ 150
+ *
+
20 20
20
50 50
50 50
10 10
10 10
0
0
0
0
0
ts en
AD
ts
sc
le
l du
sc
e ol
Normal
A
A
n do A re ld hi C ts n fa In s lt du ents
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
d n A re ld hi C ts n fa In
ts en
AD
ts
sc
le
l du
sc
e ol
Normal
A
A
n do A re ld hi C ts n fa In s lt du ents
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
d n A re ld hi C ts n fa In
ts en
AD
ts
sc
le
l du
sc
e ol
Normal
A
n do A re ld hi C ts n fa In s lt du ents
A
ts en
ts
sc
le
l du
sc
e ol
AD
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
d n A re ld hi C ts n fa In
A
n do A re ld hi C ts n fa In s lt du ents
A
d n A re ld hi C ts n fa In
Normal
0
0
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
Figure 2
Frequency (%)
A
CD4 T cells
CD4 IFNg+CLA+
60
60
40
** **
**
IFN-
*** ***
+
+*
**
B
CLA
100
*** ***
75
+
75
40
*** *** *** ***
50 50
**
C
CLA
**
IFN-
CLA
CD8 IFNg+CLA+
* **
*** *** *** *** *** ***+ + + + ** **
** *
+ +
75 75
D
+
CD8 IFNg+CLA
IFN-
**
*** *** ** *** *** ***
100 100
CLA *** *** ** ** *** + ** * **
50
+
50
50 50
20
lts du
ts en
le
ts
en
lts
sc
AD
CLA /IL-13 CLA
CD8 IFNg+CLA /IL13+CLA
1000
+
**
**
1000
**
du
s
IFN-
do
nt
n re ld s nt
hi
lts
s
*** *
A
A
C
fa
du
In
nt
ce
es
ol
lts
s
+
A
du
ce
es
ol
fa
lts
nt
L
CLA /IL-13 CLA
CD8 IFNg+CLA+/IL13+CLA+
75
Infants.Normal Children.Normal Adolescents.Normal Adults.Normal Infants.AD Children.AD Adolescents.ADAdults.AD
d n A re ld hi C ts n fa In
A
n d A re ld hi s nt C
du
In
A
K IFN75 50
** **
A
sc
*
Normal
50
*
500
500
25 25
0
0
0
0 Adults.AD
lts
du ts
n
ts
AD
en
sc
le
do
A
A e r ld hi s nt fa
Children.AD Adolescents.AD
C
In
en
n Normal
Infants.AD
lts
du
sc
le
do
A
A
nt
re ld s
fa
hi
C
Infants.Normal Children.Normal Adolescents.NormalAdults.Normal
In
ts
en
lts
n
ts
AD
du
sc
le
do
lts
du
C
Adults.AD
A
Children.AD Adolescents.AD
A re ld hi s nt fa
Infants.AD
In
n
ts
Normal
en
sc
le
do
A
A
nt
re ld s
fa
hi
C
lts
du
n
en
sc
le
do
ts
en
AD
Infants.Normal Children.NormalAdolescents.NormalAdults.Normal
In
Adults.AD
A
C
In
A re ld hi s nt fa
Children.AD Adolescents.AD
lts
n
ts
Normal
Infants.AD
du
A
sc
le
do
A
nt
re ld s
fa
hi
C
Infants.Normal Children.NormalAdolescents.NormalAdults.Normal
In
lts
du
n
ts
AD
en
sc
le
do
A
A re ld hi s nt fa
C
lts
du
In
n
en
sc
le
do
A
A
nt
re ld s
fa
hi
C
In
Normal
ce
*** * *** * ** +*
0
AD
*
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
es
*** ***
ol
*** ***
+*
*** *** ** **
d n A re ld hi C ts n fa In
ts
0
en
0
lts
sc
10
le
20
**
**
+ *
*** *** *** ***
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
du
20
40
do
** *
40
+*
0
Normal
CLA /IL-13 CLA
+
*** *** * *** * * *** *
1
AD
CD4 IFNg+CLA /IL13+CLA
60
e ol
10
1
2
A
n re ld s nt
fa
hi
60
A
C
s
*
nt
20
lts
J IFN-
40
*
du
*
In
*** *
ce
80
es
s
30
ol
nt
s
*** *
*** **
* +
120
A
lts
nt
ce
es
du
ol
fa
lts
Normal
CLA /IL-13 CLA
CD4 IFNg+CLA+/IL13+CLA+
40
d n A re ld hi C ts n fa In
A
n d A re ld hi s nt C
du
In
A
ce
es
ol
d n A re ld hi C ts n fa In
IFN-
AD
n d A re ld hi C ts n fa In s lt du
3
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
ts en
3 2
+ *
+ *
4
20
AD
IL-13 CLA
CD8 IL13+CLA
4
10
0
sc
30
0 Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
e ol
5
*** *** ** ** *** ***
30
A
5
*** ***
+
40
5
d n A re ld hi C ts n fa In
lts du
ts en
sc
H
IL-13 CLA
40 +
Normal
20
Normal
0
e ol
*
**
A
50
CD8 IL13+CLA+
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
AD
50
10
0
I
n d A re ld hi C ts n fa In s lt du
10
ts en
**
A
G
5
0
sc
15
e ol
Normal
** **
** *
** **
15
d n A re ld hi C ts n fa In
lts du
ts en
sc
le
sc
*** + *** +
AD
IL-13 CLA
CD4 IL13+CLA
0
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
A
n do A re ld hi C ts n fa In s lt du ents
A
e ol
F
*
**
d n A re ld hi C ts n fa In
Normal
IL-13 CLA
40
* **
lts du
AD
*** ***
40
ts en
sc
e ol
ts en
CD4 IL13+CLA+
0 0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
A
n d A re ld hi C ts n fa In s lt du
A
sc
e ol
d n A re ld hi C ts n fa In
Normal
20
0 0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
20
25
25
0
E
25
25
20
0
Frequency (%)
IFN-
CD4 IFNg+CLA
100
*** *** *** ***
**
CD8 T cells
Figure 3
A
CD4 T cells ** *
** *
Frequency (%)
B
IL-9 CLA
CD4 IL9+CLA+
*** **
40
+*
90
*** *
0
ts en
sc
le
lts du
A
n do A re ld hi C ts n fa In s lt du ents
A
lts
0
ts
0
en
sc
2
le
2
du
**
0
*** *** *** * ***
*** *** *** *** ** *** * **
4
+*
AD
IL-22 CLA
CD8 IL22+CLA
4
do
**
A
s
n re ld s nt
hi
fa
nt
ce
lts
L
Adults.AD
A
C
In
Children.AD Adolescents.AD
Normal
*** + *** *** *
Infants.AD
du
**
A
lts
ts
en
sc
es
ol
le
du
do
d n A re ld hi C ts n fa In
Infants.Normal Children.Normal Adolescents.NormalAdults.Normal
A
s
n re ld s nt
hi
fa
nt
**
25
0
sc
le
lts du ents
Normal
A
sc
n re
s
n do A re ld hi C ts n fa In s lt du ents
A
le
do
A
ld
nt
fa
hi
C
AD
Infants.Normal Children.Normal Adolescents.Normal Adults.Normal Infants.AD Children.AD Adolescents.ADAdults.AD
In
sc
le
lts du ents
Normal
A
n do A re ld hi C ts n fa In s lt du ents
A
n re
sc
le
do
A
s
nt
ld
fa
hi
C
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
In
sc
le
sc
le
lts du ents
A
n do A re ld hi C ts n fa In s lt du ents
do
A
A
s
n re
ld
nt
fa
hi
C
AD
sc
0.5
Adults.AD
50
Normal
e ol
ts en
C
lts
ce
es
du
In
A
Children.AD Adolescents.AD
50
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
d n A re ld hi C ts n fa In
lts du
A
sc
0.5
AD
+
25
In
sc
le
lts du ents
A
n do A re ld hi C ts n fa In s lt du ents
A
s
n re
sc
le
do
A
ld
nt
fa
hi
C
In
AD
ol
0
Infants.AD
*
0
Normal
**
IL-22 CLA
75
10
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
CD8 IL22+CLA+
75
5
0
A
ts
en
lts
sc
*** *** *** *** *** *** ** *** ** **
*** *** * *** *** *** **
5
0
1.0
0.0
d n A re ld hi C ts n fa In
le
du
10
do
10
A
s
n re ld s nt
fa
hi
lts
nt
*
15
10
1
**
0 Infants.Normal Children.Normal Adolescents.NormalAdults.Normal
K
+
*** *** *** * **
1.5
Normal
IL-22 CLA
CD4 IL22+CLA
+*
A
C
In
du
ce
es
ol
20
1.5
+ +
AD
15
+ +
20
A
lts
ts
en
sc
+
d n A re ld hi C ts n fa In
le
J
*** * *** ** * **
2.0
0.0
Infants.Normal Children.Normal Adolescents.Normal Adults.Normal Infants.AD Children.AD Adolescents.ADAdults.AD
du
do
s
30
+ +
le
0
0
A
nt
n re ld s nt
hi
fa
lts
40
+
AD
IL-17 CLA
CD8 IL17+CLA
2
2.5
Normal
IL-22 CLA
*** *** *** ** +* *** ***
**
2.5
AD
40 30
A
C
du
In
A
ce
es
ol
CD4 IL22+CLA+
H
+
5
0
d n A re ld hi C ts n fa In
50
n do A re ld hi C ts n fa In s lt du ents
7.5
2
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
50
+ +
7.5
2
0
I
sc
10
Normal
IL-17 CLA
CD8 IL17+CLA+
10.0
5
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
AD
5.0
5
Normal
e ol
** **
A
G
10
0
d n A re ld hi C ts n fa In
4
lts du ents
sc
sc
le
e ol
**
4
**
Normal
*** *** + *** ** ** *
6
10
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
AD
IL-17 CLA
CD4 IL17+CLA
+*
0
0
A
n do A re ld hi C ts n fa In s lt du ents
A
d n A re ld hi C ts n fa In
ts en
sc
le
sc
lts du
A
n do A re ld hi C ts n fa In s lt du ents
A
e ol
d n A re ld hi C ts n fa In
F **
**
4
0
Infants.Normal Children.Normal Adolescents.Normal Adults.Normal Infants.AD Children.AD Adolescents.ADAdults.AD
6
15
8
4
Normal
+
15
8
*
30
AD
IL-17 CLA
20
+
*** *** ** * *** ***
30
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
20
+
12
+ +
+
IL-9 CLA
CD8 IL9+CLA
12
60
2
CD4 IL17+CLA+
+
** *
90
2
Normal
Frequency (%)
**
+
60
0
Frequency (%)
+
**
120
D
IL-9 CLA
CD8 IL9+CLA+
120
4
20
E
+
** *
4
20
0
+*
+ +
6
*** ***
** **
C
IL-9 CLA
CD4 IL9+CLA
6
*** ***
*** **
40
CD8 T cells
AD
Figure 4 Color Key 1.5
0.5 0.5 1.5 Row Z Score
Normal
AD
AD/Normal
AdolesInfants Children Adolescents Adults Infants Children cents Adults CD4⁺ Tem ICOS⁺/CLA⁺ CD8⁺ Tcm HLA-DR⁺/CLA⁻ CD8⁺ Tem HLA-DR⁺/CLA⁺ CD4⁺ IL22⁺CLA⁺ CD8⁺ IL22⁺CLA⁺ CD8⁺ IL13⁺ CD8⁺ IL13⁺CLA⁺ CD4⁺ IL13⁺CLA⁺ CD8⁺ Effector CD4⁺ Effector CD8⁺ Tem ICOS⁺/CLA⁺ CD4⁺ Tem HLA-DR⁺/CLA⁺ CD8⁺ Tcm HLA-DR⁺/CLA⁺ CD4⁺ Tcm ICOS⁺/CLA⁺ CD8⁺ Naïve CD8⁺ Tcm ICOS⁺/CLA⁺ CD8⁺ Tcm ICOS⁺/CLA ⁻ Tregs CCR4⁺CLA⁺ CD4⁺ IL13⁺ CD4⁺ IL13⁺CLA⁻ CD4⁺ Tcm HLA-DR⁺/CLA⁺ CD4⁺ Tcm HLA-DR⁺/CLA⁻ CD4⁺ Tem HLA-DR⁺/CLA⁻ T Regs (CD25⁺CD127⁻) CD4⁺ IL9⁺ CD4⁺ IL9⁺CLA⁻ CD4⁺ IL9⁺CLA⁺ CD4⁺ Tcm ICOS⁺/CLA⁻ CD8⁺ IL9⁺CLA⁻ CD4⁺ Tem CD8⁺ IL17⁺CLA⁺ CD8⁺ IL17⁺ CD8⁺ IL17⁺CLA⁻ CD4⁺ Naïve CD8⁺ Effector CLA⁺ CD8⁺ Tem CLA⁺ CD8⁺ Tcm CLA⁺ CD8⁺ Tem ICOS⁺/CLA⁻ CD4⁺ Tem CLA⁺ CD4⁺ Tcm CLA⁺ CD8⁺ IL9⁺CLA⁺ CD8⁺ IL9⁺ CD4⁺ Tem ICOS⁺/CLA⁻ CD8⁺ IFN-γ⁺CLA⁻ CD8⁺ IFN-γ⁺ CD4⁺ IFN-γ⁺CLA⁻ CD4⁺ IFN-γ⁺ CD4⁺ IFN-γ⁺CLA⁺ CD4⁺ Tcm CD4⁺ IFN-γ⁺CLA⁻/IL13⁺CLA⁻ CD8⁺ IFN-γ⁺CLA⁺ CD4⁺ IL22⁺ CD4⁺ IL22⁺CLA⁻ CD4⁺ IL17⁺ CD4⁺ IL17⁺CLA⁻ CD8⁺ IL13⁺CLA⁻ CD8⁺ IL22⁺ CD8⁺ IL22⁺CLA⁻ CD8⁺ Tem HLA-DR⁺/CLA⁻ CD4⁺ IL17⁺CLA⁺ CD4⁺ Effector CLA⁺ CD4⁺ CLA⁺ CD8⁺ IFN-γ⁺CLA⁻/IL13⁺CLA⁻ CD8⁺ Tcm CD8⁺ Tem CD8⁺ IFN-γ⁺CLA⁺/IL13⁺CLA⁺ CD4⁺ IFN-γ⁺CLA⁺/IL13⁺CLA⁺
: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :
Infants Children Adolescents Adults 1.48⁺ 1.52 1.14 1.16 2.51 1.67 1.21 3.69** 1.09 1.16 1.12 1.62⁺ 1.08 1.45⁺ 1.23* 1.24 1.10 1.38 1.21 1.04 2.04⁺ 1.98 1.40 1.12 1.05 1.03 1.17 1.91* 1.40 1.18 1.53 1.24 1.32 1.02 1.22 1.12 1.11 1.57 1.43** 1.51** 1.20 1.39 2.03** 1.11 1.10 1.02 1.04 1.54* 1.07 1.73⁺ 1.16 1.00 1.17 1.35 1.38 1.13 2.12⁺ 1.86 1.08 1.41⁺ 1.10 1.56* 1.27 1.65* 1.39⁺ 1.65 5.97*
1.03 1.01 1.55 1.62⁺ 2.79* 2.33⁺ 2.02⁺ 1.38 1.24 1.33 1.81* 1.34 1.09 1.02 1.06 1.71⁺ 2.27* 2.88** 4.04** 4.20** 1.87* 2.52⁺ 1.64 1.74** 2.10⁺ 2.22⁺ 1.05 1.13 1.33 1.29 1.60 1.01 1.47 1.08 1.13 1.92** 1.89** 1.61 1.46* 1.66* 1.60 1.19 1.13 1.43* 1.42* 1.46* 1.46* 1.40⁺ 1.08 3.28** 1.16 2.28* 2.34* 1.19 1.22 2.54* 1.90 1.76 1.94⁺ 1.39 1.35 1.57 2.58* 1.50** 1.15 1.25 3.31*
1.40 1.98* 1.66 2.21** 1.88 1.44 1.13 1.64⁺ 1.15 1.14 1.73* 1.95 3.24* 1.26 1.33** 1.08 1.52 1.40 1.71* 1.78* 1.82 1.72 1.72 1.01 1.38 1.48 1.08 1.24 1.46 1.80* 4.65* 8.49 8.88 1.30** 1.11 1.08 1.10 1.24 1.28 1.05 1.37 1.24 1.11 1.16 1.17 1.14 1.14 1.36⁺ 1.41⁺ 1.61 1.31 1.16 1.15 1.25 1.29 1.66 1.10 1.11 1.12 1.00 1.38 1.21 1.55 1.79** 2.40** 1.41 1.76⁺
1.99** 1.89** 3.18** 2.04** 3.59** 1.90** 2.85** 2.43** 1.57** 2.03⁺ 1.95** 2.13** 2.84** 2.42** 1.09 1.47* 1.03 1.28⁺ 2.31** 1.85** 3.01** 2.12⁺ 1.02 1.39** 1.86* 1.38 1.08 1.65** 1.23 1.23 1.51⁺ 1.03 1.08 1.02 1.52* 1.02 1.14 1.22 1.16 1.00 1.01 1.45⁺ 1.44⁺ 1.04 1.03 1.05 1.07 1.14 1.23⁺ 1.76 1.07 1.16 1.31⁺ 1.25 1.24 1.50* 1.16 1.04 1.66** 1.13 1.23 1.72** 1.43⁺ 1.05 1.01 2.24* 3.49**
CD8−CLA− CD8⁺CLA⁺
**
**
**
TEWL−LS TEWL LS
+
**
***
*
+
*
**
*
**
**
*
*
***
**
*
*
***
***
*
*
+
*
*
***
***
***
***
***
***
**
+
+
***
***
***
***
***
***
**
**
***
*
***
***
**
***
***
***
***
***
***
***
***
***
*
***
***
***
***
***
***
***
**
***
***
***
***
***
CD8−IL13−CLA−−1 CD8⁺IL13⁺CLA⁻
***
***
***
***
***
CD8−IL−17−CLA− CD8⁺IL17⁺CLA⁺
***
***
CD8−IL13−CLA− CD8⁺IL13⁺CLA⁺
***
CD8−IL22−CLA− CD8⁺IL22⁺CLA⁺
*
***
***
***
***
***
***
***
***
X−CD8−IL22− CD8⁺IL22⁺ CD8−IL22−CLA−−1 CD8⁺IL22⁺CLA⁻ X−CD8−IL13− CD8⁺IL13⁺
X−CD8−IL−17− CD8⁺IL17⁺
***
CD8−IL−17−CLA−−1 CD8⁺IL17⁺CLA⁻
***
*
*
**
**
**
*
+
**
**
+
*
***
**
*
***
***
***
***
***
***
***
*
+
** +
+
*
CD8⁺IL9⁺CLA⁻
CD8⁺IL9⁺
CD8⁺IL9⁺CLA⁺
CD4⁺IL9⁺CLA⁻
+
***
*
+
+
*
*
*
*
***
**
**
*
+
***
***
***
***
*
***
***
***
***
***
**
*
***
***
***
***
*
*
+
***
***
***
***
***
***
**
**
*
*
CD4⁺IL9⁺ *
** *
+
CD4⁺IL9⁺CLA⁺
CD4⁺IL13⁺CLA⁻
CD4⁺IL13⁺
CD4⁺IL13⁺CLA⁺
CD4⁺IL22⁺CLA⁺
+
+ *
*
**
+
TEWL−NL TEWL NL CD4−IL−17−CLA− CD4⁺IL17⁺CLA⁺
Total ADQ
Pruritus ADQ
Eosinophils CD8⁺IFNγ⁺CLA⁻
CD8⁺IFNγ⁺
CD8⁺IFNγ⁺CLA⁺
CD4⁺IFNγ⁺CLA⁻
CD4⁺IFNγ⁺
CD4⁺IFNγ⁺CLA⁺
Age
CD4⁺IL17⁺CLA⁻
CD4⁺IL17⁺
CD4⁺IL22⁺CLA⁻
CD4⁺IL22⁺
*
EASI
***
SCORAD
CD4−CLA− CD4⁺CLA⁺
CD8⁺IL17⁺CLA⁻
CD8⁺IL17⁺
CD8⁺IL17⁺CLA⁺
CD8⁺IL13⁺CLA⁻
CD8⁺IL13⁺
CD8⁺IL22⁺CLA⁻
Distance: spearman / average
Duration of Disease
−1.0
CD8⁺IL22⁺
−0.5
CD8⁺IL22⁺CLA⁺
0.0
CD8⁺IL13⁺CLA⁺
TEWL NL
0.5
CD8⁺CLA⁺
CD4⁺CLA⁺
1.0
TEWL LS
cor
CD4⁺IL17⁺CLA⁺
Figure 5
+
+
+ +
+
*
+
*
+
***
***
*
+
*
*
**
***
**
**
* +
**
***
* **
**
***
***
***
+
***
***
*
*
***
***
***
***
***
*
***
***
*
**
***
***
***
***
***
**
***
***
**
**
+
+
**
***
***
***
***
***
***
***
***
***
***
+
*
**
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
X−CD4−IL22− CD4⁺IL22⁺ CD4−IL22−CLA−−1 CD4⁺IL22⁺CLA⁻ X−CD4−IL−17− CD4⁺IL17⁺ CD4−IL−17−CLA−−1 CD4⁺IL17⁺CLA⁻ Age Age Duration−of−disease Duration of Disease CD4−IFNg−CLA− CD4⁺IFNγ⁺CLA⁺
+ *
+
***
***
***
***
***
***
***
***
***
***
**
**
**
***
***
***
***
***
***
***
***
***
***
***
***
***
***
*
***
***
+
***
***
***
***
+
***
*
***
***
***
***
***
***
+
***
***
**
**
+
***
***
**
**
***
***
+
*
+
*
***
*
+
**
+
*
***
*
+
**
CD4⁺IFNγ⁺ CD4−IFNg− CD4−IFNg−CLA−−1 CD4⁺IFNγ⁺CLA⁻ CD8−IFNg−CLA− CD8⁺IFNγ⁺CLA⁺ CD8−IFNg− CD8⁺IFNγ⁺
+
+
* +
**
*
CD8−IFNg−CLA−−1 CD8⁺IFNγ⁺CLA⁻
Eosinophils Eosinophils
**
Pruritus−ADQ Pruritus ADQ
*
*
**
***
***
***
***
***
Total−ADQ Total ADQ SCORAD SCORAD
***
EASI EASI
+ +
*
** *
+
*
+
+
*
***
**
***
*
+
**
**
**
**
**
**
** **
*
*
***
*
* *
*
*
+
CD4−IL22−CLA− CD4⁺IL22⁺CLA⁺
** ***
CD4−IL13−CLA− CD4⁺IL13⁺CLA⁺
***
***
+
***
+
*
*
**
*
**
+
*
*
+
+
*
***
**
***
***
*
***
***
+
*
***
***
***
***
*
**
***
***
***
*
**
***
***
***
**
**
***
***
***
***
***
***
***
***
***
X−CD4−IL13− CD4⁺IL13⁺ CD4−IL13−CLA−−1 CD4⁺IL13⁺CLA⁻ CD4−IL9−CLA− CD4⁺IL9⁺CLA⁺ X−CD4−IL9− CD4⁺IL9⁺ CD4−IL9−CLA−−1 CD4⁺IL9⁺CLA⁻ CD8−IL9−CLA− CD8⁺IL9⁺CLA⁺ X−CD8−IL9− CD8⁺IL9⁺
***
***(p<0.001) **(p<0.01) *(p<0.05) +(p<0.1)
CD8⁺IL9⁺CLA⁻ CD8−IL9−CLA−−1
CD8⁺IL9⁺ X−CD8−IL9−
CD8⁺IL9⁺CLA⁺ CD8−IL9−CLA−
CD4⁺IL9⁺CLA⁻ CD4−IL9−CLA−−1
CD4⁺IL9⁺ X−CD4−IL9−
CD4⁺IL9⁺CLA⁺ CD4−IL9−CLA−
CD4⁺IL13⁺CLA⁻ CD4−IL13−CLA−−1
CD4⁺IL13⁺ X−CD4−IL13−
CD4⁺IL13⁺CLA⁺ CD4−IL13−CLA−
CD4⁺IL22⁺CLA⁺ CD4−IL22−CLA−
EASI EASI
SCORAD SCORAD
Total ADQ Total−ADQ
Pruritus ADQ Pruritus−ADQ
Eosinophils Eosinophils
CD8⁺IFNγ⁺CLA⁻ CD8−IFNg−CLA−−1
CD8⁺IFNγ⁺ CD8−IFNg−
CD8⁺IFNγ⁺CLA⁺ CD8−IFNg−CLA−
CD4⁺IFNγ⁺CLA⁻ CD4−IFNg−CLA−−1
CD4⁺IFNγ⁺ CD4−IFNg−
CD4⁺IFNγ⁺CLA⁺ CD4−IFNg−CLA−
Age
Age
CD4⁺IL17⁺CLA⁻ CD4−IL−17−CLA−−1
CD4⁺IL17⁺ X−CD4−IL−17−
CD4⁺IL22⁺CLA⁻ CD4−IL22−CLA−−1
CD4⁺IL22⁺ X−CD4−IL22−
CD8⁺IL17⁺CLA⁻
CD8−IL−17−CLA−−1
CD8⁺IL17⁺ X−CD8−IL−17−
CD8⁺IL17⁺CLA⁺ CD8−IL−17−CLA−
CD8⁺IL13⁺CLA⁻ CD8−IL13−CLA−−1
CD8⁺IL13⁺ X−CD8−IL13−
CD8⁺IL22⁺CLA⁻ CD8−IL22−CLA−−1
Duration of Disease Duration−of−disease
***p<0.001 **p<0.01 *p<0.05 +p<0.1
CD8⁺IL22⁺ X−CD8−IL22−
CD8⁺IL22⁺CLA⁺ CD8−IL22−CLA−
CD8⁺IL13⁺CLA⁺ CD8−IL13−CLA−
CD4⁺IL17⁺CLA⁺ CD4−IL−17−CLA−
TEWL NL TEWL−NL
TEWL LS
TEWL−LS
CD8⁺CLA⁺ CD8−CLA−
CD4⁺CLA⁺ CD4−CLA−
CD8−IL9−CLA−−1 CD8⁺IL9⁺CLA⁻
Figure 6
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p=0.88
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r=−0.37 (CI: −0.52−−0.2) (CI: -0.52–-0.2) ● 100 r=-0.37 p=5.2e−05
15
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40
Age
80
p=0.095
●
75 ●
50
25
●
60
r=-0.17 (CI: -0.53–-019)
r=−0.17 (CI: −0.53−−0.19) p=0.095
p=5.2e-05
● ● ● ● ●
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0
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● ●●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ●● ● ● ● ●● ●● ● ● ● ●●● ● ●●● ● ● ●● ●● ● ● ● ● ●●●●●● ● ●●●● ●● ●● ●● ●● ● ● ●● ● ● ●● ●● ●● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ●● ● ●●
0
Age
10
L Age vs. CD4 EM CLA+
r=-0.015 (CI: -0.51–-0.17)
●
● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ●● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ●● ●● ● ● ●● ● ● ● ●● ● ●●● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●● ●● ● ● ●● ● ●●● ●● ●● ● ● ●● ● ● ● ● ●● ● ●● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ●● ●● ● ●● ● ● ●● ● ●●●● ● ● ● ●
5
Age Age
r=−0.015 (CI: −0.51−−0.17) p=0.88
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15
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Age Age
●
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p=0.00013
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40
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80
SCORAD SCORAD
r=0.27 (CI: 0.028−0.47) r=0.27 (CI: 0.028–0.47) p=0.029 p=0.029
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20
20
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r=−0.35 (CI:(CI: −0.5−−0.18) r=-0.35 -0.5–-0.18) ● p=0.00013
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H Age vs. Eosinophils ●
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0
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40
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20
80
K Age vs. CD4 CM CLA+
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0 60
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40
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10
5
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10
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15
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r=0.081 (CI: −0.12−0.27) r=0.081 (CI: -0.12–0.27) ● p=0.42 p=0.42
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30
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J Age vs. TEWL.NL
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Age
TEWL NL TEWL.NL
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60
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150
● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●
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Age
r=0.32 (CI: 0.14−0.49) r=0.32 (CI: 0.14–0.49) ● p=0.00097 p=0.00097
50
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10
0 0
80
Age vs. TEWL.LS
TEWL.LS TEWL LS
● ●
●
●
Age Age
●
●●
r=0.19 (CI: 0.0017−0.36) r=0.19 (CI: 0.0017–0.36) ● ●● p=0.048 p=0.048 ● ● ●
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●
20
100
●
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20
0
0
0
●
G Age vs. Pruritus.ADQ
●
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SCORAD
●
●
●
● ●
SCORAD
● ●
EASI EASI
SCORAD SCORAD
● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ●●● ● ●●● ●● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ●●● ● ● ● ● ● ● ● ● ● ● ● ●
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I
20
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100
●
20 ● ● ● ● ● ●● ●
●●
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80
r=0.34 (CI: 0.17−0.49) r=0.34 p=0.00015 (CI: 0.17–0.49) ● p=0.00015
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●
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●
●
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20
●
● ●
F Age vs. EASI
p=0.0016 ●●
●
●
SCORAD
r=0.25 (CI: 0.096−0.39) r=0.25 (CI: 0.096–0.39) ● p=0.0016
●
●
SCORAD
EAge vs. SCORAD
40
●
40
EASI
60
●
● ● ● ● ● ● ●● ●●● ● ● ● ●● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ●● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ●●
EASI
80
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0
100
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20
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5
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Pruritus ADQ Pruritus.ADQ
60
●
●
15
r=0.25 (CI: 0.01−0.47) r=0.25 (CI: 0.01–0.47) p=0.0011 p=0.0011
● ● ● ●
30
CLA⁺CD4⁺ CD4 CMTcm CLA+ cells
SCORAD SCORAD
●
p=0.82e-14
Pruritus ADQ Pruritus.ADQ
●● ●
r=0.54 (CI: 0.39–0.66)
●
●
●
● ●
r=0.24 (CI: 0.064–0.41) p=0.0016
r=0.54 (CI: 0.39−0.66) ● p=8.2e−14
Eosinophils Eosinophils
r=0.74 (CI: 0.64–0.81) p=1.4e-21
CD4⁺CLA⁺ CD4 CLA+
100
D SCORAD vs. Eosinophils
C SCORAD vs. Pruritus.ADQ
r=0.24 (CI: 0.064−0.41) p=0.0016
Eosinophils Eosinophils
B SCORAD vs. CD4 CLA+
r=0.74 (CI: 0.64−0.81) p=1.4e−21
CD4 EM Tem CLA+ cells CLA⁺CD4⁺
A EASI vs. SCORAD
●
● ●
●
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●
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●
20
40
Age Age
60
80
CD4⁺ T cells r=0.11 (CI: 0.01−0.38) ● p=0.28
●
● ●
● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ●● ● ● ●●● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ●● ● ●●●● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ●● ●● ●● ● ●● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ●●●● ●●●● ● ● ● ● ● ●●●●● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●● ● ●● ● ●● ● ● ● ●● ● ●●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●●● ● ● ●
0
20
40
●
●
60
0
80
12
p=0.51
20
40
●
60
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0
●
●
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●
● ● ● ● ● ● ● ● ● ●● ● ●● ●● ● ● ● ● ●● ● ● ●● ●●● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ●●●●●● ●●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ●● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ●●●
0
80
●
● ●● ● ● ●● ● ● ● ●● ●● ● ● ●●●● ● ● ● ● ● ●● ● ● ● ● ●●●●● ● ● ●●● ●● ●● ●● ● ●● ● ● ● ● ● ● ● ●
20
40
●
60
p=0.53
30
p=0.63
p=0.38
●
2000 ●
●
● ● ● ●
1000 ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ●● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ●● ● ●● ● ● ● ● ● ● ●● ● ●●● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ●●●● ● ●● ● ● ● ● ●● ●● ●● ●● ● ● ● ●● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●●●● ● ●●● ● ●● ● ●● ● ● ● ●● ●● ● ● ● ●●●● ● ●●●●● ● ● ● ●●● ● ●
0
●
80
G Age vs. CD8 IL13+CLA+
Age vs. CD8 IFNg+CLA−/IL13+CLA− r=−0.058 (CI: −0.23−0.12) (CI: −0.25−0.14) r=-0.058 (CI: -0.23–0.12)r=−0.089 r=0.089 (CI: -0.25–0.14) p=0.53 ● p=0.38
0
Age Age
r=0.072 (CI: −0.11−0.25) (CI: −0.13−0.26) r=0.072 (CI: -0.11–0.25) r=−0.048 r=0.048 (CI: -0.13-0.26) p=0.44● p=0.63
p=0.44
20
40
80
H Age vs. CD8 IL13+CLA−
r=0.36 (CI: 0.19−0.51) r=0.14 (CI: 0.17−0.52) r=0.36 (CI: 0.19–0.51) ●r=0.14 (CI: 0.17-0.52) p=6.5e−05 p=0.16 ● p=6.5e-05 p=0.16
3
r=0.65 (CI: 0.53−0.74) r=0.45 (CI: 0.52−0.75) r=0.65 0.53–0.74) r=0.45 (CI: 0.52-0.75) ● p=3.7e−15 (CI: p=1.9e−06 p=3.7e-15 p=1.96e-06
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20
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●
● ●
● ● ● ● ● ● ●● ● ●● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ●● ● ● ●●● ●● ● ● ●● ●●● ● ● ●● ● ● ● ●● ● ●● ● ● ● ● ● ●●●● ● ● ●●● ●● ● ●● ●●●● ●●●●● ● ● ● ● ● ● ● ●● ● ● ●
0
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0
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40
60
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6
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3
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80
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9
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●
●
● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ●● ● ● ●● ● ● ● ●● ● ● ●●● ● ● ● ● ●● ● ●● ● ● ●●● ● ●● ● ● ● ●●● ● ●● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ●●● ● ●●● ●● ● ● ●●● ●● ● ● ● ●● ● ●● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ●●●● ●●●● ● ● ● ● ●● ● ●●● ● ● ● ● ●● ● ● ●● ● ● ● ●●● ● ● ● ● ●● ● ● ● ●●● ●
20
0
20
40
●
●
●
● ● ●
10
●
●
0
60
●
●
● ●
Age
●
●
80
●
● ●
●
● ●
● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ●● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ●● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ●●● ● ● ●● ●● ●● ●● ● ●● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ● ● ● ● ● ● ●● ●● ● ●● ● ●● ●● ● ● ●●●● ● ● ●●●●● ● ●●●● ● ●● ● ● ●●
0
20
40
Age
●
CD8 IL13+CLA−
●
IL-13⁺CLA⁻
●
2
●
●
1
●
●
● ●
●
● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ●●● ● ● ●● ●● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●●● ●● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●●●● ● ● ●● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ●
●
0
60
0
80
20
40
60
Age
Age
Age
I Age vs. CD4 IL22+CLA+
J Age vs. CD4 IL22+CLA−
K Age vs. CD8 IL22+CLA+
L Age vs. CD8 IL22+CLA−
r=0.31 (CI: 0.14–0.47) r=0.22 (CI: 0.12-0.48) ● p=0.00052 p=0.03 r=0.31 (CI: 0.14−0.47) p=0.00052
r=0.22 (CI: 0.12−0.48) ● p=0.03
10.0
r=0.66 (CI: 0.48−0.73) r=0.66 (CI: 0.48-0.73) p=1e−13 p=1e-13
r=0.62 (CI: 0.5−0.72) r=0.62 p=3.9e−14●(CI: 0.5–0.72) p=3.9e-14
●
●
80
Age
Age
Age 30
60
Age Age
● ●
● ●
IL-13⁺CLA⁻ CD4 IL13+CLA−
IL-13⁺CLA⁺ CD4 IL13+CLA+
● ●
F Age vs. CD4 IL13+CLA−
●
0
●
p=0.38
Age Age
r=0.16 (CI: −0.018−0.33) (CI: −0.034−0.35) r=0.16 (CI: ●-0.018–0.33) r=0.067 r=0.067 (CI: -0.034–0.35) p=0.077 p=0.51
10
●
● ●●
● ● ●
● ●● ● ● ● ● ●● ●● ● ● ● ●●● ● ● ●● ● ● ●● ● ●● ● ● ● ●● ● ●● ● ● ● ● ●● ●● ● ● ●● ● ● ● ● ●● ●●● ●● ●● ●●● ●● ●● ● ● ●● ●● ● ● ● ● ● ● ● ● ●●● ● ● ● ●●●● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●
0
EAge vs. CD4 IL13+CLA+ 30
●
●
● ● ●
●
r=0.088 (CI: −0.21−0.17) p=0.38
p=0.81
100
● ●
Age Age
p=0.077
r=-0.022 (CI: -0.2–0.16) r=0.088 (CI: -0.21–0.17) r=−0.022 (CI: −0.2−0.16) ● p=0.81
200
200
●
0
●
400
●
40
20
r=0.54 (CI: 0.4−0.67) p=3.5e−09
600
●
60
D
C Age vs. CD8 IFNg+CLA+/IL13+CLA+
r=0.55 (CI: 0.41–0.66) r=0.54 (CI: 0.4–0.67) ● p=5.9e-11 p=3.5e-09 r=0.55 (CI: 0.41−0.66) p=5.9e−11
CD8 IFNg+CLA+/IL13+CLA+ IFN-γ⁺CLA⁺/IL-13⁺CLA⁺
B Age vs. CD4 IFNg+CLA−/IL13+CLA−
r=0.2 (CI: 0.026–0.37) r=0.11 (CI: 0.01–0.38) p=0.025 p=0.028 r=0.2 (CI: 0.026−0.37) ● p=0.025
IL-13⁺CLA⁺ CD8 IL13+CLA+
IFN-γ⁺CLA⁺/IL-13⁺CLA⁺ CD4 IFNg+CLA+/IL13+CLA+
80
CD4 IFNg+CLA−/IL13+CLA− IFN-γ⁺CLA⁻/IL-13⁺CLA⁻
A Age vs. CD4 IFNg+CLA+/IL13+CLA+
CD8⁺ T cells IFN-γ⁺CLA⁻/IL-13⁺CLA⁻ CD8 IFNg+CLA−/IL13+CLA−
Figure 7
r=0.43 (CI: 0.27−0.56) r=0.3 (CI: 0.25−0.57) r=0.43 (CI: 0.27–0.56) r=0.3 (CI: 0.25-0.57) ● p=1.1e−06 p=0.0023 p=1.1e-06 p=0.0023
4
r=0.55 (CI: 0.41−0.66) r=0.55 (CI: 0.39−0.67) r=0.55 (CI: 0.41–0.66) r=0.55 (CI: 0.39-0.67) p=1.4e−10 ● p=2.6e−09 p=1.4e-10 p=0.2.6e-09
●
●
●
●
● ●
● ● ●
10
0
● ●
●
● ●
●
●
●
●
●
● ● ● ● ● ● ●● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●●● ● ● ● ●● ● ●● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ●●●● ● ● ● ● ●●●● ● ● ● ● ● ●● ●● ●● ● ● ●● ● ● ●●● ● ●● ● ● ● ●● ● ● ●● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ●●
0
20
40
Age Age
60
7.5
●
●
●
●
●
●
5.0
●● ● ●
● ●
0.0
●
●
●
●
● ● ●●●
● ●●● ●●●●● ● ● ●●● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ●● ● ●● ●● ●● ●● ● ● ●● ● ●● ● ●● ● ●● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ●●●●●●●●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ●● ● ● ●● ● ● ● ● ● ●
0
●
●
●
●
2.5
80
● ●
20
●
●
●
● ●
● ●●
●
●
● ● ●●
●
● ●
40
●
●
20
●
● ● ●●
60
●
80
0
●
●
● ●
●
●
Age Age
●
● ●
● ●
● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ●
40
● ● ●● ●
●
20
●
●
40
Age Age
●
60
●
● ● ●
● ●● ●
● ● ● ● ●● ● ● ● ● ●●
●
● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ●● ● ●● ●● ● ● ●● ● ● ● ● ●● ● ● ●● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●●●●●●●●●●● ● ● ● ● ●● ● ● ● ● ●● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ●● ● ●●● ● ● ● ● ● ● ● ● ●● ●●● ● ● ● ● ● ●
●● ●
●
0 80
●
●
●
● ●●
●
2
● ●
●
3
1
●
● ●
● ● ● ●●●●● ● ● ● ●● ●● ●●● ● ● ● ● ● ●● ● ● ●● ● ● ●●●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ●● ● ● ● ●● ● ●●● ●● ● ●●●● ● ● ●● ●● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ●● ● ● ●● ● ●● ● ● ● ●●● ● ●● ● ● ● ● ● ●●● ● ● ● ● ● ●● ●● ● ● ● ●● ● ● ● ● ●
0
CD8 IL22+CLA−
●
IL-22⁺CLA⁻
●
20
●
●
IL-22⁺CLA⁺ CD8 IL22+CLA+
CD4 IL22+CLA− IL-22⁺CLA⁻
IL-22⁺CLA⁺ CD4 IL22+CLA+
● ●
●
0
20
40
Age Age
60
80
Figure 8 cluster3
Figure E8. Healthy controls cluster3
AD patients
Normal patients ●
CD4+/CD8+ IFN-γ+ CD4+/CD8+ IL-22+ CD4+ IL-9+CLA+ Total Tregs Tcm HLA-DR+CLA+ Tem ICOS+CLA+ CD4+/CD8+ Tem/Tcm CLA+
10
CD4+/CD8+ IFN-γ+ CD8+ Tem HLA-DR+ CD8+ IL-13+CLA+ Th1/Th2 ratio CD8+ Tem cells CD4+ IL-9+CLA+ Total Tregs
5
●
labels ●
1
●
2
●
3
●
Children and Adolescents PC2
cluster3
● ●
Infants
●● ●
●
●
●
●
● ●● ● ● ● ● ●
●
0
●
● ● ● ●
● ● ● ●
●
●
●
cum.df$AgeGrp
●
● ● ●
●
Adults
●
labels ●
1
●
2
PC2
−5
0
CD4+/CD8+ IFN-γ+ CD4/+CD8+ IL-22+ Th1/Th2 ratio CD8+ Tem HLA-DR+ CD4+ Tem/Tcm CLA+ CD4+ Tem ICOS+CLA-
●
● ● ● ●
● ●● ● ● ●
●
●
● ●● ● ● ● ● ●
●
● ●
●
3
● ● ● ●
●
●
●
● ● ● ● ●● ● ●
●
●
● ●
● ●
●
● ●
●
●
cum.df$AgeGrp ●
●
●
Adolescents Adults
●
Children ●
●
Infants
●
−5
● ●
● ●
● ●
●
● ●
0
●
●
●
Infants
●
●
Adolescents
Children
●
●
●
Adults
● 5
●
5
PC2
●
●
0
5
PC1
−5 10
−5
0
5
PC1
10
Table E1. Patient demographic and clinical severity scores Characteristic
Age
Parameter
Normal Infants (n=24)
Normal Children (n=27)
Normal Adolescents (n=28)
Normal Adults (n=30)
AD Infants (n=40)
AD Children (n=26)
AD Adolescents (n=23)
AD Adults (n=43)
Mean ± SD
2.45 ± 1.44 2.00 (0.564.93)
9.16 ± 2.12 9.01 (5.0511.87)
14.36 ± 1.69
1.93 ± 1.44
7.54 ± 1.94
15.06 ± 1.90
14.30 (12.1317.46)
39.27 ± 11.97 37 (1966)
1.56 (0.414.96)
7.47 (5.0811.51)
14.29 (12.1917.99)
43.30 ± 15.09 43 (1881)
Male
13
7
11
18
22
13
13
24
Female
11
20
17
12
18
13
10
19
White
18
18
16
24
19
9
16
34
Hispanic
3
4
10
1
3
6
2
0
Black
3
3
1
2
8
6
2
6
Median (range) Sex
Race
Asian
0
2
1
3
10
5
3
3
4.04 ± 4.05 2.15 (0.012.6)
2.25 ± 1.95 1.9 (0.710.0)
3.19 ± 2.38
NA
6.42 ± 3.44
6.71 ± 3.85
9.72 ± 5.90
NA
2.4 (0.5-8.9)
NA
6.4 (2.2-15)
5.6 (0.7-15.6)
8.0 (1.923.7)
NA
Mean ± SD
NA
NA
NA
NA
52.48 ± 13.15
58.43 ± 14.91
Median (range)
NA
NA
NA
NA
52.5 (20.883.6)
57.3 (25.489.0)
58.95 ± 21.78 53.5 (21.391.2)
EASI
Mean ± SD
NA
NA
NA
NA
14.04 ± 8.64
17.76 ± 10.21
64.90 ± 15.64 65 (32.597) NA
NA
NA
NA
NA
TEWL LS
Median (range) Mean ± SD
NA
NA
NA
NA
13.0 (2.8-38.6) 36.24 ± 31.34
16.6 (3.7-51.4) 47.13 ± 24.78
NA
NA
NA
NA
TEWL NL
Median (range) Mean ± SD
NA
NA
NA
NA
28.6 (8.3-176.8) 19.08 ± 15.27
42.7 (16.6-103.4) 19.02 ± 11.00
NA
NA
NA
NA
14.5 (2.9-76.7)
16.5 (5.9-49.2)
Eosinophils (%)
Mean ± SD
Median (range) Disease Severity Scores SCORAD
Median (range)
27.93 ± 20.56 18.0 (4.6-68.4) 58.30 ± 29.68 57.6 (13.5-96.6) 22.07 ± 13.66 18.8 (5.4-53.2)
NA NA NA NA NA
P-Value b/w Normal and AD Infants 0.1656
P-Value b/w Normal and AD Children 0.005724
P-Value b/w Normal and AD Adolescents 0.1699
P-Value b/w Normal and AD Adults 0.2263
1
0.1275
0.3446
0.9082
0.03131
0.1289
0.1007
0.4775
0.09849
1.03E-05
4.70E-05
Pruritus ADQ
Mean ± SD
NA
NA
NA
NA
16.95 ± 7.51
21.96 ± 8.11
20.82 ± 8.73
NA
NA
NA
NA
NA
16.0 (1-32)
20.5 (7-35)
22.0 (5-35)
NA
Total ADQ
Median (range) Mean ± SD
NA
NA
NA
NA
33.65 ± 13.62
43.38 ± 13.36
NA
Median (range) Mean ± SD
NA
NA
NA
NA
34.0 (5-61)
46.5 (18-70)
42.75 ± 13.67 44.4 (19-65)
NA
NA
NA
NA
1.61 ± 1.38
6.33 ± 2.45
14.23 ± 2.45
NA
NA
NA
NA
NA
1.2 (0.2-4.7)
5.1 (2.0-10.5)
NA
0
1
5
0
18
16
13.7 (9.617.7) 13
0
0
0
0
1
3
3
2
0
0
0
0
1
6
4
10
Disease Duration
Median (range) History of other atopic disorder (allergic rhinitis,
NA
7
asthma, allergic conjunctivitis, environmental)
Food Allergies (FA) FA + other atopic disorder
AD: Atopic dermatitis; SCORAD: Scoring of Atopic Dermatitis; SD: Standard deviation; NA: not available; EASI: Eczema Area and Severity Index; TEWL LS: Transepidermal Water Loss Lesional Skin; TEWL NL: Transepidermal Water Loss Non-Lesional Skin; Pruritus ADQ: Pruritus Atopic Dermatitis Quickscore; Total ADQ: Total Atopic Dermatitis Quickscore
Table E2A: Sensitivity analysis with race-matched infants CD4+IL13+CLA+
N Infant (mean) 2.575
AD Infant (mean) 8.551
N Infant (median) 2.040
AD Infant (median) 6.094
P-value (means)Infant AD vs. N 0.00284
P-value (median)Infant AD vs. N 0.00174
CD4+EM+CLA+
26.698
40.595
25.365
38.290
0.00296
0.00398
CD4+Tem ICOS+CLA-
8.604
18.119
7.009
15.642
0.00413
0.000722
CD4+CM+CLA+
13.4755
21.267
12.420
20.200
0.0163
0.0177
CD4+IFNγ+CLA-/IL13+CLA-
15.278
7.346
10.116
4.707
0.0187
0.03243
CD4+Tcm ICOS+CLA-
3.909
6.841
3.151
4.548
0.0222
0.0348
CD4+IFNγ+CLA+/IL13+CLA+
9.386
1.518
6.051
1.156
0.0239
0.000335
CD4+Tem HLADR+CLA+
10.207
5.309
8.512
4.078
0.0303
0.0225
CD4+Tcm HLADR+CLA+
11.102
4.954
6.438
4.326
0.0638
0.0868
CD4+IFNγ+CLA+
11.945
8.274
10.730
8.046
0.0952
0.146
Table E2B: Sensitivity analysis with age-matched children CD4+IFNγ+CLA-/IL13+CLA-
N Child (mean) 32.881
AD Child (mean) 9.934
N Child (median) 35.858
AD Child (median) 6.515
P-value (mean)Child AD vs. N 1.11E-05
P-value (median)Child AD vs. N 1.51E-05
Tregs (CCR4+CLA+)
7.598
21.365
5.030
18.20
0.00232
0.00113
CD8+Tem ICOS+CLA+
11.481
28.929
4.974
23.208
0.00572
0.00850
CD4+IL13+CLA-
0.647
2.925
0.672
2.105
0.00698
0.0105
CD8+Tem CLA+
4.826
9.588
5.066
7.690
0.00753
0.0163
Tregs (CD25+CD127-)
6.656
11.630
6.470
9.530
0.0121
0.0368
CD8+Tcm ICOS+CLA-
3.285
7.970
2.958
7.313
0.0144
0.0236
CD8+Tcm CLA+
5.812
11.146
4.025
8.480
0.0182
0.0169
CD4+Tcm HLADR+CLA+
9.344
17.661
7.158
16.245
0.0263
0.0458
CD4+IL22+CLA-
0.775
2.395
0.729
1.682
0.0353
0.0253
Table E2C: Sensitivity analysis using only adolescent controls with no atopic history CD4+IL22+CLA+
N Adol (mean) 4.137
AD Adol (mean) 8.021
N Adol (median) 4.441
AD Adol (median) 6.400
P-value (mean)Adol AD vs. N 0.00518
P-value (median)Adol AD vs. N 0.00203
CD8+Tem ICOS+CLA+
12.689
29.184
10.301
26.667
0.00580
0.0137
CD4+IFNγ+CLA+
20.417
13.482
18.937
12.608
0.0101
0.0136
CD4+IL13+CLA-
1.065
1.660
0.938
1.403
0.0424
0.0504
CD8+Tcm HLADR+CLA+
7.686
23.516
4.006
19.950
0.0428
0.0677
CD4+Tem ICOS+CLA+
19.775
31.052
18.330
29.024
0.0502
0.103
CD4+IFNγ+CLA+/IL13+CLA+
5.738
3.104
3.663
2.616
0.0634
0.0173
CD8+IFNγ+CLA-
42.573
32.263
42.495
33.569
0.0687
0.0736
CD4+Tem ICOS+CLA-
9.488
14.020
8.163
12.741
0.0688
0.138
CD4+Tem HLADR+CLA+
6.108
12.230
6.259
6.503
0.0978
0.557
Sensitivity analyses for demographic parameters that were significantly different between AD and N within an age-group, with means, medians, and respective Pvalues for the top 10 most significant comparisons. These analyses were done for race-matched infants (A), age-matched children (B), and adolescent AD versus adolescent controls without atopic history (C). Abbreviations AD: Atopic dermatitis; Adol: Adolescent; Child: Children; CLA: Cutaneous lymphocyte antigen; HLADR: Human leukocyte antigen-DR; ICOS: Inducible co-stimulator; IFNγ: Interferon-gamma; IL: Interleukin; N: Normal; Tcm: Central memory T; Tem: Effector memory T; Treg: Regulatory T
Table E3A. Mean values for normal and atopic dermatitis infants, children, adolescents, and adult patients and respective p-values P- Values
CD4+IFNγ+ CD4+CLA+ CD4+IFNγ+CLA+ CD4+IFNγ+CLACD4+IL13+
N Infants
AD Infants
N Child
AD Child
N Adol
AD Adol
N Adults
AD Adults
Infants AD vs. N Infants
7.648
7.365
20.242
13.973
22.037
18.974
25.870
24.200
0.871
2.383 11.945 7.517 1.168
3.726 7.769 7.380 1.411
2.066 19.511 20.273 0.757
3.398 13.690 14.006 3.067
2.035 18.326 22.132 1.024
1.610 13.734 19.072 1.804
2.934 19.588 26.068 0.831
5.053 17.228 24.780
0.044 0.047 0.938
Child AD vs. N Child
Adol AD vs. N Adol
Adults AD vs. N Adults
N Infants vs. N Child
N Infants vs. N Adol
N Infants vs. N Adults
N Child vs. N Adol
N Child vs. N Adults
N Adol vs. N Adults
AD Infants vs. AD Child
AD Infants vs. AD Adol
AD Infants vs. AD Adults
AD Child vs. AD Adol
AD Child vs. AD Adults
0.040
0.371
0.501
8.17E06
2.51E05
1.12E12
0.604
0.037
0.223
0.010
3.30E05
4.59E10
0.093
0.001
0.071
0.076
1.58E04
0.107 0.058 0.040 0.004
0.469 0.075
0.009
0.641
0.010
0.010
0.378
0.765
0.478
0.164
0.666
0.367
0.036
0.215
0.130
0.106
0.142
0.750
0.009
0.010
0.033
0.481
0.203
0.479
0.858
0.659
0.409
0.780
0.300
0.215
0.341
0.692
0.365
0.141
0.920
0.211
0.022
0.026
0.375
0.139
0.041
0.132
1.916
0.625
0.173
0.109
6.99E06
0.01
0.007
0.006
0.084
0.010
0.841
0.255
0.311
0.276
0.197
0.021
0.244
0.095
0.056
0.501
0.250
0.564
0.010
0.001
0.689
0.007
0.006
0.456
0.155
0.012
0.249
0.015
0.056
0.104
0.071
0.044
0.160
0.006
0.121
0.061
0.685
0.437
0.085
0.047
0.678
2.850
0.934
1.719
0.693
1.283
0.922
9.386
1.572
8.546
2.457
5.470
3.206
14.262
4.088
0.025
0.032
0.141
0.137
1.48E04
0.474
0.036
0.591
CD4+IFNγ+CLAIL13+CLA-
15.278
8.853
34.346
10.760
24.806
12.470
39.339
26.164
0.053
1.94E06
CD4+IL9+
0.491
0.514
0.406
0.881
0.906
1.194
0.250
0.464
0.877
0.054
CD4+IL9+CLA+
3.824
3.256
6.701
7.052
9.708
10.259
2.162
2.332
0.676
0.862
0.845
0.788
0.154
CD4+IL9+CLA-
0.399
0.409
0.333
0.766
0.743
1.051
0.214
0.295
0.930
0.049
0.372
0.188
0.615
CD4+IL22+CLA-
0.467
0.399
0.870
2.063
2.313
7.910 2.595
4.746 2.239
0.051
0.217
1.077
3.627
0.090
0.034
1.124
6.750
0.099
0.594
CD4+IL13+CLA-
4.139
0.236 3.96E04
0.609
CD4+IFNγ+CLA+ IL13+CLA+
5.261
0.987
2.93E10
4.95E04
11.055
4.552
0.030
0.376
4.548
CD4+IL22+CLA+
0.028
3.37E05
8.483
2.314
0.127 7.63E06
0.001
5.338
2.654
0.005 3.94E04
8.77E13
9.114
2.340
0.608
0.679
0.029
6.428
2.169
0.970
0.057
2.30E05
9.492
0.935
0.675
0.189
0.004
2.575
0.594
0.960
6.83E06
CD4+IL13+CLA+
0.593
0.311
0.242
9.20E05
CD4+IL22+
0.488
AD Adol vs. AD Adults
2.675 9.663 2.943
0.995 0.595 0.562
0.031 0.073 0.037
0.024
0.217
0.271
0.118
0.073
8.11E05
1.28E07
0.594
0.437
0.049
0.002
3.78E05
0.753
0.452
0.874
0.006
3.72E05
3.28E08
0.635
0.085
0.010
0.002
0.010
0.064
0.243
0.013
0.087
0.001
6.13E06
0.952
0.008
0.510
0.433
0.068
0.304
0.001
5.10E06
0.005
0.865
0.182
0.147
0.001
2.53E04
0.037
0.172
0.405
0.024
0.015
2.57E04
5.45E06
0.491
0.445
0.972
0.052
0.001
0.496
0.077
0.253
1.49E04
1.08E09
0.453
0.164
0.544
7.54E07
0.719
0.145
0.254
0.001
CD4+IL17+
0.510
0.377
0.805
0.973
0.883
1.087
1.120
1.403
0.270
0.529
0.452
0.241
0.083
0.067
0.003
0.712
0.137
0.317
0.020
0.004
CD4+IL17+CLA+
2.906
2.062
3.012
2.166
2.633
2.608
2.605
2.953
0.083
0.265
0.964
0.441
0.892
0.657
0.585
0.632
0.585
0.960
0.804
0.193
0.013
0.392
0.096
0.455
0.002
1.11E06
0.693
0.192
0.342
0.720
0.001
0.002
CD4+IL17+CLACD8+IFNγ+
0.442 18.328
0.320 20.179
0.765 38.883
0.946 28.485
0.846 37.309
1.072 30.301
1.076 44.737
1.336 46.063
0.261 0.647
0.504 0.052
0.401 0.217
0.284
0.048
0.038
0.002
0.782
6.51E05
0.001
2.00E07
0.693
0.139
0.323
0.017
0.785
0.245
0.186
0.081
0.029
1.37E07
3.39E01
0.415
N/A
0.758
N/A
N/A
0.113
0.165
N/A
0.965
N/A
N/A
0.631
0.174
0.062
0.933
0.233
0.152
0.259
0.124
0.008
6.71E08
0.684
0.001
0.002
0.822
0.060
0.031
0.008
3.78E04
CD8+CLA+
2.744
3.770
1.684
1.939
1.924
1.979
N/A
N/A
0.452
0.752
0.950
N/A
CD8+IFNγ+CLA+
21.484
24.946
27.724
24.546
25.570
19.225
33.830
31.589
0.441
0.494
0.166
0.605
0.156
0.337
0.005
0.696
4.44E05
0.001
1.51E07
0.781
0.245
0.187
0.073
0.023
0.948
0.290
0.009
0.207
0.001
0.179
0.549
0.675
0.028
0.765
3.97E04
CD8+IFNγ+CLA-
18.014
19.931
39.176
28.670
37.558
30.772
45.066
46.953
0.642
0.051
0.240
CD8+IL13+
0.225
0.376
0.219
0.528
0.345
0.480
0.485
0.920
0.488
0.067
0.425
0.005
0.839
7.15E05
CD8+IL13+CLA+
2.936
3.544
2.091
4.429
2.989
3.223
3.094
8.822
0.627
0.041
0.452
0.968
0.892
0.310
0.111
0.911
0.475
0.364
CD8+IL13+CLA-
0.114
0.101
0.179
0.467
0.287
0.461
0.419
0.630
0.694
0.046
0.291
0.028
0.151
0.032
4.73E06
0.175
2.34E04
0.139
0.012
0.023
6.47E08
0.975
0.297
0.312
CD8+IFNγ+CLA+ IL13+CLA+
13.535
10.690
19.937
7.252
10.347
10.440
18.629
8.298
0.449
0.766
0.488
0.010
0.718
0.500
0.630
0.579
0.825
0.666
0.865
0.479
0.219
0.549
0.400
0.610
CD8+IFNγ+CLAIL13+CLA-
44.484
31.193
54.077
45.658
61.714
47.565
73.607
49.528
0.559
0.038
0.227
0.097
0.183
0.817
0.181
0.107
0.011
0.184
0.739
0.254
0.020
0.577
0.168
0.186
CD8+IL9+
0.732
1.020
0.626
0.624
0.895
0.849
0.363
0.528
0.324
0.996
0.906
0.075
0.743
0.669
0.069
0.524
0.334
0.120
0.183
0.573
0.041
0.433
0.650
0.159
0.092
0.004
0.002
CD8+IL9+CLA+
13.578
16.237
6.775
8.695
8.720
14.186
3.847
3.881
0.580
0.430
0.141
0.972
0.133
0.296
CD8+IL9+CLA-
0.297
0.414
0.410
0.426
0.800
0.673
0.289
0.355
0.413
0.945
0.730
0.225
0.580
CD8+IL22+ CD8+IL22+CLA+
0.160 1.434
0.341 3.598
0.206 1.564
0.395 4.147
0.670 2.375
0.593 4.692
0.649 2.398
0.753 8.611
0.096 0.190
0.152 0.023
0.170
0.053
0.015
0.599
5.47E05
0.306
0.526
0.133
0.947
0.209
0.635
0.248
0.610
0.078
0.006
3.25E05
0.901
0.725
0.283
0.002
0.416
0.017
0.481
0.024
0.522
0.147
0.936 3.75E06
0.764
0.372
0.472
0.002
0.066
4.16E05
0.824
0.214
0.112
0.227
0.088
0.972
0.768
0.565
0.017
0.714
0.010
0.025
0.004
2.45E05
0.916
0.400
0.136
0.001
0.321
0.017
0.686
0.295
0.491
0.038
0.531
CD8+IL22+CLA-
0.120
0.224
0.186
0.329
0.620
0.545
0.604
0.630
0.244
0.172
0.749
0.814
0.213
0.001
1.18E06
CD8+IL17+
0.237
0.294
0.219
0.229
2.883
0.325
0.422
0.410
0.636
0.940
0.326
0.884
0.893
0.309
0.063
0.307
0.128
0.344
0.581
0.839
CD8+IL17+CLA+
2.382
1.552
1.579
0.974
4.694
1.023
1.383
2.094
0.301
0.353
0.046
0.058
0.376
0.223
0.180
0.096
0.737
0.067
0.233
0.274
0.270
0.914
0.019
0.024
0.510
0.047
0.472
CD8+IL17+CLATregs (CD25+CD127-)
0.180 6.403
0.137 5.697
0.146 7.037
0.222 12.212
2.925 11.527
0.315 11.642
0.384 5.177
Tregs (CCR4+CLA+)
8.118
11.242
8.272
23.786
9.468
13.293
13.998
CD4+CM+CLA+
13.475
20.412
11.350
18.839
11.218
11.750
12.870
0.416
0.481
0.392
0.336
0.729
0.650
0.005
0.333
0.105
0.004
0.456
12.824
0.007
0.011
0.863
0.979
0.309
0.366
0.749
0.957
0.410
0.472
0.615
0.007
0.002
0.041
0.033
0.691
0.608
0.330
0.843
0.317
0.013
0.012
0.026
0.907
0.586
0.641
0.022
0.025
3.66E04
0.365
0.462
0.322
0.439
0.322
0.945
0.077
0.140
0.954
0.045
0.357
0.097
0.363
0.021
0.260
1.57E06
0.014
0.011
0.496
0.741
0.003
0.003
0.338
0.024
0.020
0.167
0.281
0.556
0.701
3.57E04
0.002
0.357
0.087
0.352
0.518
0.103
0.013 0.392
5.538
7.478
5.662
4.117
8.248
10.135
0.682
0.350
3.738
0.162
0.033
7.607
11.385
0.018
0.006
6.932
9.197
0.010
0.595
0.318
9.625
0.003
0.014
0.030
8.496
0.002
0.592
0.002
7.457
0.770
0.950
23.511
3.909
0.007
0.083
20.343
8.953
0.033
0.158
20.683
23.955
0.004
0.230
19.273
18.971
0.165 1.96E04
17.906
27.768
18.018
0.266 1.75E04
0.950
18.972
17.700
0.349 5.88E05
0.003
38.082
14.789
0.005
0.306
4.88E04
26.698
10.221
0.019
0.573
7.197
CD4+Effector cells CLA+
CD4+Tcm ICOS+CLA-
0.312
4.43E04
CD4+EM+CLA+
CD4+Tcm ICOS+CLA+
0.656
1.50E04
21.656 6.174
0.072 0.013
CD4+Tem ICOS+CLA+
18.372
27.272
19.461
18.874
22.129
31.052
18.027
35.898
0.052
CD4+Tem ICOS+CLA-
8.604
17.477
11.149
9.824
12.679
14.020
8.851
12.783
0.003
0.931 0.618
0.360
0.003
0.860
0.111
1.91E06
0.001
0.002
0.835
0.779
0.356
0.924
0.438
0.622
0.194
0.040
0.516
0.048
0.031
2.65E05
0.516
0.617
0.062
0.139
0.052
0.886
0.489
0.234
0.091
0.016
0.305
0.133
0.105
0.179
0.630
0.451
0.361
0.015
0.800
0.003
0.009
0.001
0.030
0.026
0.739
0.015
0.156
CD4+Tcm HLADR+CLA+
11.102
5.452
8.450
15.817
7.852
14.303
2.862
8.602
0.088
0.020
0.125
3.52E05
CD4+Tcm HLADR+CLA-
6.051
3.063
2.844
7.171
2.753
4.730
1.181
2.502
0.305
0.066
0.247
0.058
0.251
0.237
0.084
0.907
0.015
0.018
0.099
0.383
0.642
0.370
0.049
0.198
CD4+Tem HLADR+CLA+
10.207
6.294
8.139
10.876
6.277
12.230
5.419
11.540
0.072
0.455
0.103
0.001
0.517
0.066
0.023
0.500
0.319
0.467
0.107
0.106
0.005
0.751
0.824
0.853
CD4+Tem HLADR+CLA-
4.400
3.133
2.537
4.169
2.607
4.494
2.019
1.980
0.435
0.122
0.241
0.949
0.206
0.236
0.087
0.951
0.576
0.554
0.411
0.426
0.288
0.832
0.008
0.082
0.557
9.13E05
3.78E04
0.657
0.037
0.670
0.115
0.943
0.072
0.019
0.053
0.008
0.195
0.387
0.392
0.069
0.831
0.078
0.481
0.001
0.602
0.125
0.185
CD8+Tcm CLA+
12.795
11.574
5.653
10.673
6.408
7.069
12.344
10.814
0.654
0.008
0.724
0.386
0.009
0.017
0.868
CD8+Tem CLA+
15.463
13.867
4.904
9.409
7.466
8.036
11.186
10.925
0.623
0.003
0.683
0.877
0.001
0.009
0.147
0.040
2.13E05
CD8+Effector cells CLA+
9.152
7.482
4.253
4.797
5.333
5.898
4.992
3.278
0.469
0.635
0.812
0.035
0.025
0.121
0.055
0.497
0.461
CD8+Tcm ICOS+CLA+
10.895
8.773
13.133
22.439
18.967
20.423
12.525
18.407
0.613
0.093
0.776
0.020
0.669
0.115
0.689
0.222
0.869
0.068
0.005
0.012
0.001
0.727
0.396
0.659
CD8+Tcm ICOS+CLA-
3.927
4.334
3.127
7.089
5.013
7.632
4.366
4.476
0.737
0.013
0.114
0.899
0.427
0.421
0.655
0.102
0.058
0.560
0.100
0.036
0.900
0.776
0.110
0.039
CD8+Tem ICOS+CLA+
17.315
15.412
14.538
26.308
16.905
29.184
13.409
26.175
0.711
0.033
0.039
1.55E04
0.626
0.943
0.431
0.618
0.759
0.360
0.028
0.012
0.003
0.649
0.979
0.577
CD8+Tem ICOS+CLA-
5.846
3.734
2.508
4.036
2.920
3.632
3.248
3.950
0.146
0.140
0.414
0.298
0.019
0.041
0.066
0.553
0.266
0.650
0.788
0.915
0.785
0.725
0.933
0.701
0.034
5.64E08
0.055
1.09E04
0.128
0.052
0.574
0.046
1.94E04
1.18E06
0.061
0.245
3.50E08
CD8+Tcm HLADR+CLA+ CD8+Tcm HLADR+CLACD8+Tem HLADR+CLA+ CD8+Tem HLADR+CLACD4+Naïve cells CD4+Tcm
9.883 4.954 7.410 2.979 76.014 11.930
9.115 3.978 8.462 3.216 77.562 11.100
10.513 4.440 8.486 4.474 66.520 18.075
11.498 4.381 13.154 2.306 61.472 19.438
7.252 3.830 7.839 3.474 52.718 24.907
18.053 7.602 13.029 3.904 68.480 17.675
6.880 5.921 5.729 4.577 60.613 26.410
19.539 11.174 18.233 7.613 59.677 21.455
0.786 0.508 0.691 0.819 0.666 0.715
0.823 0.958 0.304 0.055 0.255 0.601
0.780 0.003 0.059
0.004 0.803 0.068
0.849 0.726 0.738
0.388 0.471 0.889
0.212 0.539 0.423
0.225
0.615
0.141
0.032
5.47E05
2.59E04
0.001
1.60E06
0.016
0.331 0.602 0.853 0.363 0.012 0.060
0.194 0.217 0.307 0.930 0.148 0.002
0.877 0.112 0.402
0.558 0.406 0.262
0.235
0.310
0.115
7.36E05
0.683
0.001
0.988
0.344
0.080
0.275
2.06E04
0.981
0.223
0.213
0.663
3.26E05
0.309
6.73E07
0.029
0.007
1.81E06
0.085
0.665
0.022
0.012
2.39E05
0.540
0.455
0.176
0.051
0.546
0.048
0.372
0.574
0.264
0.041
0.003
3.26E07 4.34E05
CD4+Tem
6.514
5.500
9.947
12.796
15.585
8.660
9.414
11.536
0.457
0.253
0.011
0.163
0.101
0.002
0.077
0.057
0.793
0.023
0.001
0.012
6.77E06
CD4+Effector cells
5.041
5.854
4.729
6.299
5.947
5.202
3.623
7.360
0.392
0.184
0.531
0.075
0.772
0.381
0.304
0.286
0.447
0.105
0.672
0.560
0.41
CD8+Naïve cells
51.973
64.082
60.900
64.812
55.577
73.750
56.350
51.734
0.031
0.394
0.001
0.189
0.106
0.568
0.386
0.338
0.257
0.880
0.874
0.025
0.003
CD8+Tcm
25.817
15.653
16.197
10.792
17.599
9.835
19.878
18.906
0.026
0.010
0.006
0.626
0.024
0.068
0.146
0.612
0.078
0.391
0.070
0.035
0.237
0.616
1.04E04
CD8+Tem
11.849
8.531
10.927
9.493
15.175
6.317
13.680
13.604
0.088
0.457
0.002
0.965
0.646
0.245
0.352
0.123
0.122
0.577
0.599
0.194
0.003
0.101
0.034
1.36E04
CD8+Effector cells
10.340
11.280
11.979
14.896
11.626
10.095
10.036
15.749
0.637
0.141
0.329
0.008
0.325
0.475
0.863
0.825
0.214
0.352
0.111
0.507
0.056
0.016
0.724
0.006
Table E3B. Median values for normal and atopic dermatitis infants, children, adolescents, and adult patients and respective p-values
P- Values
CD4+IFNγ+ CD4+CLA+
N Infants
AD Infants
N Child
AD Child
N Adol
AD Adol
N Adults
AD Adults
Infants AD vs. N Infants
5.663
3.992
17.861
12.174
21.903
17.790
26.391
23.214
0.388
1.581
2.927
1.500
2.190
1.484
0.718
2.302
3.870
0.078
Child AD vs. N Child
Adol AD vs. N Adol
Adults AD vs. N Adults
N Infants vs. N Child
N Infants vs. N Adol
N Infants vs. N Adults
N Child vs. N Adol
N Child vs. N Adults
N Adol vs. N Adults
AD Infants vs. AD Child
AD Infants vs. AD Adol
AD Infants vs. AD Adults
AD Child vs. AD Adol
AD Child vs. AD Adults
0.052
0.302
0.364
6.40E06
6.41E05
2.57E10
0.500
0.029
0.103
0.003
3.38E06
9.57E11
0.088
0.001
0.097
0.628
1.66E04
0.127
4.12E06
0.022
0.085
0.042
0.283
0.570
0.265
0.528
0.013
0.086
0.214
0.006
AD Adol vs. AD Adults
CD4+IFNγ+CLA+
10.730
6.255
19.172
12.946
18.538
12.720
19.349
15.540
0.042
0.011
0.113
0.136
0.002
0.026
8.30E05
0.500
0.891
0.448
0.017
2.09E04
2.63E05
0.461
0.154
0.433
CD4+IFNγ+CLA-
5.645
3.714
18.088
12.198
21.919
17.845
26.398
23.216
0.379
0.054
0.324
0.400
4.28E06
4.58E05
2.05E10
0.538
0.028
0.096
0.003
3.75E06
5.58E11
0.093
4.73E04
0.066
CD4+IL13+
0.521
1.086
0.779
1.803
0.847
1.582
0.529
1.369
0.123
0.007
0.009
0.001
0.810
0.381
1.000
0.365
0.769
0.342
0.097
0.127
0.241
0.767
0.443
0.634
CD4+IL13+CLA+
2.040
6.758
4.588
6.114
4.115
6.701
3.455
9.400
0.001
0.123
0.108
2.39E05
0.022
0.017
0.185
0.893
0.360
0.551
0.749
0.939
0.148
0.786
0.293
0.090
CD4+IL13+CLA-
0.464
0.891
0.673
1.459
0.777
1.504
0.437
1.023
0.289
0.013
0.005
0.009
0.810
0.418
0.769
0.345
0.497
0.180
0.058
0.014
0.399
1.000
0.161
0.060
1.497
5.73E05
4.08E04
0.056
6.41E06
0.901
0.433
0.467
0.644
0.263
0.107
0.039
0.007
0.043
0.415
0.994
0.350
0.001
0.001
3.09E04
0.006
1.58E06
0.213
0.006
0.008
2.40E07
0.260
8.06E05
0.006
0.564
0.363
0.393
0.115
0.185
0.020
0.115
0.561
0.022
0.002 3.73E07
CD4+IFNγ+CLA+ IL13+CLA+
6.051
0.843
4.175
1.304
3.389
2.624
5.464
CD4+IFNγ+CLAIL13+CLA-
10.116
4.970
35.306
7.101
22.904
11.877
34.706
24.109
0.043
1.37E06
CD4+IL9+
0.267
0.351
0.214
0.523
0.481
0.507
0.208
0.211
0.435
0.046
0.001
0.129
0.518
0.001
0.214
0.071
0.005
1.23E05
0.140
0.228
1.70E04
CD4+IL9+CLA+
1.961
1.865
2.652
5.394
5.230
8.935
1.101
1.656
0.652
0.346
0.315
0.485
0.118
0.008
0.367
0.247
0.003
6.44E05
CD4+IL9+CLA-
0.253
0.296
0.164
0.394
0.410
0.472
0.167
0.147
0.582
0.039
0.291
0.597
0.314
0.211
0.090
0.018
0.609
0.002
0.226
0.022
0.055
0.401
0.011
1.66E04
CD4+IL22+
0.435
0.444
0.807
1.512
2.260
2.116
1.840
1.989
0.980
0.085
0.812
0.375
0.012
7.99E05
6.31E09
0.002
4.71E06
0.847
4.39E04
5.27E10
4.14E08
0.165
0.378
0.351
0.035
2.46E04
8.19E05
0.662
0.651
0.062
0.939
0.145
0.089
0.304
0.054
0.001
0.345
0.043
0.383
0.003
1.57E05
4.00E10
0.002
1.53E06
0.913
1.26E04
5.31E12
3.73E14
0.165
0.034
0.477
0.007
0.939
0.286
0.317
0.011
8.70E05
4.30E08
0.282
0.045
0.312
0.537
0.869
0.894
0.819
0.889
0.160
0.023
0.211
0.042
0.701
0.005
2.16E05
2.25E09
0.271
0.055
0.593
0.670
0.003
0.016
CD4+IL22+CLA+ CD4+IL22+CLA-
3.442 0.377
5.174 0.263
3.170 0.763
5.982 1.450
3.606 2.218
6.283 2.099
4.395 1.727
8.241 2.307
0.137 0.362
0.141
0.919
0.281
CD4+IL17+
0.385
0.271
0.651
0.582
0.780
0.762
0.996
0.936
0.426
0.844
0.434
0.349
0.049
8.96E02
CD4+IL17+CLA+
2.638
1.825
2.091
1.865
2.235
2.711
2.141
2.628
0.140
0.351
0.591
0.230
0.675
0.440
CD4+IL17+CLACD8+IFNγ+
0.355 15.667
0.220 16.053
0.605 45.807
0.512 26.526
0.731 40.483
0.751 29.900
0.884 46.182
0.811 41.604
0.260 0.967
0.776 0.060
0.462 0.251
0.318
0.021
0.037
0.008
0.907
1.36E04
0.003
1.72E06
0.773
0.425
0.229
0.060
0.025
4.76E07
1.13E01
0.406
N/A
0.271
N/A
N/A
0.034
0.002
N/A
0.560
N/A
N/A
0.434
0.012
0.764
0.154
0.052
0.640
0.514
0.116
0.181
0.161
0.006
0.002
1.34E06
0.021
2.00E07
0.633
0.002
0.010
0.303
4.63E04
0.034
6.71E12
0.928
0.001
0.001
CD8+CLA+
0.855
2.059
0.719
0.927
1.417
0.753
N/A
N/A
0.097
0.166
0.833
N/A
CD8+IFNγ+CLA+
23.358
19.786
25.432
19.330
21.156
13.050
34.618
26.705
0.791
0.526
0.081
0.506
0.229
0.960
7.16E05
CD8+IFNγ+CLACD8+IL13+ CD8+IL13+CLA+ CD8+IL13+CLA-
15.342 0.103 1.310 0.060
16.881 0.141 2.247 0.078
45.703 0.140 1.438 0.122
26.485 0.208 3.121 0.184
40.481 0.183 1.638 0.158
31.094 0.270 2.421 0.264
46.282 0.467 2.460 0.419
44.073 0.692 6.702 0.536
0.915 0.226 0.237
0.060 0.044 0.164
0.261 0.261
0.007
0.449
4.44E05
0.431 0.843
0.923
0.334
0.818
0.415
0.525
4.67E04
0.351
0.224 0.030
0.056 0.042
0.774
0.123
0.712
0.114
0.134
0.655
0.824
1.16E04
0.583
0.010
3.90E04
0.577
3.51E04
0.023
2.14E04
4.82E05
2.48E15
0.867
0.025
0.024
0.169
0.096
0.070
0.074
4.46E06
0.037
0.982
9.33E05
0.363
0.395
0.129
0.055
0.729
0.007
0.786
0.700
0.802
1.000
0.954
0.924
0.512
0.021
0.224
0.113
0.132
0.804
0.685
0.147
0.017
0.240
0.745
0.743
0.212
0.908
0.607
0.474
0.388
0.054
0.777
0.419
0.085
0.431
0.682
0.003
0.054
0.115
0.312
0.953
0.202
0.271
0.994
0.357
0.173
0.002
2.10E05
0.181
0.492
0.252
0.426
CD8+IFNγ+CLA+ IL13+CLA+
7.190
5.842
13.043
4.218
4.559
3.723
12.751
4.242
0.465
CD8+IFNγ+CLAIL13+CLA-
38.959
29.056
56.026
41.155
59.395
45.281
77.830
48.033
CD8+IL9+
0.297
0.451
0.111
0.306
0.471
0.534
0.360
0.369
0.057
CD8+IL9+CLA+
7.202
9.416
2.744
7.499
5.078
12.186
2.299
1.883
0.233
0.118
0.036
0.578
0.074
0.346
0.004
0.347
0.664
0.052
0.185
0.859
4.98E06
CD8+IL9+CLA-
0.216
0.181
0.091
0.297
0.361
0.394
0.291
0.278
0.640
0.129
0.743
0.467
0.245
0.094
0.282
0.002
0.028
0.240
0.928
0.150
0.301
CD8+IL22+
0.100
0.192
0.128
0.184
0.357
0.332
0.580
0.668
0.123
0.067
0.937
0.406
0.444
0.005
8.05E07
0.013
6.32E06
0.360
0.370
0.005
1.77E07
0.104
1.83E04
0.015
CD8+IL22+CLA+
0.674
1.438
0.835
2.976
0.982
2.916
1.732
5.576
0.061
0.039
0.039
1.59E05
0.459
0.216
0.003
0.425
0.043
0.261
0.227
0.029
4.70E06
0.548
0.026
0.067
CD8+IL22+CLA-
0.068
0.140
0.114
0.160
0.345
0.318
0.536
0.457
0.224
0.166
0.830
0.905
0.219
0.001
1.35E07
0.013
7.66E06
0.486
0.162
2.79E04
1.44E08
0.093
0.001
0.085
CD8+IL17+
0.107
0.089
0.081
0.114
0.264
0.083
0.297
0.301
0.987
0.081
0.162
0.914
0.431
0.054
0.003
0.006
1.34E05
0.455
0.475
0.899
5.81E05
0.651
0.001
0.003
CD8+IL17+CLA+
0.799
0.674
0.538
0.364
1.661
0.606
1.251
1.204
0.508
0.481
0.033
0.962
0.369
0.290
0.454
0.057
0.099
0.522
0.233
0.786
0.093
0.431
0.009
0.041
0.006
1.02E05
0.351
0.274
0.355
8.64E07
0.887
0.001
0.003
7.92E05
1.13E05
0.004
0.918
0.010
0.002
0.008
0.077
0.476
0.117
CD8+IL17+CLATregs (CD25+CD127-)
0.103 5.150
0.078 5.420
0.072 6.630
0.084 9.530
0.211 10.900
0.079 11.200
0.272 5.260
Tregs (CCR4+CLA+)
5.820
7.630
6.230
18.200
7.925
11.900
11.850
CD4+CM+CLA+
12.420
20.090
9.514
16.810
10.289
8.830
12.297
0.245
0.967
0.166
0.191
0.135
15.300
0.156
0.094
0.144
0.912
0.193
0.001
0.367
0.002
0.002
0.004
0.287
9.650
0.007
0.023
0.990
0.779
0.325
0.103
0.876
0.759
0.256
0.181
0.479
0.001
0.002
0.019
0.042
0.342
0.335
0.283
0.910
0.850
0.031
0.003
0.038
0.776
0.621
0.666
0.018
0.002
1.78E04
0.647
0.318
0.116
0.102
0.629
0.974
0.033
0.021
0.928
0.124
0.466
0.188
0.368
0.030
0.261
1.68E07
0.037
0.033
0.392
0.775
0.005
0.013
0.248
0.026
0.003
0.145
0.164
0.440
0.992
1.95E04
0.001
0.268
0.018
0.637
0.258
0.283
0.015 0.340
19.290
18.250
19.660
18.330
0.005
0.092
0.852
7.651
5.440
4.320
4.045
3.202
3.450
7.387
4.999
0.378
0.497
3.151
4.463
8.001
7.264
19.187 8.328
22.861 8.355
6.401 2.454
19.259 5.857
0.169 0.039
CD4+Tem ICOS+CLA+
16.725
24.570
17.600
18.115
25.072
29.024
15.782
32.419
0.042
CD4+Tem ICOS+CLA-
7.010
14.018
11.264
8.993
10.428
12.741
7.049
10.065
0.003
CD4+Tcm HLADR+CLA+ CD4+Tcm HLADR+CLA-
6.438 2.061
4.326 0.911
6.473 2.262
15.789 4.716
6.279 1.996
9.549 2.209
1.862 0.520
6.813 1.190
0.086 0.250
1.000 0.788
0.358
0.002
0.974
0.189
1.38E06
0.463
0.164
0.302
0.669
0.024
0.194
0.264
3.98E09
0.881
1.09E04
0.709
0.769
0.148 0.285
0.058 0.071
CD4+Tem HLADR+CLA+
8.512
4.757
3.968
7.464
6.259
6.503
3.799
9.215
0.059
0.243
0.579
1.23E04
CD4+Tem HLADR+CLA-
1.529
1.468
1.360
2.354
1.271
2.606
0.944
1.510
0.507
0.038
0.360
0.102
CD8+Tcm CLA+
11.109
9.430
4.050
8.480
6.160
5.735
12.660
0.015
0.001
24.100
15.758
0.005
0.782
16.490
18.023
0.818
8.76E07
0.017
36.470
10.720
0.001
3.99E04
25.365
8.761
0.025
0.561
6.970
CD4+Effector cells CLA+
CD4+Tcm ICOS+CLA-
0.810
2.45E04
CD4+EM+CLA+
CD4+Tcm ICOS+CLA+
0.898
8.890
0.942
0.009
0.659
0.002
1.000
0.005
0.550
0.613
0.111
0.109
0.420
0.020
0.042
9.36E05
0.536
0.087
0.044
0.017
0.679
0.149
0.107
0.182
0.429
0.724
2.13E04
0.029
0.003
0.152
0.019
0.461
0.044
0.538
0.034
0.841
4.24E05
1.43E05
0.001
0.303
0.223
0.242
0.005
0.873
0.157
0.060
0.657
0.945
0.408
0.162
0.330
0.001
0.991
0.352
0.365
0.565
0.162
0.716
0.965
0.265
0.036
0.278
0.824
0.530
0.028
0.256
0.296
1.33E04
0.001
0.653
0.016
0.700
0.071
0.863
0.012
0.019
0.159
0.115
0.605
0.602
0.610
0.256
0.804
0.283
0.094
0.666
0.620
1.67E05
0.324
0.010
0.025
0.526
0.009
0.419
0.079
5.54E05
CD8+Tem CLA+
11.382
9.080
5.102
7.690
6.023
6.645
10.313
9.412
0.531
0.009
0.560
0.554
5.98E05
CD8+Effector cells CLA+
6.460
5.543
4.110
3.606
4.030
3.030
3.621
3.260
0.566
0.846
0.762
0.183
0.023
0.023
0.041
0.831
0.435
0.713
0.225
0.214
0.021
0.599
0.376
0.924
CD8+Tcm ICOS+CLA+
3.735
7.295
5.822
19.633
16.667
24.721
12.136
15.659
0.558
0.091
0.921
0.088
0.628
0.014
0.023
0.080
0.219
0.255
0.013
0.029
0.001
0.782
0.751
0.686
CD8+Tcm ICOS+CLA-
1.961
3.105
2.459
5.675
3.218
6.940
4.356
3.548
1.000
0.055
0.068
0.484
0.965
0.174
0.161
0.090
0.043
0.876
0.145
0.027
0.474
0.575
0.242
0.029
3.46E04
0.805
0.664
0.624
0.456
0.584
0.831
0.049
0.019
0.003
0.667
0.818
0.544
0.036
0.087
0.148
0.529
0.298
0.748
0.869
0.806
0.250
0.943
0.337
0.786
0.049
1.39E05
0.111
0.001
0.958
0.032
1.96E06
0.089
4.47E06
0.017
CD8+Tem ICOS+CLA+
9.841
9.695
13.601
21.448
16.689
26.667
11.837
21.531
0.597
0.040
0.052
CD8+Tem ICOS+CLA-
4.818
3.143
2.042
2.679
2.364
3.850
2.686
3.106
0.141
0.435
0.496
0.140
0.042
2.29E09
0.048
1.39E04
CD8+Tcm HLADR+CLA+ CD8+Tcm HLADR+CLA-
9.502 2.673
6.457 2.016
6.354 3.448
7.056 2.393
4.270 2.903
18.549 5.044
5.924 4.121
18.648 11.376
0.749 0.664
0.972 0.691
0.938 0.462
0.432 0.953
0.663 0.205
0.560 0.332
0.827 0.396
0.388 0.101
0.865 0.352
CD8+Tem HLADR+CLA+
4.361
6.255
4.416
4.751
5.331
9.719
4.596
15.959
0.728
0.438
0.361
3.06E10
0.990
0.981
0.650
0.990
0.649
0.802
0.597
0.430
2.39E05
0.989
0.016
0.024
CD8+Tem HLADR+CLA-
1.786
2.302
3.203
1.638
2.874
1.806
3.300
6.633
0.842
0.046
0.383
0.001
0.139
0.194
0.027
0.741
0.642
0.382
0.376
0.826
2.31E06
0.559
7.00E07
0.002
CD4+Naïve cells
80.750
79.750
66.200
63.000
58.100
69.250
61.750
63.700
0.706
0.240
0.005
1.000
0.038
3.37E05
1.49E04
0.015
0.144
0.176
5.04E05
0.006
7.57E06
0.150
0.719
0.056
CD4+Tcm
9.370
8.510
17.400
15.700
22.950
16.550
25.500
17.900
0.460
0.869
0.072
0.049
0.007
2.41E04
3.65E06
0.049
0.004
0.442
3.95E04
0.006
3.84E05
0.723
0.349
0.248
0.159
3.44E04
0.027
0.767
0.013
2.39E04
0.007
2.27E05
0.205
0.958
0.121
0.286
3.90E04
0.008
2.93E04
0.597
0.494
0.041
0.309
0.058
0.217
CD4+Tem CD4+Effector cells
4.560 4.400
4.290 4.718
8.950 2.940
10.100 4.970
12.350 5.260
8.345 4.600
7.860 1.195
10.500 2.490
0.633 0.500
0.162 0.125
0.016 0.365
0.141 0.014
0.314
0.030 0.150
CD8+Naïve cells
53.350
69.800
61.500
65.200
61.250
78.500
57.850
53.300
0.026
0.497
0.003
0.317
0.130
0.496
0.550
0.624
0.257
0.511
0.824
0.058
0.002
0.044
0.005
9.57E06
CD8+Tcm
22.600
10.200
15.800
10.600
15.200
10.030
19.700
17.200
0.026
0.015
0.007
0.532
0.110
0.213
0.566
0.915
0.086
0.090
0.200
0.144
0.013
0.689
0.001
3.02E04
CD8+Tem
9.875
6.160
9.460
7.660
11.900
4.655
13.400
11.400
0.046
0.237
0.002
0.818
0.806
0.330
0.265
0.220
0.106
0.846
0.640
0.197
0.002
0.098
0.015
1.10E04
CD8+Effector cells
9.095
8.285
11.800
15.100
11.250
11.050
7.325
14.100
0.801
0.181
0.450
0.018
0.130
0.311
0.585
0.765
0.056
0.185
0.046
0.410
0.034
0.024
0.952
0.056
Mean expression of all T-cell subset frequencies in healthy and AD individuals, as well as P-values (measured by Welch’s t-test) for all AD versus healthy comparisons and comparisons within the healthy group and within the AD group (A). Median expression of all T-cell subset frequencies in healthy and AD individuals, as well as P-values (measured by Wilcoxon-Mann-Whitney test) for all AD versus healthy comparisons and comparisons within the healthy group and within the AD group (B). Abbreviations AD: Atopic dermatitis; Adol: Adolescent; Child: Children; CLA: Cutaneous lymphocyte antigen; HLADR: Human leukocyte antigen-DR; ICOS: Inducible co-stimulator; IFNγ: Interferon-gamma; IL: Interleukin; N: Normal; Tcm: Central memory T; Tem: Effector memory T; Treg: Regulatory T
Table E4. Selected Spearman correlations and their P-values in atopic dermatitis patients AD Age Marker Duration of Disease CD4+IFNγ+CLACD8+IL13+CLACD4+IFNγ+ CD8+IL13+ CD8+IL22+CLACD8+IL22+ CD8+IL17+CLACD4+IFNγ+CLA+ CD8+IL22+CLA+ CD8+IL9+CLA+ CD8+IL17+ CD8+IL13+CLA+ EASI CD4+IL22+CLA+ CD8+CLA+ SCORAD Total ADQ Eosinophils CD4+IL13+CLA+ Pruritus ADQ CD8+IFNγ+CLA+ CD4+IL9+CLACD8+IL17+CLA+ CD4+IL9+ CD8+IL9+CLACD4+IL13+ CD8+IL9+ CD4+IL9+CLA+ CD4+IL13+CLACD4+CLA+ CD8+IFNγ+CLA-IL13+CLACD8+IFNγ+CLA+IL13+CLA+
EASI r 0.980 0.647 0.646 0.638 0.581 0.546 0.501 0.462 0.439 0.428 -0.405 0.372 0.358 0.340 0.312 -0.370 0.250 0.237 0.238 0.163 0.155 0.150 -0.145 0.143 -0.122 0.111 0.098 -0.093 -0.088 0.072 0.061 -0.058 -0.022
P-value 1.12E-56 1.39E-15 3.67E-15 4.76E-15 6.66E-12 1.36E-10 8.84E-09 1.06E-07 5.45E-07 1.11E-06 5.03E-06 3.15E-05 6.46E-05 1.52E-04 5.16E-04 9.35E-04 1.38E-03 1.14E-02 3.48E-02 7.74E-02 1.02E-01 1.03E-01 1.15E-01 1.21E-01 1.85E-01 2.30E-01 2.86E-01 3.17E-01 3.41E-01 4.37E-01 5.07E-01 5.26E-01 8.09E-01
Marker r P-value CD4+IL9+CLA+ 0.331 3.44E-03 CD8+IL9+CLA0.316 5.45E-03 CD8+IL9+CLA+ 0.265 2.05E-02 CD8+IL9+ 0.260 2.35E-02 CD4+IL9+CLA0.245 3.30E-02 CD4+IL22+CLA+ 0.224 5.18E-02 CD4+IL9+ 0.221 5.52E-02 CD4+CLA+ -0.213 6.50E-02 CD8+CLA+ -0.209 6.94E-02 CD8+IFNγ+CLA-IL13+CLA0.112 3.24E-01 CD4+IL13+CLA+ 0.093 4.24E-01 CD4+IL13+ 0.081 4.88E-01 CD4+IL13+CLA0.071 5.41E-01 CD8+IFNγ+CLA+IL13+CLA+ 0.019 8.67E-01 CD8+IFNγ+CLA+ -0.002 9.86E-01 SCORAD Marker
r EASI 0.738 CD8+IL9+CLA0.271 CD4+CLA+ 0.246 CD8+IL9+ 0.203 CD4+IL22+CLA+ 0.201 CD4+IL13+CLA+ 0.200 CD4+IL13+ 0.126 CD4+IL9+CLA-0.071 CD8+IL9+CLA+ 0.070 CD8+CLA+ -0.063 CD4+IL9+CLA+ -0.050 CD4+IL9+ -0.046 CD8+IFNγ+CLA+ 0.044 CD4+IL13+CLA0.029 CD8+IFNγ+CLA+IL13+CLA+ -0.017 CD8+IFNγ+CLA-IL13+CLA0.013
P-value 1.42E-21 3.32E-03 7.53E-03 2.89E-02 2.95E-02 3.17E-02 1.75E-01 4.45E-01 4.56E-01 5.93E-01 5.97E-01 6.19E-01 6.41E-01 7.54E-01 8.58E-01 8.86E-01
Total ADQ Marker
r SCORAD 0.576 EASI 0.532 CD8+IL9+CLA0.261 CD4+IL9+CLA+ 0.240 CD8+CLA+ -0.192 CD8+IL9+ 0.192 CD4+IL13+CLA+ 0.182 CD4+IL13+ 0.162 CD4+IL9+ 0.136 CD4+IL9+CLA0.121 CD4+IL22+CLA+ 0.120 CD4+IL13+CLA0.113 CD4+CLA+ -0.107 CD8+IFNγ+CLA+IL13+CLA+ -0.095 CD8+IL9+CLA+ 0.088 CD8+IFNγ+CLA+ 0.070 CD8+IFNγ+CLA-IL13+CLA- -0.039
P-value 2.54E-11 1.59E-09 2.68E-02 4.26E-02 1.05E-01 1.07E-01 1.26E-01 1.74E-01 2.56E-01 3.12E-01 3.15E-01 3.44E-01 3.69E-01 4.20E-01 4.64E-01 5.59E-01 7.39E-01
Eosinophil Count Marker r Pruritus ADQ 0.350 EASI 0.335 Total ADQ 0.288 SCORAD 0.267 CD4+CLA+ -0.133 CD4+IL9+CLA0.086 CD4+IL13+CLA+ 0.080 CD4+IL9+ 0.070 CD8+CLA+ 0.063 CD8+IFNγ+CLA+ 0.048 CD4+IL22+CLA+ -0.048 CD8+IFNγ+CLA+IL13+CLA+ 0.046 CD8+IL9+ 0.044 CD4+IL13+ 0.036 CD8+IL9+CLA0.032 CD4+IL13+CLA0.030 CD8+IL9+CLA+ 0.025 CD8+IFNγ+CLA-IL13+CLA- 0.020 CD4+IL9+CLA+ -0.012
P-value 2.42E-03 2.76E-03 1.35E-02 1.88E-02 3.46E-01 5.46E-01 5.74E-01 6.21E-01 6.56E-01 7.36E-01 7.37E-01 7.43E-01 7.56E-01 8.00E-01 8.22E-01 8.30E-01 8.62E-01 8.85E-01 9.33E-01
Pruritus ADQ Marker r Total ADQ 0.945 SCORAD 0.547 EASI 0.456 CD8+IL9+CLA0.185 CD8+CLA+ -0.185 CD4+IL13+CLA+ 0.170 CD4+IL9+CLA+ 0.151 CD4+CLA+ -0.138 CD4+IL13+ 0.133 CD8+IL9+ 0.121 CD8+IFNγ+CLA+IL13+CLA+ -0.108 CD4+IL13+CLA0.087 CD8+IFNγ+CLA+ 0.062 CD4+IL22+CLA+ 0.044 CD4+IL9+ 0.032 CD8+IL9+CLA+ 0.031 CD4+IL9+CLA0.021 CD8+IFNγ+CLA-IL13+CLA- -0.002
TEWL LS
TEWL NL Marker EASI
P-value 9.72E-56 3.80E-10 4.45E-07 1.20E-01 1.21E-01 1.54E-01 2.06E-01 2.49E-01 2.65E-01 3.11E-01 3.62E-01 4.68E-01 6.07E-01 7.17E-01 7.88E-01 7.97E-01 8.62E-01 9.89E-01
r 0.294
P-value 2.71E-03
Marker Age
r 0.333
P-value 5.89E-04
Significant Spearman correlations (P<0.05), with accompanying correlation coefficient (r) and P-value between various T-cell subsets and age or clinical scores (e.g. EASI, SCORAD, pruritus ADQ, TEWL NL, TEWL LS, eosinophil count). Abbreviations ADQ: Atopic dermatitis Quickscore; Adol: Adolescent; Child: Children; CLA: Cutaneous lymphocyte antigen; EASI: Eczema Area and Severity Index; HLADR: Human leukocyte antigen-DR; IFNγ: Interferon-gamma; IL: Interleukin; LS: Lesional; N: Normal; NL: Non-lesional; SCORAD: SCORing Atopic Dermatitis; Tcm: Central memory T; Tem: Effector memory T; TEWL: Transepidermal water loss; Treg: Regulatory T
Duration of Disease Marker r CD4+IFNγ+ 0.584 CD4+IFNγ+CLA0.583 CD4+IL9+CLA+ 0.570 CD8+IL13+CLA0.507 CD4+IFNγ+CLA+ 0.406 CD8+IL22+CLA0.392 CD4+IL9+CLA0.381 EASI 0.376 CD4+IL9+ 0.350 CD8+IL22+ 0.317 CD4+CLA+ -0.304 CD8+IL13+ 0.299 CD8+CLA+ -0.299 CD4+IL13+CLA0.289 CD8+IL22+CLA+ 0.281 Total ADQ 0.284 CD4+IL22+CLA+ 0.276 CD8+IL9+CLA0.235 Pruritus ADQ 0.222 CD4+IL13+ 0.217 Eosinophils 0.234 CD8+IL17+CLA0.155 SCORAD 0.153 CD4+IL13+CLA+ 0.149 CD8+IFNγ+CLA-IL13+CLA0.143 CD8+IL17+CLA+ -0.093 CD8+IL9+ 0.055 CD8+IL17+ 0.049 CD8+IFNγ+CLA+IL13+CLA+ 0.039 CD8+IL9+CLA+ 0.028 CD8+IL13+CLA+ 0.016 CD8+IFNγ+CLA+ -0.006
P-value 2.45E-08 2.74E-08 6.39E-08 2.50E-06 2.51E-04 4.28E-04 6.34E-04 6.46E-04 1.81E-03 4.96E-03 7.22E-03 8.15E-03 8.34E-03 1.09E-02 1.35E-02 1.41E-02 1.50E-02 3.95E-02 5.68E-02 5.84E-02 9.18E-02 1.77E-01 1.80E-01 1.96E-01 2.05E-01 4.26E-01 6.35E-01 6.75E-01 7.30E-01 8.07E-01 8.89E-01 9.61E-01
Table E5: Pairwise comparisons between age group associated clusters P-values Adults vs. Infants
Children and Adolescents vs. Infants
Children and Adolescents vs. Adults
Adults vs. Infants
Children and Adolescents vs. Infants
Children and Adolescents vs. Adults
P-value ANOVA
FDR ANOVA
CD4+IFNγ+
23.985
11.339
-12.646
2.82E-14
1.03E-08
6.47E-08
2.56E-19
1.74E-17
CD4+IFNγ+CLA-
24.326
11.726
-12.600
2.88E-14
7.95E-09
1.44E-07
4.46E-19
2.99E-17
CD4+IFNγ+CLA+
16.491
5.763
-10.728
1.30E-13
1.00E-03
1.85E-07
2.49E-13
1.64E-11
CD8+IFNγ+CLA-
37.265
13.395
-23.870
2.22E-13
8.51E-04
4.17E-07
5.10E-13
3.31E-11
CD8+IFNγ+
36.183
12.373
-23.810
3.67E-13
1.86E-03
2.93E-07
9.04E-13
5.78E-11
CD4+IL22+CLA-
2.477
2.543
0.067
3.26E-07
3.52E-10
0.987
5.75E-11
3.62E-09
CD4+IL22+
1.010
2.831
1.821
7.52E-02
3.82E-11
2.49E-04
6.00E-11
3.72E-09
CD4+IL9+CLA+
-0.975
5.543
6.518
6.81E-01
2.10E-07
2.57E-07
2.51E-09
1.53E-07
CD8+IL13+CLA-
0.701
0.334
-0.367
5.93E-08
1.44E-03
4.61E-03
8.43E-08
5.06E-06
CD4+Naïve cells
-22.633
-8.536
14.097
5.04E-08
1.05E-02
3.47E-04
1.12E-07
6.60E-06
CD8+IL22+CLA-
0.780
0.311
-0.469
7.63E-08
1.11E-02
1.01E-03
1.63E-07
9.45E-06
CD4+IL17+CLA-
1.167
0.843
-0.324
2.34E-06
3.36E-05
0.300
3.66E-07
2.09E-05
CD4+IL17+
1.174
0.816
-0.358
1.75E-06
5.30E-05
0.228
3.79E-07
2.12E-05
CD8+Tem HLADR+CLA-
5.887
0.329
-5.558
1.62E-06
9.26E-01
2.96E-06
5.29E-07
2.91E-05
CD4+IL13+
0.068
1.791
1.723
0.987
6.20E-06
2.73E-04
1.50E-06
8.11E-05
CD4+IL9+
-0.135
0.734
0.869
0.782
6.58E-05
6.80E-05
3.48E-06
1.84E-04
CD4+IL9+CLA-
-0.092
0.603
0.695
0.848
7.50E-05
1.24E-04
5.57E-06
2.90E-04
CD4+Tcm
12.321
7.113
-5.208
1.07E-05
1.25E-03
8.09E-02
7.32E-06
3.73E-04
CD4+IL13+CLA-
0.141
1.539
1.398
0.934
1.85E-05
1.47E-03
7.95E-06
3.97E-04
CD8+IL22+
0.767
0.292
-0.474
4.99E-06
0.047
5.16E-03
9.67E-06
4.74E-04
-21.405
-12.280
9.124
2.02E-05
0.002
0.090
1.47E-05
0.001
CD4+Tem
8.138
2.779
-5.358
8.69E-06
0.081
0.003
1.70E-05
0.001
CD4+IFNγ+CLA+IL13+CLA-
73.154
5.044
-68.110
6.60E-05
0.924
1.73E-04
3.91E-05
0.002
CD4+IL22+CLA+
2.017
4.986
2.969
0.295
4.58E-05
0.065
7.44E-05
0.003
Tregs CD25+CD127+
1.378
4.627
3.249
0.561
7.94E-05
0.028
7.69E-05
0.003
CD8+Tcm ICOS+CLA+
6.025
13.250
7.225
0.257
7.12E-05
0.123
1.20E-04
0.005
CD8+IL17+CLA-
0.395
0.152
-0.243
7.12E-05
0.102
0.018
1.28E-04
0.005
CD8+Naïve cells
-14.301
3.940
18.241
4.73E-03
0.490
9.87E-05
1.67E-04
0.007
CD4+Tem CLA+
CD4+IFNγ+CLA+IL13+CLA+
5.002
0.293
-4.709
3.34E-04
0.955
6.48E-04
2.05E-04
0.008
CD4+Tcm CLA+
-10.615
-6.947
3.668
5.60E-04
0.004
0.345
2.68E-04
0.010
CD8+IFNγ+CLA+
13.375
-4.151
-17.526
0.010
0.496
3.61E-04
5.62E-04
0.021
CD8+Tem CLA+
-4.363
-6.058
-1.696
0.105
0.001
0.683
0.002
0.059
CD4+Tcm HLADR+CLA+
2.583
7.579
4.996
0.629
0.002
0.148
0.003
0.101
CD8+IL13+CLA+
5.232
1.302
-3.930
0.002
0.546
0.026
0.003
0.118
CD8+Effector cells
6.634
-0.497
-7.131
0.012
0.958
0.004
0.004
0.135
CD8+Tem ICOS+CLA+
5.485
12.108
6.622
0.453
0.003
0.280
0.004
0.143
CD8+Tcm HLADR+CLA+
10.335
10.257
-0.079
0.033
0.007
1.000
0.005
0.145
CD8+Tem
5.247
-0.345
-5.592
0.017
0.970
0.006
0.006
0.198
CD4+IL17+CLA+
1.288
0.507
-0.781
0.004
0.272
0.113
0.007
0.198
CD8+Effector cells CLA+
-4.570
-2.774
1.796
0.010
0.055
0.440
0.008
0.226
CD4+IL13+CLA+
-0.035
4.409
4.444
1.000
0.015
0.049
0.008
0.229
CD8+IL17+CLA+
0.731
-0.664
-1.395
0.286
0.220
0.010
0.012
0.321
CD8+IL9+CLA+
-8.800
-2.116
6.684
0.009
0.649
0.055
0.012
0.321
CD8+IL13+
0.709
0.244
-0.464
0.009
0.405
0.115
0.013
0.328
CD4+Tem ICOS+CLA-
-8.238
-4.029
4.209
0.019
0.201
0.308
0.023
0.542
CD8+Tcm CLA+
-2.498
-4.210
-1.713
0.420
0.019
0.638
0.026
0.597
CD8+Tem HLADR+CLA+
7.009
7.170
0.161
0.123
0.031
0.999
0.027
0.597
CD4+Tcm ICOS+CLA-
-1.975
2.717
4.693
0.571
0.177
0.034
0.028
0.597
CD4+Tcm ICOS+CLA+
4.248
7.190
2.942
0.434
0.023
0.639
0.031
0.617
CD8+Tcm ICOS+CLA-
-1.177
2.186
3.363
0.702
0.136
0.047
0.031
0.617
CD4+Tem HLADR+CLA+
1.993
5.482
3.489
0.753
0.034
0.387
0.040
0.716
CD8+IL22+CLA+
4.546
1.976
-2.571
0.062
0.450
0.383
0.076
1.00
CD8+Tcm
2.806
-2.710
-5.517
0.558
0.407
0.087
0.090
1.00
CD4+Effector cells
2.203
-1.360
-3.563
0.406
0.564
0.077
0.092
1.00
CD8+IL9+CLA-
0.036
0.240
0.203
0.965
0.107
0.313
0.101
1.00
CD8+IL17+
0.226
0.031
-0.194
0.123
0.938
0.194
0.125
1.00
CD4+Tem HLADR+CLA-
-1.824
0.474
2.298
0.297
0.873
0.123
0.144
1.00
-101.376
-49.219
52.157
0.167
0.526
0.609
0.184
1.00
-0.238
0.178
0.415
0.592
0.640
0.190
0.213
1.00
CD8+CLA+
3.071
-1.244
-4.315
0.519
0.494
0.283
0.227
1.00
CD8+IFNγ+CLA+IL13+CLA+
-8.738
-7.932
0.807
0.386
0.310
0.992
0.257
1.00
CD8+Tcm HLADR+CLA+
3.841
0.516
-0.885
0.943
-4.726
0.335
0.364
1.00
Tregs CCR4+CLA+
3.026
0.678
3.157
0.502
0.131
0.999
0.499
1.00
CD4+Effector cells CLA+
-2.420
0.647
-0.254
0.992
2.167
0.683
0.642
1.00
CD4+Tcm HLADR+CLA+
-0.908
0.887
-0.975
0.797
-0.067
0.999
0.798
1.00
CD4+CLA+
-0.589
0.801
-0.168
0.974
0.421
0.887
0.816
1.00
CD4+Tem ICOS+CLA+
2.145
0.902
0.733
0.980
-1.412
0.952
0.910
1.00
CD8+Tem ICOS+CLA-
0.128
0.990
-0.225
0.951
-0.353
0.922
0.914
1.00
CD8+IFNγ+CLA+IL13+CLACD8+IL9+
P-values, as measured by Welch’s t-test, that compare the three clusters that were obtained by k-means unsupervised clustering of all AD patients, as well as both raw and FDR-adjusted ANOVA score among all clusters. Abbreviations ANOVA: Analysis of variance; CLA: Cutaneous lymphocyte antigen; FDR: False discovery rate; HLADR: Human leukocyte antigen-DR; ICOS: Inducible co-stimulator; IFNγ: Interferon-gamma; IL: Interleukin; Tcm: Central memory T; Tem: Effector memory T; Treg: Regulatory T
Table E6. Evolution of pathologic T-cell subsets in atopic dermatitis from infancy to adulthood Healthy controls
0-5y/o IFN-γ CLA+>CLACD4+>CD8+
IL-13 CLA+>CLACD4+>CD8+
IL-9
6-11y/o
12-17y/o
Atopic Dermatitis
≥ 18y/o
0-5y/o
6-11y/o
12-17y/o
CLA->CLA+
CLA->CLA+
≥ 18y/o
---
-----
IL-17 ---
CLA-
CLA-
CLA-
---
CLA-
CLA-
CLA-
IL-22 ---
---
---
---
---
CLA-
---
CLA-
---
CLA+
CLA+
Tregs (CLA+)
---
---
---
Arrows show developmental changes in healthy controls and AD across ages, as well as AD vs. control changes in the AD groups. CLA, Cutaneus Lymphocyte Antigen; Tregs, T-regulatory cells
Figure E1
A
B 10
C
5
5
10
5
10 4
10
4
10
3
10
10 4
76.6
12.6
65.2
10
0
0
50K
FSC-A
Frequency (%)
D
100K
150K
CLA CD4 Tcm cells
CD4 CM CLA+
** **
100 100
** **
** **
200K
50
0
CD8
200
**
**
10 2
10 3
CLA CD4 Tem cells
CD4 EM CLA+ 200
** *
**
150 150
100 100
** **
**
*** ***
F
AD N
Infants
H
AD N
AD
N
0
AD
Children Adolescents
Adults
HLA-DR CLA CD4 Tcm cells CD4 CM HLDR+/CLA+
** ***
100
100
+ +
*** * + *
50
50
0
*
**
100
**
AD N
AD N AD
Children Adolescents
CD4 CM HLADR+/CLA
N
0
AD
Adults
** *
*** +
+* + +
50
50
*** ***
N
AD N
AD N
AD
N
AD
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
Infants
Children Adolescents
Adults
+*
*** *
** ***
AD N
40
AD N
AD
N
AD
AD N
AD N
AD
N
AD
HLA-DR CLA CD4 Tem cells CD4 EM HLADR+/CLA+
50
0
Infants
Children Adolescents
Adults
0
Adults
**
** **
N
AD N
AD N
Infants
K HLA-DR
Children Adolescents
N
AD
Adults
CLA CD4 Tem cells
CD4 EM HLADR+/CLA
* **
60
*
+ *
AD
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
60 +
+
40 40
*** ***
+ +
20
*
20
0 0
0
N
+ *
40
Children Adolescents
+
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
*** ***
** **
+
*** ***
0
N
*** * *** +
**
+
60
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
100
25
CLA CD8 Tem cells
20
100
25
5
CD8 EM CLA+
20
Infants
J
G
10
60
** **
50
0
+ *** ***
**
10
4
80
*** **
0
0
**
10
3
80
0
N
75
* **
10
20
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
75 *** **
** **
40
HLA-DR CLA CD4 Tcm cells
100
0
20
Infants
I
4.84 2
CLA CD8 Tcm cells
40
0
N
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
5.95
CD45RO
CD8 CM CLA+
60
+ *
0
10 5
60
50
0
10 2
10 4
**
** *
50 0
Frequency (%)
41.3
0
250K
E
**
50
51.9
10 2
CD4
CD3
10 2
3
CCR7
10
3
N
AD N
AD N
AD
N
AD
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
Infants
Children Adolescents
Adults
N
AD N
AD N
AD
N
AD
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
Infants
Children Adolescents
Adults
Figure E2
CD4⁺ T cells
A
B
Infants Normal
Normal
CD4 Infants
Normal
CD4 Children
Normal
CD8 Infants
CD8 Children
Naive 56%
Naive 62%
Naive 56%
Naive 62%
Naive 64%
Naive 68%
AD
CD8 Children
Naive 64%
Naive 50%
Naive 55%
Children
AD
CD8 Infants
Naive 50%
Naive 55%
Naive 68%
D
Infants
AD
CD4 Children
Naive 78%
Naive 78%
C
Children
AD
CD4 Infants
Naive 78%
Naive 78%
Tcm 12%
m Tc
13
12 %
%
%
%
%
15
%
m Tc
10
17
9%
28
%
H
Adults Normal
AD
CD8 Adults
CD8 Adults
Naive 56%
Naive 54% Naive 54%
Naive 56%
Naive 77%
Naive 70%
Tcm 15%
m Te
% 11
m Tc
m Te
m Tc
19 Naive 60%
or ct fe Ef 20%
Tem 10%
CD8 Adolescents
Naive 60%
Naive 60%
Effector 20%
Tem 13%
r to
Tcm 17%
CD8 Adolescents
Naive 60%
Effector 13%
Adolescents Normal AD
AD
Naive 61%
c fe Ef
%
m Tc
% 11
%
% 11
G
Normal
Tem 9%
Tcm 28%
CD4 Adults
Naive 61%
Effector 11%
m Te or ct fe Ef 13%
or ct fe Ef 10%
12
m Te
Tcm 19%
CD4 Adults
Naive 70%
Naive 57%
Effector 10%
Tem 12%
Adults
CD4 Adolescents
Naive 57%
16
m Te
F
Adolescents Normal AD CD4 Adolescents
Tem 17%
m Tc
6%
Tem 11%
Effector 8%
m Te
Tcm 16%
5%
6%
% 11
m Te
% 11
m Te
Tem 5%
m Tc
5%
Tem 6%
r to
c fe Ef
r to
m Tc
Tcm 11%
Effector 8%
Effector 5%
Effector 6%
c fe Ef
Tcm 11%
or ct fe Ef 5%
Effector 5%
E
CD8⁺ T cells
Naive 77%
0
20
40
60
80
Age Age
M
r=0.42 (CI: 0.26–0.56)
r=0.56 (CI: 0.25-0.57)
p=2.7e-06
p=2.4e-09
r=0.42 (CI: 0.26−0.56) ● p=2.7e−06
0
●
●
25
●
p=0.11
● ●
●
●●●
●
●
●
● ● ● ● ●
●●
●
●
●
●●
●
● ●
●
●
●
●
●
●
●
●
●
●● ●
● ●
●
Effector
● ●
●
●
●
●
● ●
●●
●
●
●
● ●
●
●
●
40
●
●
●
● ●
●
60
80
0
0
20
40
r=0.24 (CI: 0.21-0.54)
p=2e-05
p=0.018
r=0.24 (CI: 0.21−0.54) p=0.018
60
Age Age
OAge vs. CD8 CM
r=0.39 (CI: 0.22–0.53) r=0.39 (CI: 0.22−0.53) ● p=2e−05
20
10
●
P
r=0.008 (CI: 0.022-0.4)
p=0.019
p=0.94
r=0.008 (CI: 0.022−0.4) p=0.94
40
40 ●
60
●
20
0
20
●
● ● ●
●
●
●
● ●
● ●
● ●●
●● ●●
●●
●● ● ● ● ● ●
● ● ●
●
●
●
●
●● ● ●● ● ● ●
● ● ● ●
●
10
●
●
0 40
Age Age
60
●● ●●
80
0
● ●
● ●
● ● ●
●
● ●
●
● ●
●
● ● ●● ● ● ●●● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ●● ● ● ● ● ●●●● ● ● ●● ● ● ● ● ● ● ● ●●● ●● ● ● ● ● ● ● ●● ●● ● ● ●● ● ● ● ● ●● ●● ● ● ● ● ● ● ●●● ● ●● ●● ● ●● ● ● ● ● ●● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●●
●
●
● ●
20
●
●
● ●● ●●● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ●● ● ●● ●●●● ●● ● ● ● ●● ● ●● ● ● ● ●● ●● ●● ● ●●● ●●● ● ● ● ● ● ● ● ●●●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ●● ● ●●● ● ● ● ● ● ●●● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●
● ● ● ●
●
●
●●
● ●
● ● ●
●
● ●
●
● ●
● ●
● ●● ● ●● ●
20
● ●● ●
● ●●● ● ● ●● ● ●● ● ● ● ● ● ●
40
60
80
Age Age
Age vs. CD8 EM r=0.16 (CI: 0.056−0.43) r=0.16 (CI:p=0.11 0.056-0.43)
r=0.25 (CI: 0.07−0.42) r=0.25 (CI: 0.07–0.42) ● p=0.0071
p=0.11
● p=0.0071 ●
●
●
●
●
20
●
●
● ● ● ● ●
● ●
● ●
● ●
● ● ●● ●
● ● ●
●
● ●●
●
●
20
●
● ● ● ● ● ● ● ●● ● ● ● ● ●
0 40
Age Age
60
80
● ● ● ● ● ● ●
●
●● ●
40
● ●
●
●
● ● ●
● ●
● ●●
●
● ● ●
● ●
●
●
● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ●●●●● ● ● ● ● ●● ●● ● ●●● ● ● ●● ● ● ●● ● ●● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●●● ●●● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ●● ● ●● ● ●● ● ●● ●● ● ●● ● ●● ● ● ● ●
0
20
● ●● ● ● ●
● ● ● ● ● ● ●
●
●● ●
● ● ●
● ●
● ● ●
●● ● ●
● ●
●●
●●
●●
●
20
● ●
●
●
●●
●
● ● ●●
CD8 EM
●
●
Tem
●
● ● ● ● ● ● ● ●● ●● ●● ●● ● ●● ● ● ● ● ● ● ● ●● ● ● ●●●● ●● ● ● ● ●● ● ●● ● ● ●● ●● ●● ● ●● ● ● ●●● ●●● ● ● ●● ● ●● ● ●● ●● ●●● ● ●● ● ●● ● ● ● ●● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ●●● ● ● ● ●● ● ●● ● ● ●● ● ● ● ● ●● ● ● ●● ●●
0
●
●
●
●
30
● ●
Tcm CD8 CM
●● ●
● ●
●
●
●
Tem CD4 EM
Tcm CD4 CM
40
● ●
●
●
●
30
● ●
●● ●
●
● ●
●
● ●
●
●
60
●
●
0
80
r=0.22 (CI: 0.036–0.39) r=0.22 (CI: 0.036−0.39) ● p=0.019
● ●
●
●
● ●● ● ● ● ● ● ● ● ●
● ● ●
●
20
30
●
● ● ● ●●● ● ● ● ● ● ●
p=0.32
40 ●
●
Age vs. CD4 EM
r=0.56 (CI: 0.25−0.57) p=2.4e−09
r=0.15 (CI: −0.033−0.33) (CI: −0.047−0.34) r=0.15 (CI: -0.033–0.33) r=−0.1 r=-0.1 (CI:p=0.32 -0.047-0.34)● p=0.11
p=0.85
CD8 Effector
Naive ●
●
● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ●● ●●● ● ● ● ● ● ● ●● ● ●
Age Age
N
Age vs. CD4 CM
CD8 Naïve
Effector
Naive
CD4 Naïve
CD4 Effector
0
● ●
%
● ●
●
18
●
●● ● ● ● ● ● ● ●● ● ●● ● ● ●● ● ●● ● ● ●● ●● ● ●●●● ● ● ● ●● ●● ●● ● ●● ●●● ●● ●●● ● ● ●● ● ●● ● ● ● ●●● ● ● ●● ●● ● ● ● ●●● ●● ●● ● ●●● ● ● ●● ●● ● ● ● ● ●●● ●●●● ●● ● ● ● ● ● ● ● ●●● ●● ● ●● ●● ●● ●● ● ● ● ● ● ●
or ct fe Ef 15%
●
25
●
50
m Tc
●
● ●
●
●
●
● ●
10
●
● ● ●
%
●
●
●
13
● ●
●
●
●
Tcm 18%
%
● ●●
●
●
20
●
●
m Te or ct fe Ef 10%
●
● ●●
●
20
m Tc
●
●
●
●
%
●
●●
14
●
75
●
m Te
●
% 11
●
●
r to
●
8% m Tc 5%
● ●
●
●
c fe Ef
● ● ●● ●
●
●
● ●
m Te
●
●
Tcm 20%
Age vs. CD8 Effector
r=−0.24 (CI: −0.4−−0.056) (CI: −0.42−−0.042) r=0.019 (CI: -0.42--0.042) r=-0.24 (CI: -0.4–-0.056)r=0.019 p=0.011 ● p=0.85 ● ● ● ●● ● ● ●● ●●● ●● ● ● ● ● ● ● ●● ● ● ●● ●● ● ●●● ●● ●● ●●● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ●● ● ●● ● ●●● ● ● ● ● ● ●●● ●● ●●● ● ● ● ●●● ● ●● ● ● ●● ● ●● ● ●● ● ●● ● ● ● ●● ● ●● ● ● ● ● ● ●● ● ● ●
Effector 15%
Tem 13%
Tem 14%
L
p=0.011 ●
p=0.0021
●
●
● ●
%
●●
●
●
Tem 5%
15
● ●
●
●●
● ● ● ●
●
●
●● ● ● ● ● ●
100
r=−0.22 (CI: −0.39−−0.039) (CI: −0.4−−0.025) r=p=0.018 -0.22 (CI: -0.39–-0.039) r=−0.31 r=-0.31 (CI: -0.4--0.025) ● p=0.0021
p=0.018
m Tc
%
50
●
%
21
%
%
%
75
●● ●●● ● ● ● ● ●● ● ●● ●● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ●● ● ● ● ●● ● ● ●● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ●●● ● ●● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●●● ● ●● ● ● ● ● ●●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●
30
p=1.6e-05
Tcm 15%
Tcm 8%
K Age vs. CD8 Naïve
Age vs. CD4 Effector
r=−0.41 (CI: −0.55−−0.24) (CI: −0.56−−0.23) r=-0.41 (CI: -0.55–-0.24) r=−0.42 r=-0.42 (CI: -0.56--0.23) ●● p=7.1e−06 p=1.6e−05 ●● ●● ● ● ● p=7.1e-06 ● ● ●● ●
Tem 12%
12
m Tc
12
26
10
J
Age vs. CD4 Naïve
or ct fe Ef 13%
Tcm 21%
Effector 10%
Effector 11%
Effector 13%
m Te
m Te
m Tc
%
9%
15
m Te
%
%
20
16
I
Effector 7%
or ct fe Ef 4%
Effector 7%
Tem 12%
Tcm 26%
m Te
Tem 10%
Tem 9%
5%
m Tc
7%
m Tc
Tcm 20%
Tcm 15%
r to
c fe Ef
r to
c fe Ef
m Te
Tem 16%
Effector 4%
Effector 5%
Effector 7%
● ● ●
10 ●●
●
●
●
● ●
0 40
Age Age
60
80
● ●
●
● ● ●
●
●● ●
●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ●● ● ● ● ●●● ● ●● ●●● ● ●● ● ●● ● ●● ● ● ● ●● ●●● ● ● ● ●● ●●● ● ● ● ● ●● ● ● ● ●● ● ● ●● ● ●● ● ● ● ● ●● ● ● ● ●● ●●● ● ● ● ● ● ● ● ● ● ●●●● ●●● ● ● ● ●● ● ● ● ● ●
0
●
●
20
● ●
● ● ● ●
●
●
● ●●●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ●
●
●●
●
●
●
40
Age Age
60
●
80
Figure E3 Non-Activated T-cells 10 5
0.0128
0
10 5
10 4
10 3
10 3
10 2
10 2
0
0
IFNγ
10 4
99.1 0
10
5
0.924 10 2
10 3
10 4
0.0257
5
10 4
10 3
10 3
0
0
IL-13
10 4
0.977
10
5
10 2
10 3
10 4
0.0129
10 5
10 4
10 3
10 3
0
0
CLA
0.926 10 2
10 3
10 4
3.16
10 5
10 2
10 3
10 4
10.8
10 5 0.267
3.91
0
10 4
0
78.4
85
10 5
6.43e-3
99.1
0.322
0
6.43e-3
99
18.1
10 5
10
0
IL-22
Activated T-cells
10 2
10 3
10 4
10 5
3.47
0.187
93.3
3.02
0
10 2
10 3
10 4
10 5
Figure E4
A
CD4 IFNg+CLA+
100 100
Frequency (%)
CD4 T cells IFN*** ***
**
75 75
50 50
**
** **
*** ***
+
*** ***
**
B
CLA
CD4 IFNg+CLA
100
+*
IFN-
+
50 50
C
CLA *** ***
*** *** + + ** ** *** *** ** *** *** *** *** **
100
25
IFN-
+
*** *** + +
** *
* **
100 100
D
CLA
CD8 IFNg+CLA+
100
50
N
AD N
Infants
E
AD N
AD
N
AD
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
0
CD4 IL13+CLA+
40
* ** *** ***
40
30 30
Children Adolescents
Adults
*
**
Infants
F
IL-13 CLA
CD4 IL13+CLA
20 20
+ + *** ***
AD
Children Adolescents
N
AD
0
**
15
AD N
AD N
Infants
AD
N
CD8 IL13+CLA+
Children Adolescents
*** ***
60
5
10
0
6 *** *** *** ***
N
AD N
AD N
AD
N
AD
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
0
Infants
I
IFN-
Children Adolescents
*
50 40
40 30
30
+*
*** *
AD N
Infants
J
IFN-
*
*** **
N
AD
0
*** *
** *** ***
100
100
*** ***
** *
*** *
** **
+*
** **
AD N
Infants
AD N
AD
K IFN-
Children Adolescents
N
AD
4
0
AD
Adults
IL-13 CLA * * *
+ *
+
N
AD N
AD N
Infants
L
IFN-
Children Adolescents
*** *
*
N
AD
Adults
CLA /IL-13 CLA
CD8 IFNg+CLA /IL13+CLA
*
**
75
AD
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
Adults
CLA /IL-13 CLA
CD8 IFNg+CLA+/IL13+CLA+
75
1000
1000
**
50
**
N
0
N
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
Adults
CLA /IL-13 CLA
CD4 IFNg+CLA /IL13+CLA
150
*** *
AD
Children Adolescents
150
*
20
50
** +
500
500
20
50
25
50
+*
10
0
Adults
CLA /IL-13 CLA
CD4 IFNg+CLA+/IL13+CLA+
AD N
AD
2
0
N
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
AD N
Children Adolescents
*** *** *** * *** * *** *** *** *** + * + + *
2
+
20
0
0
**
AD N
CD8 IL13+CLA
4
20
** **
** *
5
10
N
6
** **
10
20
** ***
* **
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
Infants
H
40
** **
0
Adults
40
15
AD
IL-13 CLA
60
+
*** ***
0
N
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
G
*
**
** ***
50
Adults
IL-13 CLA
10
20
AD N
*** ***
50
0
N AD N
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
*** ***
CLA ** **
+
100
50
0
0
IFN-
150
+ +
+
CD8 IFNg+CLA
150
25
0
Frequency (%)
CD8 T cells
0
0
25
*** ***
+*
**
AD N Infants.AD
Infants
AD N
AD N
Children.Normal Children.AD Adolescents.NormalAdolescents.AD Adults.Normal
AD Adults.AD
Children Adolescents Adults
0 0
0
N
Infants.Normal
N
Infants.Normal
AD N Infants.AD
Infants
AD N
AD N
Children.Normal Children.AD Adolescents.NormalAdolescents.AD Adults.Normal
Children Adolescents
AD
Adults.AD
Adults
0
0
N
Infants.Normal
AD N Infants.AD
Infants
AD N
AD
N
Children.Normal Children.AD Adolescents.NormalAdolescents.AD Adults.Normal
Children Adolescents
AD
Adults.AD
Adults
N
Infants.Normal
AD N Infants.AD
Infants
AD N
AD N
Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal
AD
Adults.AD
Children Adolescents Adults
Figure E5
A
CD4 T cells
CD4 IL9+CLA+
80
Frequency (%)
80
** **
60 60
*** ** ** *
** *
40
B
IL-9 CLA
*** ***
CD4 IL9+CLA
*** ***
7.5
N
AD N
AD N
Frequency (%)
AD
CD4 IL17+CLA+
Children Adolescents
N
100
0
AD
**
N
AD N
Infants
CD4 IL17+CLA
7.5
20
**
5
5.0
+
10
AD N
AD
N
Children Adolescents
AD
**
*** **
*** *** +
AD N
Infants
AD N
AD
CD4 IL22+CLA+
Children Adolescents
N
AD
*** ***
+ + +
40 40
+* 20
AD N
AD
N
AD
CD4 IL22+CLA
*** *** *** ** *** ***
15
** **
10
10
*** ***
*
** ***
*** ***
AD N
AD N
AD
N
AD
Infants
Children Adolescents
Adults
N
0
AD N Infants.AD
Infants
CD8 IL22+CLA+
AD N
0
AD
*** *
+
AD
N
AD
Infants
Children Adolescents
Adults
0
N
AD
**
N
Infants.Normal
Adults.AD
Adults
*** **
**
**
AD N
Infants.AD
CD8 IL22+CLA
*** *** *** *** ***
6 6
** ***
4 4
+
AD Adults.AD
Adults
IL-22 CLA
8
**
AD N
Children Adolescents
8
+*
**
AD N
Children.Normal Children.AD Adolescents.NormalAdolescents.AD Adults.Normal
Infants
L
*** *
*** ***
* **
2 2
0
AD N
*** *
0.5
*** ***
30
AD N
AD
Adults
0.5
IL-22 CLA
30
N
*
1.0
Children Adolescents
**
60
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
*** *
1
*
60
+
*** ***
1.5
Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal
90
*** ***
N
IL-17 CLA
CD8 IL17+CLA
1.5
+
90
0
N
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
**
AD
0.0
120
*
AD N
Children Adolescents
2.0
+
**
AD N
Infants
2
+
Infants.Normal
120
5
0
0
K
IL-22 CLA
N
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
**
+
+ +
Adults
5
0
AD
Adults
H
5.0
Children Adolescents
0
IL-17 CLA
5
AD N
Infants
20
*** **
20
N
20
*** ***
AD N
0.0
15
+ +
CD8 IL17+CLA+
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
J
+*
AD N
2.5
0
+
3
2.5
Adults
IL-22 CLA
60
6
3
Children Adolescents
7.5
0.0
N
AD N
7.5
2.5
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
N
10
2.5
0
6
+
10.0
** **
**
9
0
Infants
G
*
9
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
Adults
IL-17 CLA ** *
7.5
20
10
*** ***
+ +
+
IL-9 CLA
CD8 IL9+CLA
+
0
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
F
+*
+
50 0
** *
** *
50
Adults
IL-17 CLA
30
Frequency (%)
100
0.0
30
60
*** *
2.5
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
*** ***
150
** *
D
IL-9 CLA
CD8 IL9+CLA+
150
2.5
Infants
I
**
+*
0
0
** + + +*
5
20
E
+
5.0
20 0
C
IL-9 CLA +
7.5
*** **
40
CD8 T cells
0 0
N
AD N
AD N
AD
N
AD
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
Infants
Children Adolescents
Adults
N
AD N
AD N
AD
N
AD
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
Infants
Children Adolescents
Adults
Figure E6
●●
●
● ● ●● ●
● ●
● ●
●
● ● ●● ●
●
0
●● ● ●●●
●
●
● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●●● ● ● ● ● ● ●●●●● ● ● ●● ●● ●● ● ● ● ● ● ● ●● ●● ●● ● ● ● ● ● ●● ●● ● ● ● ● ●● ● ● ● ● ● ●●● ● ● ●● ● ●● ● ● ● ●●● ● ● ●● ● ●● ● ●●●● ●● ● ● ●● ● ● ●
20
●
● ●● ● ●
●
●
● ●●
● ● ●
●
● ●
● ●
● ● ● ●
●
●
● ● ●
●
●
●● ●
●● ● ● ●
●
● ● ●
●
● ● ● ●● ● ●●
●
● ● ●
0
20
40
60
80
r=−0.038 (CI: 0.045−0.43) ● p=0.71
p=0.71
p=0.0093 ●
●
● ●
●●
60
●
●
●
● ● ●
● ●
40
●
● ● ● ● ●
● ● ●
●
● ●
● ●
0
●● ● ● ● ● ●● ●● ● ● ●● ●● ● ● ●● ● ●● ●● ● ● ●● ● ● ●● ●● ●● ●● ● ● ● ● ● ● ● ● ● ●● ● ●●●● ● ● ● ● ● ● ●●● ● ●● ●● ● ●●● ● ● ●● ● ● ● ●● ● ●● ● ●● ● ● ●● ●● ● ● ●● ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ●
0
80
● ●
● ● ● ● ●
●
20
●
●
●
● ●
●
●●
●
● ●
● ●
●
●
●
●
●
● ● ●●●
●●
●
●
●
● ● ●
● ● ●
●
●
● ●
●
●
●
●
●
●
●
●
●
20
40
Age Age
60
100
● ●
15
50
●
● ●
25
0
● ●
●
● ●
●
● ● ●●● ●● ●● ● ● ● ●● ● ● ● ● ● ●
●
●
● ● ●● ●● ● ● ●●● ●●
● ●
● ●● ● ●● ● ● ● ●●
40
●
● ●
60
40
● ● ●
●
●
●
●●
20
●
●
● ●● ● ● ●● ●
● ● ● ●● ● ● ● ● ● ●●● ● ● ●● ● ●●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●
● ● ●
0
60
● ● ●
●
● ●
●
●
●
●
●
●
● ●
● ●
●
●
●
●
0
●
●
●
●
●
●
● ●●● ●● ● ● ● ● ● ● ● ●
●● ● ● ● ● ●
●
●
40
6 ●
15
●
20
●
●
● ●
● ●●
3
● ● ●
10
r=0.28 (CI: 0.056−0.47) r=0.28 (CI: 0.056–0.47) p=0.015 p=0.015
● ●
●
● ● ● ●● ● ●
0
● ● ●● ● ● ● ● ● ● ● ● ● ●●
0
●●
● ● ● ● ●●
●
● ● ●● ●
●
●
5
●● ●
●
● ● ●
●
●
● ●
●
●
●
●
0
Duration.of.disease
● ● ●
●
●
10
●
●
●
●
●
●
●
●
15
Duration of Disease
●● ● ● ● ● ● ● ●● ● ●● ● ● ●● ● ● ●● ● ●●● ●●
0
●● ● ● ● ● ●● ● ● ●
●
● ●
●
●
5
●
● ●
●
● ● ●●
● ●
● ●
● ●
● ● ●
10
●
●
● ● ●●
●
●
● ● ●
● ●
●
0
15
5
10
15
Duration.of.disease
L
Duration.of.disease vs. CD4 IFNg+CLA−
●
40
r=0.58 (CI: 0.41−0.71) r=0.58 (CI: 0.4–0.7) p=2.7e−08 ● p=0.000002
●
●
● ●
●
● ● ● ●
●
0 5
K Duration.of.disease vs. CD4 IL22+CLA+ ●
●
●
●
● ●
●
Duration of Disease
●
2
●
●
Duration.of.disease
●
●
●
● ●
●●
●
●
0
●
●
●
● ●
●
● ● ●
10
Duration.of.disease
10
● ●
● ●
Duration of Disease
● ●
● ●●
●●
0 15
●
● ● ● ●●
0
●
●
●
● ● ●● ● ● ● ● ● ●●
● ●
●
●
●
● ●
●
20
●
●
● ●
● ● ●
●
●
● ● ● ●
●
●
● ● ●
●
● ● ●
●
● ●
●● ●●●
● ●●
●
●
●
●
●
● ●
●
● ●
●
CD4⁺IFNγ⁺CLA ⁻ CD4 IFNg+CLA−
●
80
●
● ●
●
20
10
CD4⁺IL22⁺CLA⁺ CD4 IL22+CLA+
CD4⁺IL9⁺ CD4 IL9+
CD4⁺IL13⁺ CD4 IL13+
●
60
●
●
4 ●
●
Duration.of.disease vs. Pruritus.ADQ
● ● ●●● ● ●
●
p=0.0018 ●
●
Age Age
●
9
●
● ●
● ●● ● ● ● ●
●
●
● ●
● ●
● ●
●
●
20
30
●
●
●
● ●
20
● ● ● ●● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ●● ●●● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ●● ●●●●● ●●● ● ●● ●●●● ●● ●
●
●
●
r=0.35 (CI: 0.14−0.53) r=0.35 (CI: 0.14–0.53) p=0.0018
p=0.058
●
●
● ● ●
●
J Duration.of.disease vs. CD4 IL9+
r=0.22 (CI: −0.0077−0.42) r=0.22 (CI:● -0.0077–0.42) p=0.058
● ●●
●
●●
Duration of Disease
Duration.of.disease vs. CD4 IL13+
●
●●
●
●
●
●
Duration.of.disease
Duration of Disease
●
●
● ●●
●
● ●
●
●
●
●
● ●
r=0.22 (CI: −0.0064−0.43) r=0.22 (CI:●-0.0064–0.43) p=0.057 ● p=0.057 ● ●
●
● ●● ● ● ●
40
●
●
5
Duration.of.disease
● ● ●
H ●
●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●
●
0
●
●
●
0
15
40
●
●
●
●
●
80
●
●
80
●
10
●
●
● ● ● ● ●
●
●
●● ● ●
5
● ●
●
● ● ● ●● ● ●● ●
●●
●
● ●●● ● ●●● ●● ●● ●
0
●● ●
20
●
● ●
● ●
● ●● ●● ● ● ●● ● ● ● ● ●● ● ● ●●● ●● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ●● ●●●● ● ●● ●●● ● ●● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ●●● ●●● ●● ● ●● ●●● ●● ● ●●● ●● ●● ●
●
●
5
●
●
r=0.15 (CI: −0.072−0.36) r=0.15 (CI: -0.072-0.36) p=0.18 p=0.18 ●
60
EASI EASI
●
●
●
● ● ● ● ● ● ●
G Duration.of.disease vs. SCORAD
SCORAD SCORAD
● ●● ● ●
●
●
● ●
●
●
10
●
●
●
p=0.00065
● ●●
●
Age Age
●
0
●
12
●
Age vs. CD8 EM HLADR+/CLA+ r=0.41 (CI: 0.23−0.55) r=0.026 (CI: 0.22−0.56) r=0.41 (CI: 0.23–0.55) r=0.026 (CI: 0.22-0.56) ● p=1.6e−05 p=0.8 ● ● ● ● ● p=1.6e-05 p=0.8
Age Age
r=0.38 (CI: 0.17−0.55) r=0.38 p=0.00065 (CI: 0.17–0.55)
p=1.1e-56
I
75
80
F Duration.of.disease vs. EASI
r=0.98 (CI: 0.97−0.99) r=0.98 (CI: 0.97–0.99) p=1.1e−56
●
r=−0.059 (CI: 0.24−0.58) p=0.57
Age Age
EDuration.of.disease vs. Age
0
r=0.42 (CI: 0.25–0.57) r=-0.059 (CI: 0.24-0.58) p=7.3e-06 p=0.57 r=0.42 (CI: 0.25−0.57) ● p=7.3e−06
HLA-DR⁺CLA⁺CD8⁺ Tem cells CD8 EM HLADR+/CLA+
●
●●
40
p=0.16
● p=0.0056
D
C Age vs. CD8 CM HLDR+/CLA+
r=0.24 (CI: 0.062–0.41) r=-0.038 (CI: 0.045–0.43) r=0.24 (CI: 0.062−0.41) p=0.0093
Pruritus ADQ Pruritus.ADQ
60
r=−0.14 (CI: 0.064−0.44) ● p=0.16
CD8 CM HLDR+/CLA+ HLA-DR⁺CLA⁺CD8⁺ Tcm cells
B Age vs. CD4 EM ICOS+/CLA+
r=0.26 (CI: 0.078–0.43) r=-0.14 (CI: 0.064–0.44) r=0.26 (CI: 0.078−0.43) p=0.0056
ICOS⁺CLA⁺CD4⁺ Tem cells CD4 EM ICOS+/CLA+
ICOS⁺CLA⁺CD4⁺ Tcm cells CD4 CM ICOS+/CLA+
A Age vs. CD4 CM ICOS+/CLA+
●
30 ● ● ●● ●
●
●
●
●
0
5
10
15
Duration.of.disease Duration of Disease
●
●
●
● ●
●
● ●
●
●
●
●
● ●
● ●
●
●● ● ● ● ●
● ●
●
●
●
● ●
●
● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ●● ●
0
●
●
●
●
10
●
● ●
●
20
●
● ● ●
●
5
● ●
10
15
Duration.of.disease Duration of Disease
CD4⁺ T cells
A Age vs. CD4 IFNg+CLA+ r=0.44 (CI: 0.28–0.57) r=0.44 (CI: 0.28−0.57) ● p=5.5e−07 ●
p=0.013
10
0
●
●
● ●
●
p=4.7e-09
● p=0.1
●
●
●
●
● ●
●
● ● ● ● ● ● ● ● ● ● ●●●● ● ● ● ● ● ● ●● ● ●● ●● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●●● ●● ● ●● ● ● ● ● ● ● ●● ●● ● ●● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ●●● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ●●● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ●● ● ● ● ● ● ●● ● ●●● ● ● ● ● ● ● ● ●●● ● ● ●● ● ● ●● ● ●
0
20
40
●
●
●
40
●
● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●●● ●● ● ● ● ●●● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●●●●● ●●● ● ● ● ●● ● ●● ●● ● ●●● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ●● ●●● ● ● ● ● ● ●● ● ● ●●● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ●● ● ● ● ●● ●● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ●● ● ●
20
80
0
20
40
Age Age
40
20
●
r=−0.14 (CI: −0.32−0.036) ● p=0.12
p=0.12
p=0.41
75 ●
● ●
●
● ●
●● ● ●
● ● ● ●● ●● ● ● ● ● ● ● ●●● ● ● ● ● ● ●● ● ● ● ●● ● ●● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ●●
●
●
● ●
●
●
● ●
● ●● ● ●
● ●
●
●
●
●● ●●●
● ●● ●● ● ●● ●● ●● ● ●●●●● ● ● ●●● ● ●● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ●● ● ●● ● ● ● ● ● ● ●●●● ●● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ●
● ● ● ●
● ● ● ● ●
●
●
● ●
●
50
25
● ● ● ●
0
●
40
60
80
● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ●● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ●● ●●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ●● ●● ●● ● ● ● ● ● ●● ●● ●● ● ● ● ● ● ●● ● ●
0
20
40
●
●
60
80
Age Age
H
r=-0.4 (CI: -0.54–-0.24) r=-0.21 (CI: -0.56–-0.23)
Age vs. CD8 IL9+CLA−
(CI: ● -0.071–0.29) p=0.23 8 r=-0.11 r=0.11 (CI: −0.071−0.29)
r=−0.21 (CI: −0.56−−0.23) p=0.036
p=5e-06 ●
●
●
●
●
●
●
●
●●
● ● ●●
●
●
●
●●
20
● ● ●
● ●
r=−0.4 (CI: −0.54−−0.24) ● p=5e−06
4
p=8.2e-05
●
●
G Age vs. CD8 IL9+CLA+
p=0.35
r=0.38 (CI: 0.36−0.65) r=0.38 (CI: ● 0.36-0.65) ● p=8.2e−05
●
●
Age Age
r=−0.094 (CI: −0.33−0.052) p=0.35
●
●
●
● ● ● ●
●●
0
80
r=-0.14 (CI: -0.32–0.036) r=0.-0.094 (CI: -0.33–0.052)
r=−0.082 (CI: −0.28−0.11) p=0.41
p=0.34
60
●
● ● ● ● ●
0
F Age vs. CD4 IL9+CLA−
r=-0.088 (CI: -0.26–0.093) r=-0.082 (CI: -0.28–0.11) 40
● ● ●● ●
●
p=1.3e-09
●
● ●
Age Age
EAge vs. CD4 IL9+CLA+ r=−0.088 (CI: −0.26−0.093) ● p=0.34
●
●
0
60
●● ● ●
● ● ●
Age vs. CD8 IFNg+CLA− r=0.52 (CI: 0.37−0.64) r=0.52 (CI: 0.37–0.64) ● p=1.3e−09
r=0.18 (CI: −0.047−0.34) p=0.076 ●
p=0.076
● ●
●
CD8 IFNg+CLA+ IFN-γ⁺CLA⁺
30
p=1.4e-15
●
IFN-γ⁺CLA⁻ CD4 IFNg+CLA−
IFN-γ⁺CLA⁺ CD4 IFNg+CLA+
●
r=0.15 (CI: -0.031–0.32) r=0.18 (CI: -0.047-0.34)
60
● ●
●
r=0.54 (CI: 0.52-0.75)
r=0.15 (CI: −0.031−0.32) ● p=0.1
r=0.54 (CI: 0.52−0.75) ● p=4.7e−09
60
●
D
CAge vs. CD8 IFNg+CLA+
r=0.65 (CI: 0.53–0.74) r=0.65 (CI: 0.53−0.74) p=1.4e−15
r=0.25 (CI: 0.26–0.58)
40
20
B Age vs. CD4 IFNg+CLA−
r=0.25 (CI: 0.26−0.58) p=0.013 ●
p=5.5e-07
CD8⁺ T cells
IFN-γ⁺CLA⁻ CD8 IFNg+CLA−
Figure E7
p=0.23
p=0.036
r=0.21 (CI: −0.088−0.3) r=0.21 (CI:p=0.033 -0.088–0.3)
p=0.033
60
10
0
0
20
40
2
● ●
● ● ●●
●
0
60
●
● ●
1
●
●
80
●
●
● ● ●
● ● ● ● ● ● ● ●● ●●●● ● ● ●●● ● ● ●● ● ●● ● ● ● ● ● ● ●●● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ●● ●●● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●●●●●●● ● ●●● ● ● ● ●●● ● ● ●● ● ●●● ●● ● ● ●●● ● ●● ● ●● ● ●● ●●●●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●●●●● ● ●● ●● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ●●● ● ● ● ● ●● ● ● ●
●
0
20
40
Age
Age vs. CD4 IL17+CLA+
r=0.2 (CI: 0.019−0.36) 15 r=0.2 ● (CI: 0.019–0.36) p=0.031
p=0.031
p=0.37
p=7.6e-08
●●
5
0
IL-17⁺CLA⁻ CD4 IL17+CLA−
IL-17⁺CLA⁺ CD4 IL17+CLA+
●
●
● ● ●
●
● ●
● ●
●
● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ●● ● ● ● ●● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ●● ● ●● ● ●●● ● ● ●● ● ●● ● ● ● ● ● ● ●●●● ● ●● ●● ●●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ●●● ● ● ●● ● ●● ● ● ●● ● ● ●● ● ● ●● ● ● ●●● ● ● ● ● ● ●● ● ●● ● ●●● ● ●● ● ●● ● ● ● ● ● ● ●● ● ●●●● ● ●●●● ● ● ●● ● ●● ● ● ●● ● ● ● ●● ● ● ● ●● ●● ●
0
20
40
Age
Age
60
0
80
20
40
60
r=0.27 (CI: 0.3−0.61) r=0.27 (CI: 0.3-0.61) p=0.0063
p=0.0063
●
2
● ● ● ● ● ● ●
● ● ●
1
●
0
●
●
●
●
●
● ● ●
● ● ●
20
● ●
●● ●● ●
●
●
●
●
● ●
●
● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ●●● ●● ● ● ● ●● ● ● ●● ● ● ●● ● ● ● ● ●● ● ●● ●● ●●● ●● ● ●● ●● ● ● ● ● ● ● ● ●●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●
0
●
●
●●
●
60
0
80
20
40
●
● ● ● ●
●
60
80
Age
Age
L
Age vs. CD8 IL17+CLA−
p=0.27
60
r=0.46 (CI: 0.31−0.59) r=0.42 (CI: 0.29−0.6) r=0.46 (CI: 0.31–0.59) r=0.42 (CI: 0.29-0.6) p=1.1e−07 ● p=9.6e−06 p=1.1e-07 p=9.6e-06
●
3 ●
● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ●● ●●● ●● ●● ● ● ● ● ● ●●●● ● ● ●●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●●●● ● ● ●●● ● ●● ● ● ● ●● ● ● ● ●● ● ●● ● ●● ● ● ●●●●● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ●● ●
30
●
● ● ● ● ●
● ● ● ●
●
r=0.11 (CI: −0.057−0.33) p=0.27
p=0.12
●
●
● ●
2
●
● ●
●
r=0.14 (CI: -0.038–0.31) r=0.11 (CI: -0.057-0.33) r=0.14 (CI: −0.038−0.31) ● p=0.12
●
●
80
●
●
Age
K Age vs. CD8 IL17+CLA+
r=0.47 (CI: 0.31−0.6) r=0.47 (CI: 0.31–0.6) p=7.6e−08 ●
●
4
0
J Age vs. CD4 IL17+CLA−
r=-0.091 (CI: 0.00018–0.38)
6
●
Age
r=−0.091 (CI: 0.00018−0.38) p=0.37
●
●
20
● ● ● ● ● ● ●●● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ●● ● ●● ● ● ●● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ●● ●●● ●● ●● ● ●● ● ● ● ● ●● ● ● ●●● ●● ●●● ● ●● ● ● ● ● ● ●●● ● ● ●●●● ●● ● ● ●● ●●●●● ● ● ● ●●● ● ● ● ●● ●● ● ● ●●● ●●● ● ● ● ● ● ●●● ● ● ●●● ● ● ●● ● ● ●●● ● ● ●● ● ● ● ● ● ● ●● ●●●●●●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●
Age
4
10
●
Age
Age
I
●
● ● ●
0 ●
●
● ●
40
●
●
●
IL-9⁺CLA⁻
● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ●●● ● ●● ●● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ●● ● ● ●●● ● ● ● ● ● ● ●● ● ●● ● ●● ● ● ● ● ●● ●● ● ● ●● ●● ● ● ●●●● ● ● ● ● ●●● ● ● ●●● ● ●●●● ●● ● ●● ● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●●●● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ●●
● ●● ●
●
● ●
CD8 IL9+CLA−
●●
●
IL-17⁺CLA⁺ CD8 IL17+CLA+
20
●
●
3
● ● ●●
20
●
10
● ● ● ●● ●●● ● ●
●
●●
●
40
Age
Age
●
●
● ●
● ● ● ●● ●
●● ● ● ● ●
● ●
● ●
●
60
●●
●
●
80
IL-17⁺CLA⁻ CD8 IL17+CLA−
●
● ●
IL-9⁺CLA⁺
●
●
CD8 IL9+CLA+
IL-9⁺CLA⁻
●
CD4 IL9+CLA−
IL-9⁺CLA⁺
CD4 IL9+CLA+
●
30
●
●
●
0
●
●
● ●
20
20
●
● ● ● ● ● ● ● ● ●● ● ●● ●● ● ● ●● ●● ● ●● ● ● ● ●●●● ● ● ●● ● ●●● ● ●●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ●● ●●● ●● ● ●● ●● ●● ●● ● ● ● ● ● ● ● ●●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ●●● ● ● ● ●●●● ● ● ●●●● ● ●● ● ● ●●● ● ●●
0
40
40
Age
Age
60
●
●
0 80
● ● ● ●● ● ● ● ● ●● ●●●●● ●● ● ● ● ● ● ● ●●● ● ● ● ●●● ● ●● ● ●●● ●●●● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ●● ●●●●● ●●●●● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ●●● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ●
0
20
40
Age
Age
60
●
●
80
Figure E8 A SCORAD vs. CD4 IL13+CLA+
B SCORAD vs. CD8 IL9+CLA−
●
4
●
●
p=0.0034
●
●
●
●●
● ●
● ●
●
●
●
●
●
●
● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ● ● ● ●● ●● ●● ● ●● ● ●● ● ● ●● ● ● ● ● ●
● ● ● ● ● ● ● ● ●
●
●
●
●
●
●●
●
●
●
●
●
40
60
● ●
●
●
2 ●
●
● ● ● ●
● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ●● ● ● ●● ● ● ● ● ●● ●●● ● ● ● ●● ● ● ●● ● ●●● ● ● ● ● ● ●●● ● ●● ● ● ● ● ●● ● ● ●
● ● ● ●●
●
●
●
●
0
r=0.24 (CI: 0.021−0.45) r=0.24 (CI:● 0.021–0.45) p=0.033
●
p=0.033
●
4
80
20
100
40
60
Pruritus ADQ Pruritus.ADQ
● ● ●
5.0 ● ●
● ●
●
●
●
●
● ●
● ●
20
●
● ● ●● ● ● ● ● ● ●● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ●●● ● ● ● ●● ● ●
10
●
● ●●
10.0
● ● ●
●
●
● ●●
● ● ●
●
●
● ●●
●
●
●
●
●
●
●● ●
●
● ● ● ● ●● ●● ● ● ●
●
25
●
●
●● ●
●
●
●
20
40
60
H Pruritus.ADQ vs. Eosinophils r=0.39 (CI: 0.15−0.58) r=0.39 p=2.7e−07 (CI: 0.15–0.58) p=2.7e-07
● ● ●
5.0 ● ●
●
2.5
●
0.0 40
60
●
●
0
●
● ●
●
●
●
●
● ● ● ● ● ● ● ● ● ● ●
10
● ●
●
15
● ● ● ● ● ●
●
20
●
●
●
5
● ●
●
●
●
●
●
●
●
● ● ● ●
● ●
●
●
●
●
● ●
0 10
30
20
30
Pruritus ADQ Pruritus.ADQ
L
K TEWL.NL vs. CD4 IL22+ 10.0
●
●
Pruritus ADQ ●
● ●
● ●
●
● ●
● ●
Pruritus.ADQ
r=0.15 (CI: −0.11−0.38) r=0.15 (CI: -0.11–0.38) ● p=0.06 p=0.6
●
●
10
●
● ●
●
● ● ●
● ●
● ●
● ●
●
●
● ● ●
●
● ● ● ● ● ● ●
●
●
●
● ● ●
●
r=0.16 (CI: −0.098−0.39) r=0.16 (CI: -0.098–0.39) ● p=0.045 p=0.045
10.0
● ●●
● ●
50
TEWL.LS TEWL LS
75
●
●
● ●
● ●
●
● ● ●
●
● ●
●
●
●
● ● ● ● ● ● ●● ●
● ●
0.0 100
TEWL.LS vs. CD4 IL22+ r=0.28 (CI: 0.035−0.49) r=0.28 (CI: 0.035–0.49) p=0.00025 p=0.00025
●
●
●
20
●
30
40
0.0
50
SCORAD SCORAD
●
●
● ●
● ●
● ●
●
●
●
● ●
● ● ● ●● ●● ●
●
● ● ● ●● ● ● ● ● ● ●● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ●
●●
● ● ●
● ● ● ●
●
● ● ● ● ●
●
●
●
●
●
●
●
● ●
●
●
● ●
● ●●
40
40
TEWL NL TEWL.NL
60
● ● ●
0
● ●
20
●
40
TEWL NL TEWL.NL
60
80
● ●
● ●
●
50
50
75
100
●
●
●
●
●
● ● ●●
● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●
60
●● ●
●● ● ●
40
● ●
● ● ●
●
●
● ●
●
●
●
●
●
●
●●
20 100
● ●
●
●
●
TEWL.LS TEWL LS
●
● ●
●
0 0
●
TEWL.LSLS TEWL
●
●
●
●
●
●
● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ●● ● ● ● ●● ●●● ● ● ● ●
20
●
●
● ●
● ●
●
r=−0.063 (CI: −0.26−0.13) r=-0.063 (CI: -0.26–0.13) ● p=0.42 ● ● p=0.42 ● ●
● ●
●
●
P TEWL.LS vs. SCORAD
●
●
80
25
●
●
●
●
● ● ● ● ● ●●●● ● ● ● ● ●● ● ●● ● ● ● ● ●●
50
●
●
●
●●
20
40
●
●
●● ●
●
●
●
30
TEWL.NLNL TEWL
80
● ●
20
● ●
● ●
●
0.0
●
●
● ●
●
●
●
●● ●
●
●
● ● ● ● ●
●
0 20
● ●● ●
40
●
●
● ●
● ●
r=0.076 (CI: −0.12−0.27) r=0.076 (CI: -0.12–0.27) ● p=0.45 p=0.45 ●
60
●
●
●
●
● ● ● ● ●
● ●
●
●
60
● ●
●
O TEWL.LS vs. EASI
● ●
●
●
5.0
● ● ● ● ● ●●
●●
10
● ●
2.5 ●●
● ● ●
7.5
●
●
●
● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●
●●
40
●
● ● ● ●●
●
● ●
TEWL.NL TEWL NL
●
● ●● ●
●
● ●
● ●
●
●
●
r=0.18 (CI: −0.015−0.37) r=0.18 (CI: -0.015–0.37) ● p=0.019 ● p=0.019 ● ●
80
●
●●
● ● ● ●
10
●
●
2.5
●
●
●
●
NTEWL.NL vs. SCORAD
r=0.29 (CI: 0.11–0.46)● p=0.0027
●
● ●
●● ●
●
●
5.0
●
●
● ●
●
●● ●
●
●
5.0
●
●
●
●
●
2.5 ●
7.5
● ●
r=0.29 (CI: 0.11−0.46) p=0.0027
0
● ●
●
EASI EASI
7.5
●
7.5
MTEWL.NL vs. EASI
●
●
● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ●● ● ● ●● ● ● ●● ● ●● ● ● ●●●●● ● ● ● ● ●
0
CD4 IL22+ CD4⁺IL-22⁺
● ●
20
● ● ● ● ● ●
100
SCORAD SCORAD
CD4 CLA+ CD4⁺CLA⁺
●
●
●
1
●
80
●
20
● ●
●
●
60
●
J TEWL.NL vs. CD4 CLA+ ●
●
0.0
●
●
●
●
●
EASI EASI
●
2.5
●
●
0
60
r=−0.28 (CI: −0.49−−0.035) r=-0.28 (CI:● -0.49–-0.35) p=0.00026 p=0.00026 ●
●
60
●
● ●
r=0.17 (CI: −0.067−0.38) r=0.17 (CI: -0.067–0.38) ● p=0.031 p=0.031
●
TEWL.LS vs. CD4 CLA+
5.0
40
●●
●
●
●
●
2
20
●
EASI
7.5
20
●
●
EASI
10.0
●
10.0
●
●
● ●
40
●
●
SCORAD
●
● ● ●
0
20
I
●
●
●●
●
●●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ●● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●
CD4 IL22+ CD4⁺IL-22⁺
0.0
●
●
10
G Pruritus.ADQ vs. CD4 IL22+
●
● ● ●● ● ● ● ● ● ● ● ● ●
●
●
●
●●
●
●
● ● ● ●●●
● ● ●● ● ● ● ●● ● ●● ● ● ●● ● ●● ● ● ● ● ● ●● ● ●● ● ● ● ●● ●● ● ●
●
●
●
●
100
●
30
7.5
●
● ●
● ●
3
SCORAD
r=0.46 (CI: 0.3−0.59) r=0.46 (CI: 0.3–0.59) ● ● ● ●● p=4.4e−07 p=4.4e-07 ●
●
●
80
●
●
●
●
F EASI vs. Pruritus.ADQ ●
● ●
●
SCORAD
p=0.0086
2.5
●
20
SCORAD
r=0.3 (CI: 0.079−0.49) r=0.3 (CI: 0.079–0.49) ● p=0.0086
● ● ● ● ● ●
● ●
●
●
0
EEASI vs. CD4 IL22+
● ●
●
●
●
SCORAD SCORAD
10.0
●
●
1
EASI EASI
10
● ●
●
●
●
CD4 IL22+ CD4⁺IL-22⁺
20
3
EASI vs. CD4 IL9+CLA−
●
CD4⁺IL-9⁺CLA⁻ CD4 IL9+CLA−
●
●
20
CD4 IL22+ CD4⁺IL-22⁺
r=0.23 (CI: 0.047−0.39) r=0.23 (CI: 0.047–0.39) p=0.0034
Eosinophils Eosinophils
●
●
0
CD4 CLA+ CD4⁺CLA⁺
30
CD4⁺IL-22⁺CLA⁺ CD4 IL22+CLA+
● ●
●
●
EASI EASI
D
CSCORAD vs. CD4 IL22+CLA+
r=0.28 (CI: 0.099−0.44) r=0.28 ● p=0.00031 (CI: 0.099–0.44) p=0.00031
● ●
CD8⁺IL-9⁺CLA⁻ CD8 IL9+CLA−
CD4⁺IL-13⁺CLA⁺ CD4 IL13+CLA+
30
r=0.2 (CI: 0.016−0.37) r=0.2 p=0.011 (CI: 0.016–0.37) p=0.011
150
0
50
100
TEWL.LSLS TEWL
150
Figure E9 60 60
Frequency (%)
B
CD25 CD127 Tregs
T Regs (CD25+CD127 )
*** *** *** **
40 40
** **
*** ***
* **
** **
**
** **
*** ***
20 20
*** ***
** ** 80 80
N
AD
N
AD
N
AD
Infants
Children
N
Adolescents
CAge vs. T Regs (CD25+CD127
AD
** **
*** ***
+
** * +
N
r=0.2 (CI: 0.015 0.38) r=0.2 (CI: 0.015–0.38) p=0.034
p=0.034
)
r= 0.092 (CI: 0.0017 0.39) p=0.38
r=-0.092 (CI: -0.0017–0.39)
10
N
Infants
AD
Children
N
AD
Adolescents
N
AD
Adults
D Age vs. Tregs CCR4+CLA+ 60
p=0.38
20
AD
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
Adults
CCR4 CLA Tregs Tregs CCR4+CLA+
CD25 CD127 Tregs T Regs (CD25+CD127 )
** *
40 40
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
0
** * + *
00
00
30
CCR4 CLA Tregs
Tregs CCR4+CLA+
120 120
* **
Frequency (%)
A
r=0.23 (CI: 0.043 0.4) r=0.23 p=0.016 (CI: 0.043–0.4)
r=0.4 (CI: 0.029 0.41) r=0.4 (CI:p=5e 0.029-0.41) 05
p=0.016
p=5e-05
40
20
0 0
20
40
Age
Age
60
80
0
20
40
Age
Age
60
80
1 1
Supplementary Materials and Methods
2
Isolation of Peripheral blood mononuclear cells and multiparameter flow
3
cytometry blood analyses
4
Peripheral blood mononuclear cells (PBMCs) were obtained by gradient
5
centrifugation with Ficoll-Paque Plus (Amersham Biosciences, Pittsburgh, PA).
6
The blood was laid under the Ficoll gradient; after spinning, the PBMCs were
7
collected at the interface between the plasma and the Ficoll gradient and washed
8
with PBS before staining for flow cytometry analysis. For T-cell surface staining,
9
PBMCs were washed and incubated for 30 minutes in ice with fluorochrome-
10
conjugated monoclonal antibodies (mAbs) to cell surface molecules (CD3-Pe AF
11
610, CD8-V-500, CD4-Qdot800, CD45RO-PeCy7, CLA-FITC, ICOS-
12
PerCpCy5.5, CCR7-Alexa Fluor 700, HLA-DR-APC-H7, CCR4-BV 421, CD25-
13
PE, CD127-BV 650, Live/dead-Blue Dye). Then, the cells were fixed in ice with
14
4% paraformaldehyde (BD Biosciences, San Jose, CA) for 20 minutes. For
15
intracellular cytokine staining, whole blood was activated with 25 ng/mL of
16
phorbol 12-myristate 13-acetate (PMA) and 2 µg/mL of ionomycin in the
17
presence of 10 µg/mL brefeldin A (protein transporter inhibitor) for 4 hours at
18
37°C. All reagents were purchased from Sigma Aldric h (St. Louis, MO). Non-
19
activated controls were only treated with brefeldin A. After this incubation, EDTA
20
(2 mM; Invitrogen, Grand Island, NY) was added for 15 minutes at room
21
temperature (RT) to stop activation. The blood was then incubated with FACS
22
lysing solution for 10 minutes at RT and washed with PBS twice. Then, the cells
23
were incubated for 20 minutes with fluorochrome-conjugated mAbs to cell
2 24
surface molecules (CD3-Pe AF 610, CD4-Qdot800, CLA-FITC). Subsequently,
25
the cells were permeabilized (Perm/Wash; BD Biosciences, San Jose, CA) and
26
incubated for 20 min with fluorochrome conjugated mAbs (IL-13-PerCpCy5.5, IL-
27
22-PeCy7, IL-9-Pe, IFN-γ- Alexa Fluor 700 and IL-17-APC efluor 780) in 5%
28
mouse serum (eBioscience, San Diego, CA). All incubations for cell surface and
29
intracellular staining were done at RT. Appropriate FMO-isotype controls were
30
used. Following incubations, cells were washed and subsequently acquired with
31
a BD LSR II flow cytometer (BD Biosciences) and analyzed by flow-Jo software
32
(Treestar, Ashland, OR).
33
3 34
Supplementary Figure legends
35
Figure E1. After gating on CD3+ viable T-cells using flow cytometry, CD3+CD4+
36
and CD3+CD8+ were defined and analyzed separately. CCR7 and CD45RO were
37
used to differentiate memory subsets within the CD4 and CD8 populations.
38
CCR7+CD45RO- defined naïve cells, CCR7+CD45RO+ central memory T-cells
39
(Tcm), CCR7-CD45RO+ effector memory T-cells (Tem) and CCR7-CD45RO-
40
Effector/Temra/terminally differentiated T-cells (A-C). We then further defined
41
activated ICOS/HLA-DR activated Tcm/Tem memory subset, using CLA to
42
segregate skin-homing (CLA+) versus systemic (CLA-) subsets, for each memory
43
population. Gating strategy for (A) CD3 vs. FSC-A, (B) CD4 vs. CD8, and (C)
44
CCR7 vs. CD45RO cells. (D-G) CD4+/CD8+ Tem (CD45RO+CCR7-) and Tcm
45
(CD45RO+CCR7+) subset frequencies and (H-K) HLA-DR+CLA+/-
46
CD45RO+CCR7+/- in AD and their respective controls across ages. Bar plots
47
represent means (black)/medians (red) ± SEMs. AD, Atopic dermatitis; CLA,
48
Cutaneous lymphocyte antigen; CCR, C-C Motif Chemokine Receptor 7; FSC-A,
49
Forward Scatter-Area; SEM, Standard error of the mean/median; Tcm, central
50
memory T-cells; Tem, effector memory T-cells. P-values are designated
51
as ***<0.001, **<0.01, *<0.05, and +<0.1.
52
Figure E2. (A-H) Pie charts showing the distribution of CD4+/CD8+ naïve, Tcm,
53
Tem, and effector T-cells in infants, children, adolescents and adults. (I-P)
54
Spearman correlation scatter plots (linear regression [red=AD/blue=control line]
55
with their CI [in gray]) for frequencies (%) of naïve/effector/TCM/TEM T-cells in the
56
CD4+ (I-J, M-N) and CD8+ (K-L, O-P) compartments versus age in AD patients
4 57
(red dots/line) and controls (blue dots/line). AD, Atopic dermatitis; CI, Confidence
58
interval; r, Spearman correlation coefficient with CI and associated P-value; Tcm,
59
central memory T-cells; Tem, effector memory T-cells.
60
Figure E3. Gating strategy of IFN-γ, IL-13, and IL-22 versus CLA positivity in
61
CD4+ and CD8+ non-activated and PMA/ionomycin activated T-cells in a
62
representative patient with AD. CLA, Cutaneous lymphocyte antigen.
63
Figure E4. A-D) IFN-γ+, (E-H) IL-13+, and (I-L) IFN-γ+/IL-13+ cytokine
64
frequencies in CLA+, CLA-, CD4+ and CD8+ T-cells in AD and their respective
65
controls across ages. The ratio between the % of IFN-γ+CLA+CD4+ (or CD8+) T
66
cells and the % of their IL-13+ counterpart was calculated for each sample but
67
was not multiplied by 100 and is therefore unitless. Bar plots represent means
68
(black)/medians (red) ± SEMs. AD, Atopic dermatitis; CLA, Cutaneous
69
lymphocyte antigen; SEM, Standard error of the mean/median. P-values are
70
designated as ***<0.001, **<0.01, *<0.05, and +<0.1.
71
Figure E5. A-D) IL-9+, (E-H) IL-17+ and (I-L) IL-22+ frequencies in CLA+, CLA-,
72
CD4+ and CD8+ T-cells in AD and their respective controls across ages. Bar plots
73
represent means (black)/medians (red) ± SEMs. AD, Atopic dermatitis; CLA,
74
Cutaneous lymphocyte antigen; SEM, Standard error of the mean/median. P-
75
values are designated as ***<0.001, **<0.01, *<0.05, and +<0.1.
76
Figure E6. Spearman correlation scatter plots (linear regression
77
[red=AD/blue=control line] with their CI [in gray]) for (A-D) frequencies (%) of
78
ICOS+ and HLA-DR+ activated CLA+/CD4+/CD8+ Tem (CD45RO+CCR7-) and
79
Tcm (CD45RO+CCR7+) T-cells versus age and (E-L) disease duration versus
5 80
clinical scores and various cytokine-producing T-cell subsets in AD patients (red
81
dots/line) and control subjects (blue dots/line). CI, Confidence interval; CLA,
82
Cutaneous lymphocyte antigen; EASI, Eczema area and severity index; r,
83
Spearman correlation coefficient with CI and associated P-value; SCORAD,
84
SCORing of Atopic Dermatitis.
85
Figure E7. Spearman correlation scatter plots (linear regression
86
[red=AD/blue=control line] with their CI [in gray]) for frequencies (%) of
87
CD4+/CD8+, CLA+/CLA- (A-D) IFN-γ +, (E-H) IL-9+, and (I-L) IL-17+ producing T-
88
cells versus age in AD patients (red dots/line) and controls (blue dots/line). CI,
89
Confidence interval; CLA, Cutaneous lymphocyte antigen; r, Spearman
90
correlation coefficient with CI and associated P-value.
91
Figure E8. Spearman correlation scatter plots (linear regression
92
[red=AD/blue=control line] with their CI [in gray]) for (A-C) SCORAD, (D-F) EASI,
93
(G-H) Pruritus, and (I-P) TEWL versus clinical measures and frequencies (%) of
94
various T-cell subsets. Dots colors represent different AD patient ages from
95
infancy to adulthood as shown in Figure 1. CI, Confidence interval; CLA,
96
Cutaneous lymphocyte antigen; EASI, Eczema area and severity index; LS,
97
Lesional; NL, Non-lesional; SCORAD, SCORing of Atopic Dermatitis; TEWL,
98
Transepidermal water loss.
99
Figure E9. A) Total and B) skin-homing Treg frequencies in AD and their
100
respective controls across ages and (C-D) Spearman correlation scatter plots
101
(linear regression [blue=control/red=AD lines] with their CI [in gray]) for Treg
102
frequency (%) versus age. Bar plots represent means (black)/medians (red) ±
6 103
SEMs. r, Spearman correlation coefficient with CI and associated P-value. AD,
104
Atopic dermatitis; CI, Confidence interval; CLA, Cutaneous lymphocyte antigen;
105
SEM, Standard error of the mean/median; Tregs, T-regulatory cells. P-values are
106
designated as ***<0.001, **<0.01, *<0.05, and +<0.1.
S0091-6749(19)31319-3
DOI:
https://doi.org/10.1016/j.jaci.2019.09.031
Reference:
YMAI 14212
To appear in:
Journal of Allergy and Clinical Immunology
Received Date: 21 April 2019 Revised Date:
23 August 2019
Accepted Date: 12 September 2019
Please cite this article as: Czarnowicki T, He H, Canter T, Han J, Lefferdink R, Erickson T, Rangel S, Kameyama N, Kim HJ, Pavel AB, Estrada Y, Krueger JG, Paller AS, Guttman-Yassky E, Evolution of pathologic T-cell subsets in atopic dermatitis from infancy to adulthood, Journal of Allergy and Clinical Immunology (2019), doi: https://doi.org/10.1016/j.jaci.2019.09.031. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 American Academy of Allergy, Asthma & Immunology
Evolution of polar T-cell subsets in healthy controls and atopic dermatitis patients from infancy to adulthood
Normal Cytokine Subset Development
AD Cytokine Subset Development
IFN-γ
(CLA+>CLACD4+>CD8+)
IL-13
(CLA+>CLACD4+>CD8+)
IL-9 IL-17 (CLA-)
IL-22 Tregs
(CLA+)
Blood Phenotype
0-5y/o
6-11y/o
12-17y/o
>18y/o
0-5y/o
6-11y/o
12-17y/o
>18y/o
Abbreviations: AD, Atopic dermatitis; CLA, Cutaneous lymphocyte antigen; IFN-γ, Interferon γ; IL, Interleukin; Tregs, T-regulatory cells
1 1
Evolution of pathologic T-cell subsets in atopic dermatitis from infancy to
2
adulthood
3 4
Tali Czarnowicki, MD MSc,1,3 Helen He, BSc,1 Talia Canter, BS,2 Joseph Han,
5
BS,1 Rachel Lefferdink, MD,2 Taylor Erickson, BS,2 Stephanie Rangel, PhD,2
6
Naoya Kameyama, PhD,1 Hyun Je Kim, MD PhD,1 Ana B Pavel, PhD,1 Yeriel
7
Estrada, BS,1 James G Krueger, MD PhD,3 Amy S Paller, MD,*2 Emma Guttman-
8
Yassky, MD PhD*1
9
*These authors contributed equally to the manuscript
10 11
1
12
Medicine at Mount Sinai, New York, NY, USA
13
2
14
School of Medicine, IL, USA
15
3
16
NY, USA
Department of Dermatology and the Immunology Institute, Icahn School of
Departments of Dermatology and Pediatrics, Northwestern University Feinberg
Laboratory for Investigative Dermatology, The Rockefeller University, New York,
17 18
Corresponding Author:
19
Emma Guttman-Yassky MD PhD
20
Department of Dermatology, Icahn School of Medicine at Mount Sinai Medical
21
Center, 5 E. 98th Street, New York, NY 10029
22
Email: [email protected]
23
Telephone: 212-241-9728/3288; Fax: 212-876-8961
2 24
Funding: This study was funded by a grant from the LEO Foundation. The study
25
was also supported by the Northwestern Skin Disease Research Center (NIAMS
26
P30 AR057216) and the Northwestern University Clinical And Translational
27
Sciences (NUCATS) Institute (UL1TR001422). JGK has received research
28
support (grants paid to his institution) and/or personal fees from Pfizer, Amgen,
29
Janssen, Lilly, Merck, Novartis, Kadmon, Dermira, Boehringer, Innovaderm,
30
Kyowa, BMS, Serono, BiogenIdec, Delenex, AbbVie, Sanofi, Baxter, Paraxel,
31
Xenoport, and Kineta. ASP is an investigator for Galderma, Incyte, Leo, Pfizer,
32
and Regeneron (grants paid to Northwestern Univ.) and has received honoraria
33
related to AD consulting from AbbVie, Asana, Boehringer, Dermavant, Dermira,
34
Eli Lilly, Forte, Galderma, Leo, Matrisys, Menlo Therapeutics,
35
Morphosys/Galapagos, Novartis, Pfizer, Regeneron, and Sanofi-Genzyme. EGY
36
is an employee of Mount Sinai and has received research funds (grants paid to
37
the institution) from Abbvie, Asana Biosciences, Celgene, Dermavant, DS
38
Biopharma, Galderma, Glenmark, Innovaderm, Janssen Biotech, Leo Pharma,
39
Novan, Novartis, Pfizer, Ralexar, Regeneron, and Union Therapeutics. EGY is
40
also a consultant for Abbvie, Allergan, Amgen, Asana Biosciences, Celgene,
41
Concert, DBV Technologies, Dermira, DS Biopharma, Eli Lilly, EMD Serono,
42
Escalier, Flx Bio, Galderma, Glenmark, Kyowa Kirin, Leo Pharma, Mitsubishi
43
Tanabe, Novartis, Pfizer, Regeneron, Sanofi, and Union Therapeutics.
44
Disclosures: Other authors have declared that they have no conflict of interest.
45
Word Count: 4,645
3 46
Keywords: Atopic dermatitis, endotypes, T-cell, CLA, IL-13, IL-22, IFN-γ, ICOS,
47
HLA-DR
48
4 49
Abstract
50
Background: The circulating immune phenotype was defined in adults and
51
young children with early atopic dermatitis/AD, but chronologic changes in blood
52
of AD infants and children through adolescence have not been explored.
53
Objective: To compare immune activation and cytokine polarization in blood of
54
0-5y/o (n=39), 6-11y/o (n=26), 12-17y/o (n=21) and ≥18y/o (n=43) patients with
55
AD vs. age-matched controls.
56
Methods: Flow cytometry was used to measure interferon-γ, interleukin (IL)-9,
57
IL-13, IL-17, and IL-22 cytokines in CD4+/CD8+ T-cells, with ICOS and HLA-DR
58
defining mid- and long-term T-cell activation, respectively, within skin-
59
homing/CLA+ vs. systemic/CLA- T-cells. Unsupervised clustering differentiated
60
patients based on their blood biomarker frequencies.
61
Results: While CLA+ Th1 frequencies were significantly lower in infants with AD
62
vs. all older patients (P<0.01), CLA+ Th2 T-cells were similarly expanded across
63
all AD age groups compared with controls (P<0.05). After infancy, CLA- Th2
64
frequencies were increased in AD in all age groups, suggesting systemic immune
65
activation with disease chronicity. IL-22 frequencies serially increased from
66
normal in infants to highly significant levels in adolescents and adults compared
67
to respective controls (P<0.01). Unsupervised clustering aligned the AD profiles
68
along an age-related spectrum from infancy to adulthood (ICOS, IL-22 etc.).
69
Conclusions: The adult AD phenotype is achieved only in adulthood. Unique
70
cytokine signatures characterizing individual pediatric age endotypes may require
71
age-specific therapies. Future longitudinal studies, comparing the profile of
5 72
cleared vs. persistent pediatric AD, may define age-specific changes that predict
73
AD clearance.
74 75
Capsule Summary: Decreased Th1/Th2 ratio characterizes the atopic dermatitis
76
(AD) phenotype across all age groups, while chronic activation, IL-9, IL-22, and
77
Treg changes characterize older patients. Even the adolescent AD profile differs
78
from adult AD.
79 80
Clinical Implications: Diverse immune signatures in different pediatric and adult
81
atopic dermatitis age groups argue for age-specific, rather than uniform,
82
therapeutic interventions.
83 84 85 86 87 88 89 90 91 92 93 94
6 95
Abbreviations
96
AD: Atopic dermatitis
97
ADQ: Atopic dermatitis quickscore
98
CD69: Cluster of Differentiation 69
99
CLA: Cutaneous Lymphocyte Antigen
100
EASI: Eczema area and severity index
101
HLA-DR: human leukocyte antigen DR
102
IFN-γ: Interferon gamma
103
IL: Interleukin
104
ICOS: Inducible co-stimulator molecule
105
ILC: Innate lymphoid cell
106
MHC: Major histocompatibility complex
107
PBMC: Peripheral Blood Mononuclear Cell
108
SCORAD: SCORing of Atopic Dermatitis
109
Tc1: Type 1 Cytotoxic T-cell
110
Tc17: Type 17 cytotoxic T-cell
111
Tc2: Type 2 cytotoxic T-cell
112
Tc22: Type 22 cytotoxic T-cell
113
TEWL: Transepidermal water loss
114
Tc: T cytotoxic
115
Th: T helper
116
Y/o: Years old
117
7 118
Introduction
119
Infancy, childhood and adolescence are critical periods for immune system
120
maturation.1 Early abnormal immune development may cause immune-related
121
disorders. Indeed, 85% of atopic dermatitis (AD) cases present before 5 years of
122
age.2 While young adults have a different AD phenotype from elderly patients,3
123
immune changes in AD between early childhood and adulthood are unknown.4
124
Blood studies that dissect developmental changes from infancy through
125
adulthood are limited and primarily focused on T-helper (Th)1/Th2 subsets.
126
Some studies with healthy controls showed expansion of Th1/Th2/T-cytotoxic
127
(Tc)1 subsets with age,5, 6 while others reported no changes in Th2/Tc2 over
128
time.7 Immature interferon (IFN)-γ response8 and low Th1/Tc1 frequencies9-11
129
were seen in early stages of normal development, and abnormal Th1/Th2 ratios
130
in cord blood and infants who develop AD were described.12-14 However, few
131
studies compared pediatric and adult AD populations6, 15-17 and none directly
132
compared consecutive age groups of AD with age-matched controls, which is
133
critical in understanding normal vs. pathological development of acquired
134
immunity.
135
The therapeutic arsenal available for 0-12 year old (y/o) AD patients is
136
limited to topical agents and/or broad systemic immune suppressants.4, 18 Active
137
development of targeted therapeutics is ongoing for adults and adolescents with
138
AD and will eventually move to children, further necessitating the elucidation of
139
pediatric endotypes at successive pre-adult age groups to introduce safe,
140
effective, age-tailored targeted approaches.19
8 141
We compared T-cell memory subset activation and polarized CD4/CD8
142
subset frequencies within the skin-homing/cutaneous lymphocyte antigen/CLA+
143
and systemic/CLA- compartments, in the blood of infants and toddlers (0-5y/o),
144
young children (6-11y/o), adolescents (12-17y/o) and adults (≥18y/o) with
145
moderate-to-severe AD. To differentiate pathologic from physiologic immune
146
maturation, age-matched healthy controls were included. We found that
147
decreased Th1/Th2 ratios were shared across all AD ages, but unique
148
fingerprinting characterizes individual AD age groups, differentiating them from
149
their age-matched peers. Our intracellular and T-cell blood biomarker data
150
grouped the AD cohort, but not the controls, into three unique age phenotypes
151
aligned along a spectrum.
152 153
Methods
154
Patient characteristics and blood samples
155
Blood was obtained (with signed Institutional Review Board-approved informed
156
consent from parents and patients 12 years and older) from 39 infants and
157
toddlers (0-5y/o; mean 23 months), 26 children (6-11y/o; mean 7.5yrs), 21
158
adolescents (12-17y/o; mean 14.9yrs) and 43 adults (≥18y/o; mean 43yrs) with
159
moderate-to-severe AD, as well as healthy age-matched controls (24-30 in each
160
group; demographic and laboratory data in Table E1). Sensitivity analyses on
161
patient subsets who were matched for all demographic parameters yielded
162
similar results to analysis including all subjects (Table E2A-B).
9 163
Disease severity was scored using SCORing of AD (SCORAD) for adults and
164
SCORAD, Eczema Area and Severity Index (EASI), and Atopic Dermatitis
165
Quickscore (ADQ) in those less than 18y/o. ADQ is a parent-administered tool,
166
which assesses involvement and pruritus of 7 body parts and highly correlates
167
with SCORAD.20 In subjects <18y/o, eosinophil counts and skin-barrier
168
assessment through transepidermal water loss (TEWL) of lesional (LS) and non-
169
lesional (NL) arm skin (AquaFlux Model AF200; Biox, London, England)21 were
170
measured when feasible. No patients received systemic immunosuppressive
171
treatment within 4 weeks prior to this study, thus the data present baseline
172
immune phenotyping of the studied populations. Concomitant allergic
173
manifestations were recorded, and individuals were classified as either having
174
general allergies (allergic rhinitis/asthma/allergic conjunctivitis/environmental),
175
food allergies (FA), both, and neither. Controls had no personal history of AD.
176
Only 1 child and 5 adolescent controls had history of non-cutaneous atopic
177
manifestations, however sensitivity analysis excluding these subjects did not alter
178
the results (Table E2C).
179
Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
180
PBMCs were isolated from whole blood by Ficoll-Paque Plus (GE Healthcare,
181
Sweden). Briefly, the blood was laid under Ficoll gradient; after spinning, PBMCs
182
were collected at the interface between the plasma and the Ficoll gradient (see
183
Methods in the Online Repository/OR).
184
Stimulation of blood cell populations for cytokine responses
10 185
Ex-vivo cell activation is required to detect cytokine production, as less than 1%
186
of non-stimulated cells produce cytokines. Whole blood was incubated with PMA
187
(25ng/ml) plus ionomycin (2µg/ml) in the presence of brefeldin A (10µg/ml) for 4
188
hours at 37ºC to induce cytokine responses. After stimulation, red blood cells
189
were lysed with FACS lysing solution to obtain leukocytes (see Methods in the
190
OR).
191
Cell surface staining and intracellular staining on PBMCs and stimulated
192
and non-stimulated CD4/CD8 T-cells
193
Peripheral blood mononuclear cells/PBMCs were stained with fluorochrome-
194
labeled Abs to cell surface markers (CD3, CD8, CD4, CD45RO, CCR7, ICOS,
195
HLA-DR, CLA, CCR4, CD25, CD127). Stimulated and non-stimulated blood cells
196
were also stained for cell surface markers [CD3, CD4, and CLA (CD8+ T-cells
197
were gated thorough CD3+CD4- T subpopulation] and permeabilized with
198
FACS/perm to stain for cytokines including Interleukin (IL)-13, IL-22, IL-9, IFN-γ
199
and IL-17 (see Methods in the OR).
200
Statistical analysis
201
Statistical analyses were performed with R software (www.R-projets.org). Means
202
and medians were compared using the Welch t-test and Wilcoxon-Mann-Whitney
203
test, respectively. Unsupervised hierarchical clustering of variables (T-cell subset
204
frequencies, age, and clinical scores) was performed using the R package
205
“hclust,” with a McQuitty agglomeration algorithm and Spearman coefficient as a
206
similarity metric and presented as a heat map and a dendrogram. Individual
207
scatter plots were constructed that display Spearman coefficients, 95%
11 208
confidence intervals, and p-values for AD and healthy samples. We performed k-
209
means unsupervised clustering across principal components of the frequencies
210
of all AD and separately among all healthy control subsets. We found that 3
211
clusters separated AD patients, but not controls, along a chronologic age
212
spectrum. ANOVA, in conjunction with the Tukey test, was used to find markers
213
that differentiated any two clusters. P-values and fold-changes (FCH) were
214
designated as ***(p<0.001), **(p<0.01), *(p<0.05), and +(p<0.1).
215
We also performed a power calculation based on our previous flow
216
cytometry study on moderate-to-severe adult AD patients.22 We determined that
217
39 AD patients would have greater than 90% power (at a significance level of
218
0.05) to detect differences versus controls in various inflammatory cell subsets
219
(CD4+IL-22+CLA+, CD4+IL-13+CLA-, CD8+IL-17+CLA+, CD8+ Tcm HLA-
220
DR+CLA+).
221 222
Results
223
Flow cytometry was used to measure frequencies of IFN-γ, IL-9, IL-13, IL-17A
224
and IL-22-polarized T-cells, defining Th1/Tc1, Th9/Tc9, Th2/Tc2, Th17/Tc17 and
225
Th22/Tc22 subsets in CD4+/CD8+ T-cells, respectively. Cell surface staining was
226
used to assess expression of mid (inducible co-stimulator molecule/ICOS) and
227
late (HLA-DR) activation markers in central (Tcm/CCR7+CD45RO+) and effector
228
(Tem/CCR7-CD45RO+) memory T-cells in skin homing/cutaneous/CLA+ and
229
systemic/CLA- compartments.
12 230
Patients and controls were divided into 4 consecutive age groups (infants
231
and toddlers 0-5y/o, children 6-11y/o, adolescents 12-17y/o and adults ≥18y/o).
232
To display both healthy vs. pathologic developmental changes and immune
233
abnormalities within each age group vs. controls, we present two types of
234
comparison plots; both contain similar data but focus on either AD patients vs.
235
controls for each age group or AD vs. control across all ages. To better represent
236
the effect of values distribution, the comparison plots presented below contain
237
both the mean (in black) and the median (in red) +/- standard error, and their
238
respective p-values. Results discuss the mean values, while median percentages
239
for main comparisons discussed are presented in Table E3.
240
Skin-homing memory T-cell expansion and ICOS activation feature early
241
AD
242
Tcm and Tem T-cells are main components of the adaptive immune system,
243
harboring distinct homing capacities.23 While both express the skin homing
244
marker CLA, only Tcm cells retain CCR7 positivity, which enables them to
245
migrate into lymph nodes and function as an immunologic reserve.24 After gating
246
on CD3+ viable T-cells using flow cytometry, CD3+CD4+ and CD3+CD8+ were
247
defined and analyzed separately. CCR7 and CD45RO were used to differentiate
248
memory subsets within the CD4 and CD8 populations. CCR7+CD45RO- defined
249
naïve cells, CCR7+CD45RO+ central memory T-cells (Tcm), CCR7-CD45RO+
250
effector memory T-cells (Tem) and CCR7-CD45RO- Effector/Temra/terminally
251
differentiated T-cells (Fig. E1A-C). We then further defined activated ICOS/HLA-
13 252
DR activated Tcm/Tem memory subset, using CLA to segregate skin-homing
253
(CLA+) versus systemic subsets (CLA-).
254
Normal development was characterized by a slight drop of CLA+ CD4+Tem (but
255
not Tcm) cells between infancy and childhood (infants 26.7% vs. children 19%,
256
P=0.01; Figs. 1A-B, E1D-E), but this decrease was significantly more evident in
257
AD (P<0.05; Figs. 1A-B, E1D-E). CD4+CLA+ Tem/Tcm cells were significantly
258
higher in AD infants (Tcm: 20.4% vs. 13.4%, P=0.006; Tem: 38% vs. 26.7%,
259
P=0.002; Fig. E1D-E) and children (Tcm: 18.8% vs. 11.3%, P=0.01; Tem: 27.7%
260
vs. 19%, P=0.03; Fig. E1D-E) than controls, a difference that diminished with
261
increasing age. T-cell memory subset fluctuations with age are presented in
262
Figure E2. Due to increased proportions of effector and naïve cells, CD8+
263
Tcm/Tem subsets dropped between infancy and childhood exclusively in controls
264
(Tcm: infants 12.8% vs. children 5.6%, P=0.008; Tem: infants 15.4% vs. children
265
5%, P<0.001; Fig. 1C-D), leading to higher frequencies in children with AD
266
(P<0.01), but otherwise frequencies were overall similar between AD and
267
controls over time (Fig. E1F-G). Significant CD4+/CD8+ effector cells increase
268
with age characterized AD vs. controls, who showed decrease of this subset
269
during the years (Fig. E2J, L).
270
CD4+ Tcm/Tem ICOS mid-activation continuously increased in both
271
controls and AD, however it decreased significantly between adolescence and
272
adulthood, exclusively in controls (Tcm CLA+: 24% vs. 9%, P=0.003; CLA-: 9.2%
273
vs. 3.7%, P=0.001; Fig. 1E-H). Both skin-homing/CLA+ and systemic/CLA- ICOS-
274
activated CD4+ T-cell frequencies were significantly increased in infants and
14 275
adults with AD vs. respective controls (P<0.05; Fig. 1E-H). Skin-homing CD8+
276
Tcm/Tem ICOS activation increased gradually, most notably in AD patients (Fig
277
1I-L), with frequency differences uniquely seen in adults with AD vs. controls
278
(Tcm: 18.4% vs. 12.5%, P=0.01; Tem: 26% vs. 13.4%, P<0.001; Fig. 1I-L).
279
The HLA-DR antigen, indicating chronic activation,25 had similar (or even
280
lower) expression in infants with and without AD (P>0.1; Fig. E1H-K), but started
281
to rise in AD children (Tcm+CD4+CLA+ AD: 15.8% vs. 8.4%, P=0.02; CLA-: 7.2%
282
vs. 2.8%, P=0.06; Fig. E1H-I), reaching consistently high levels across
283
CD4+/CD8+/CLA+/CLA-/Tcm/Tem cells in adults with AD compared to healthy
284
individuals (P<0.05; CD8+ Tcm/Tem data not shown; Fig. E1H-K).
285
Decreased Th1/Th2 ratio characterizes AD across ages
286
Since T-cell activation leads to cytokine polarization we next studied different
287
polar T-cell subsets. Representative flow cytometry plots and gating strategy are
288
presented in Figure E3. Congruent with past publications,9, 10 IFN-γ levels
289
increased with age in both control and AD subjects, with the lowest frequencies
290
seen in infants (Fig. 2A-D). Nevertheless, in AD IFN-γ did not reach control
291
levels, particularly within the skin homing compartment (CD4+CLA+: infants
292
control 12% vs. AD 7.7%, P=0.04; children control 19.5% vs. AD 13.6%, P=0.05;
293
adolescents control 18% vs. AD 13%, P=0.07) until adulthood (P=0.2; Figs. 2A-
294
D, E4A-D). Even in adulthood, levels trended towards lower frequencies.
295
Interestingly, the only population that showed lower systemic/CLA- CD4+/CD8+
296
IFN-γ was the 5-12y/o age group (CD4+: control 20% vs. AD 14%, P=0.04; CD8+:
297
control 39% vs. AD 28%, P=0.05; Fig. E4B, D).
15 298
In controls, IL-13+CD4+CLA+ was lowest in infants, reaching a plateau in
299
childhood. Conversely, in AD patients, levels were similarly elevated across all
300
ages (P>0.1; Fig. 2E). Systemic/CLA- Th2 was overall low in controls (Fig. 2F).
301
Levels were significantly higher in AD than controls in children (2.8% vs. 0.6%,
302
P=0.005; Fig. E4F), adolescents (1.5% vs. 0.7%, P=0.006; Fig. E4F) and adults
303
(1.3% vs. 0.7%, P=0.005; Fig. E4F). While Tc2 cells were slightly higher in
304
children with vs. without AD, differences in CD8+ subsets were more prominent in
305
adulthood (Figs. 2G-H, E4G-H). Reflecting the Th1 and Th2 imbalances
306
characterizing AD, the Th1/Th2 ratio was decreased in AD than controls in both
307
CLA+/CLA- subsets across the ages. Tc1/Tc2 was significantly lower in children
308
and adults with AD vs. controls (Figs. 2I-L, E4I-L).
309
Both healthy controls and AD patients showed Th9 increases over time,
310
peaking in adolescence and dropping in adulthood (Figs. 3A-B, E5A-B). No Th9
311
differences were observed between AD and controls, besides 5-12y/o children
312
with AD that showed significantly increased CLA- levels (0.76% vs. 0.3%,
313
P=0.04; Fig. E5B). This AD age group also showed increased CLA+/CLA- Tc9
314
levels (Figs. 3C-D, E5C-D). CLA+ Th17 cells were generally stable and similarly
315
abundant across ages among control and AD (Figs. 3E, E5E), contrary to CLA-
316
Th17 cells which showed developmental expansion in both (Figs. 3F, E5F).
317
Contrary to adolescents with AD that had lower CLA+ Tc17 than controls (1% vs.
318
4.7%, P=0.04), adults had significantly higher frequencies (2.1% vs. 1.4%,
319
P=0.05; Figs. 3G-H, E5G-H). Systemic/CLA- CD4+/CD8+ IL-22+ similarly
320
increased with age in both controls and AD (Fig. 3J, 3L, E5J, E5L), while skin-
16 321
homing Th22/Tc22 cells increased with age, primarily in AD (Figs. 3I, 3K, E5I,
322
E5K). Starting in childhood, skin-homing Th22/Tc22 were significantly higher in
323
AD vs. controls, and incrementally increased with age (Th22: children 6.7% vs.
324
4%, P=0.07; adolescents 7.9% vs. 3.6%, P=0.001; adults 8.2% vs. 4.4%,
325
P<0.0001; Figs. 3I, 3K, E5I, E5K). Polar T-cell subset development in controls
326
and AD is summarized in Table E6 and illustrated in the graphical abstract.
327
The unsupervised hierarchical clustering heatmap in Figure 4 summarizes
328
the above, displaying all polarized T-cell subsets for control and AD across age
329
groups (red: positive/increase; blue: negative/decrease). Fold-changes (FCHs) of
330
the mean frequencies of AD vs. controls for each age group are presented (stars
331
and plus signs display significance). The green cluster includes subsets that
332
were relatively low and stable among controls, but incrementally increased with
333
age in AD. Most of these subsets were significantly increased in adults with AD
334
vs. controls (FCH>1.57, P<0.05), while younger AD patients showed lower or no
335
significance. The pink box shows increased IL-9 frequencies in childhood, which
336
decline in adulthood, particularly in AD. The yellow cluster shows markers that
337
increased in both controls and AD, therefore minimizing the differences between
338
groups.
339
T-cell activation, clinical measures and IFN-γ are associated with age and
340
AD chronicity
341
We also evaluated how clinical characteristics, including AD severity (SCORAD,
342
EASI), patient age, disease duration, eosinophil counts, pruritus and TEWL relate
343
to different polar T-cell subsets. Unsupervised hierarchical clustering of all T-cell
17 344
subset frequencies, clinical scores, AD duration and age was performed using
345
Spearman correlations as a similarity metric, as displayed in the correlation
346
heatmap and dendrogram in Figure 5 (red: positive correlation; blue: negative
347
correlation, stars and plus signs display significance). Congruent with recent AD
348
data,22, 26 which showed positive correlations between IL-13- and IL-22- and
349
between IL-17- and IL-22-producing T-cells,22, 26, 27 these cytokines largely
350
grouped together (turquoise rectangle). IFN-γ-producing T-cells clustered
351
together with AD duration and patient age (yellow rectangles). A tight cluster
352
gathering multiple clinical measures (green rectangles) was located adjacent to
353
IL-9/IL-13-producing T-cells (purple rectangles). Significant correlations depicted
354
in this heatmap are listed in Table E4, with selected individual scatter plots
355
presented in Figures 6-7 and E6-E8.
356
SCORAD and EASI were positively correlated (r=0.74, P<0.001; Fig. 6A).
357
SCORAD also correlated with skin-homing CD4+ cells (r=0.24, P=0.0016; Fig.
358
6B), pruritus (r=0.54, P<0.0001, Fig. 6C) and eosinophils (r=0.25, P=0.001; Fig.
359
6D). To evaluate normal vs. pathologic development with age, we
360
comprehensively assessed age correlations in control and AD subjects,
361
presenting both on the same scatter plots for clarity, when applicable. Age
362
correlated positively with severity by SCORAD (r=0.25, P=0.0016; Fig. 6E) and
363
EASI (r=0.34, P<0.001; Fig. 6F), and with all other clinical measures, including
364
pruritus (Fig. 6G), eosinophils (Fig. 6H), and lesional TEWL (Fig. 6I-J).
365 366
While skin homing Tcm/Tem cells correlated negatively with age (red=AD, blue=controls; Fig 6K-L), the proportion of ICOS and HLA-DR-activated skin-
18 367
homing T-cells increased exclusively in AD (Fig. E6A-D). AD duration, which
368
highly correlated with patients’ age (r=0.98, P<0.0001; Fig. E6E), also
369
demonstrated significant positive correlations with EASI and pruritus (Fig. E6F-
370
H). Among the cytokine subsets (Fig. E6I-L), the most significant correlation was
371
noted between AD duration and IFN-γ-producing cells (r=0.58, P<0.0001; Fig.
372
E6L).
373
Overall IFN-γ increased with age in both AD and controls (Fig. E7A-D),
374
however, although the Th1/Th2 (but not the Tc1/Tc2) ratio, and particularly its
375
skin-homing component, increased with age in both groups, it remained
376
significantly decreased in AD across all ages vs. controls (Fig. 7A-D).
377
Conversely, differences between AD and controls were observed in skin-homing
378
Th2/Tc2 throughout development, with controls never reaching AD levels (Fig.
379
7E-H). CLA+ Th22/Tc22 showed significantly higher developmental increases in
380
AD (Fig. 7I, K), while systemic subsets generally overlapped (Fig. 7J, L). Similar
381
negative trends of IL-9+ (Fig. E7E-H), and expansion of systemic Th17 cells were
382
seen in controls and AD subjects (Fig. E7I-L).
383
Positive correlations between SCORAD and IL-13, IL-9 and IL-22-
384
producing cells were recorded (P<0.02; Fig. E8A-C). EASI correlated with Th9,
385
Th22 and pruritus (P<0.03; Fig. E8D-F), while pruritus was associated with Th22
386
and eosinophil counts (P<0.032; Fig. E8G-H). TEWL correlated with CD4+CLA+
387
cells, only in non-lesional skin (Fig. E8I-J). Th22 positively correlated with TEWL
388
in lesional tissues (Fig. E8K-L), and AD severity correlated with TEWL in non-
389
lesional skin (Fig. E8M-P).
19 390
Because Foxp3 staining requires cell permeabilization, surface markers
391
were used for Treg identification. 90% of CD25+CD127-CCR4+ cells co-express
392
Foxp3, therefore CD25+CD127-CCR4+ phenotype defined Tregs.28 In analyzing
393
T-regulatory cells (Tregs), both total Treg (CD25+CD127-) and skin-homing
394
(CCR4+CLA+ fraction) Treg frequencies were captured. The only AD age group
395
that showed significant increases of both total (Fig. E9A) and skin-homing (Fig
396
E9B) Tregs vs. control was 5-12y/o (P<0.01). Developmentally, AD showed
397
similar total Treg trends as controls, whereas discrepancies were more evident in
398
skin-homing subsets (Fig. E9C-D).
399
Cytokine polarization and T-cell activation differentiate AD patients into
400
separate age clusters along a spectrum
401
We integrated the T-cell and cytokine biomarkers to differentiate the entire AD
402
cohort based on their blood phenotype. The principal components of all
403
biomarker data for all subjects were analyzed using unsupervised k-means
404
clustering, separately for AD patients and controls. As illustrated in Figure 8, in
405
the AD cohort, the frequencies of different markers defined three meaningful
406
clusters, aligning along a spectrum. While infants (pink ellipse) clustered on the
407
far left, and adults (green ellipse) on the right, children and adolescents (blue
408
ellipse) generally clustered together, between the infants and adults. The
409
markers that best distinguished between each set of clusters appear in the boxes
410
between the two cohorts and are summarized in Table E5. Th1/Th2 ratio, CD8+
411
activation and IFN-γ-producing T-cells differentiated adults from younger groups.
412
Tregs, T-cell activation, and different cytokine subsets were able to differentiate
20 413
distinctive AD age groups. Applying the same model to the control population did
414
not distinguish between age groups (Fig. 8).
415 416
Discussion
417
This is the first comprehensive study that compares systemic immune profiles of
418
different AD age groups (0-5y/o, 6-11y/o, 12-17y/o and ≥18y/o), with appropriate
419
comparisons to controls. Since circulating CLA+ T-cells have been suggested as
420
peripheral biomarkers in AD,29 we utilized CLA to segregate between skin-
421
homing and systemic immune changes that accompany healthy and AD
422
development. Our data suggest unique endotypes in infants and toddlers,
423
children, adolescents and adults with AD, possibly advocating for personalized,
424
endotype-driven approaches rather than “one-size-fits-all” therapeutic strategies.
425
In addition, the results start to define age-specific differences between pediatric
426
AD patients and age-matched controls as a complement to our recent data
427
showing differences between elderly vs. young AD adult endotypes.3 Our data
428
suggest that even adolescents have a different profile from adults, possibly
429
contributing to the somewhat lower response to the recently approved IL-4Rα
430
antagonist, dupilumab, in adolescents vs. adults.30, 31
431
Immune responses at birth are immature10 with decreases of naïve T-cells
432
in healthy children with increasing age, paralleled by increases in Tcm/Tem
433
cells.32 Skin-homing Tcm/Tem cells are higher in infants and children with AD
434
compared to controls and decrease with age, exclusively in AD. Elevated skin-
435
homing memory cells cluster AD infants separately from other age groups,
21 436
suggesting early relevance. Excessive naïve-to-memory switch, which causes an
437
influx of memory cells into the circulation in infancy and early childhood, may play
438
a role in AD initiation, while exclusive decreases in the AD population over time
439
may suggest increased migration of CLA+ Tcm/Tem cells to skin.
440
ICOS is involved in multiple adaptive immune responses, including
441
induction of various T-cell polarizations,33-35 Treg regulation,36 antibody
442
responses (including IgE switching),37 and innate lymphoid cell (ILC)2-mediated
443
cytokine production.38, 39 ICOS has also been implicated in the pathogenesis of
444
asthma.40, 41 We have previously shown that Tcm/Tem ICOS activation is higher
445
in AD vs. control children.42 Subsequent data showed acclerated systemic/CLA-
446
ICOS T-cell activation in 0-3y/o AD infants.43 Our present data support ICOS
447
expansion in both healthy and AD development, particularly in CD4+ T-cells.
448
However, positive correlations with age were exclusively seen in AD, and
449
specifically in skin-homing memory subsets. Also, infants and adults with AD
450
have higher CD4+CLA+/CLA- ICOS activation than controls, a difference that is
451
also significant for CD8+ subsets in children. The low CD8+ frequencies in infants,
452
which increase with time,10 may explain the earlier activation of CD4+ subsets.
453
Early involvement of ICOS in AD indicates its possible role in disease induction
454
via either stimulation of T-cell subset polarization, Treg modulation, IgE induction,
455
or ILC2s activation. Furthermore, targeting ICOS/ICOS-ligand in an asthma
456
mouse model altered T-cell differentiation and ameliorated asthma.44 Since AD
457
often precedes asthma,45 CLA- ICOS expansion in AD infants may also be
458
associated with non-cutaneous atopic manifestations. These data suggest the
22 459
potential for ICOS targeting as a preventive or therapeutic approach for AD and
460
atopic associations, though this is speculative and further studies are needed.
461
The chronic T-cell activation MHC class II antigen, HLA-DR,46 increases
462
with age in healthy individuals.47, 48 HLA-DR activation has been shown in chronic
463
adult AD.42 While it is not elevated in AD infants, its activation increases with age.
464
At birth, immune responses are Th2-biased, with low Th1/IFN-γ in both
465
healthy newborns8, 9, 49, 50 and those who eventually develop AD.13 Our data
466
shows that Th1 frequencies increase with age in both AD and control infants.
467
However, and congruent with recent blood data,51 AD subjects display
468
significantly lower IFN-γ frequencies than controls, particularly among CD4+CLA+
469
subsets. Conversely, CLA+Th2 cells are similarly increased across all AD age
470
groups and are significantly higher than controls, even in infants. Systemic/CLA-
471
Th2 cells are significantly higher in AD starting in childhood, which implies
472
systemic Th2 activation with greater chronicity. Consistent with the less dominant
473
role of CD8 suggested in children with early AD42 and low CD8 abundance in
474
infants,10 Tc2 elevations are mostly prominent in adult AD. CLA+ Th1/Th2 ratio
475
correlation with age that is consistently decreased in AD compared to control,
476
and the decreased CLA+/CLA- Th1/Th2 ratio among all AD age groups best
477
reflect the immune imbalance that accompanies AD from birth to adulthood.
478
Circulating Th9 cells have skin-homing predilection.52, 53 IL-9 is elevated in
479
both skin and serum of children with AD, and was also correlated with disease
480
severity.54, 55 While the mechanism by which IL-9 contributes to AD is unknown,
481
IL-9 is linked with mast cell, eosinophil and ILC function and has been thought to
23 482
play a role in AD,56 as well as in other atopic disorders.57-60 Accordingly, IL-9
483
subsets cluster closest to all AD clinical measures
484
(eosinophils/pruritus/SCORAD/EASI/TEWL). Both normal and AD development
485
are characterized by IL-9 increases with age, which drop in adulthood. We show
486
considerably elevated systemic/CLA- T-cells exclusively among the 5-12y/o AD
487
group vs. controls, consistent with early allergy development in childhood with a
488
subsequent decline in adulthood.61, 62 CLA- systemic activation and cytokine
489
polarization with age support the systemic nature of AD and need for systemic
490
therapeutic approaches in moderate-to-severe disease, as well as the
491
association of comorbidities and increased inflammatory and cardiovascular-
492
associated proteins in more chronic AD.63-66
493
Black et al.67 showed strong Th17 skewing early in life. While similar
494
CLA+Th17 frequencies are seen in infants and older individuals regardless of AD,
495
systemic/CLA- Th17 cells are relatively low in infants of both groups and mature
496
with time. IL-17 signatures characterize pediatric AD skin,55 but these changes
497
are not mirrored in the circulation. This discrepancy may be due to increased skin
498
residence of Th17 cells, or by reciprocal peripheral regulation of Th17 and Th2
499
axes.68, 69
500
Skin-homing Th22 is elevated in children, adolescents and adults with AD
501
vs. controls. Congruent with past studies showing that IL-22 is correlated with AD
502
severity,70 we present positive correlations between IL-22-producing CD4+ cells,
503
SCORAD, EASI and pruritus. CD8+ T-cells are major producers of IL-22 in adult
504
AD skin.70 Lower proportions of CD8+ vs. CD4+ in early childhood47, 71 and the
24 505
fact that IL-22 marks AD chronicity42 may explain why skin-homing Th22/Tc22
506
cell frequencies are highest in adolescents and adults. Elevated IL-22 levels,
507
shown to negatively regulate IFN-γ,72 may also contribute to the low Th1
508
frequencies in AD. Systemic/CLA- Th22/Tc22 subset development was similar in
509
AD and controls, potentially suggesting a greater pathogenic relevance of IL-22
510
in AD skin73 and the impaired skin barrier.74 Studies have shown positive
511
correlations and cellular co-production of IL-13 and IL-2222, 75 and of IL-17 and IL-
512
22.27, 76 Indeed, these markers cluster together in the correlation heatmap. IFN-γ
513
clusters with patient age and disease duration, differentiating infants from
514
children and adolescents, and adolescents from AD adults. The association of
515
IFN-γ with disease chronicity agrees with previous chronic adult AD data,
516
suggesting that IFN-γ possibly plays a role in inflammatory disorders chronicity,
517
rather than in disease intiation.26, 42, 77 Expansion of CD8+ memory cells,
518
increases in CLA+IL-22-producing cells, and intensification of ICOS+/HLA-DR+
519
activation with maturity may account for the positive correlations between age,
520
disease duration and severity measures. Increasing severity with age was not
521
seen in a recent study that followed AD infants only between 0-11 months.78
522
TEWL increases with age in AD and correlates with IL-22 (which also increases
523
with age), likely again reflecting the contribution of IL-22 to the barrier impairment
524
in AD.
525
Besides maintaining immune tolerance,79 Tregs influence activation of
526
effector cells.80, 81 Only 5-12y/o children with AD show significantly higher total
527
and skin homing Tregs than controls. This age group is characterized by other
25 528
unique features, including increased skin-homing Tcm/Tem cells, decreased
529
systemic IFN-γ, and increased systemic IL-9 frequencies. Additionally, skin-
530
homing IFN-γ is almost doubled from infancy to childhood AD, although still
531
below age-matched controls. Multiple immunological changes occurring during
532
these years may be involved in AD clearance, or development of non-cutaneous
533
manifestations, commonly occurring during these years.82, 83
534
Profiling AD across ages is imperative for targeted therapeutic
535
development. Unlike psoriasis, in which targeted treatments lead to remarkable
536
responses in most patients, AD responses to targeted therapeutics are much
537
lower.84 This disparity may be attributed to the multi-cytokine activation in AD,
538
despite the shared Th2 activation, vs. the Th17-centered responses in
539
psoriasis.84 Additionally, AD has a highly varied endotype repertoire, with
540
different immune polarizations.3, 85, 86 Despite common features, particularly
541
elevated Th2 expression, AD is endotypically different across ages, and
542
treatments should rather be tailored to the unique age endotype. While one could
543
hypothesize that the immune changes merely reflect developmental phenomena
544
that are age-related, the lack of clear clustering in controls implicates AD as the
545
driver of the distinct, progressive age-related endotypic characteristics rather
546
than age alone. The clustering model, based on the flow cytometry biomarkers
547
splits the entire cohort into 3 separate age clusters only in AD patients.
548
Expectedly, IFN-γ, IL-22, HLA-DR increased chronologically, distinguishing older
549
subjects from infants with AD. Skin-homing Tcm/Tem cells and systemic ICOS
550
activation, both higher in AD infants, separate infants from other groups. This
26 551
model demonstrates that a limited group of blood biomarkers can distinguish
552
among various AD endotypes based on patient age, with a spectrum of age-
553
dependent vs. only ‘infantile’ or ‘adult’ phenotypes. Because our data show that
554
the ‘adult’ or ‘stable’ AD phenotype is only achieved in adulthood, it may be
555
possible to intervene before adulthood and prevent the establishment of the adult
556
AD phenotype.
557
This study has a few limitations. Despite inclusion of different age groups,
558
this study was not longitudinal and thus did not follow the same cohort with time.
559
Additionally, studies in blood do not allow one to measure T-cell subsets in skin,
560
such as tissue-resident memory/Trm T-cells. Furthermore, the study
561
characterized polyclonal T-cell responses, and not antigen-specific responses
562
induced by culprit triggers. Finally, the pathogenicity of immune axes presented
563
here cannot be further dissected without future targeted therapeutic studies to
564
check mechanism.
565
AD was initially considered an early-onset pediatric disease with 75%
566
"outgrowing" their disease by 10y/o.2, 83, 87, 88 More recent studies have
567
established AD as a disorder that often persists into adulthood.89, 90 Comparing
568
the profile of cleared vs. persistent pediatric AD, ideally through longitudinal
569
studies, will better define age-specific characteristics that predict AD clearance.
570 571 572 573
27 574
References
575
1.
576
Simon AK, Hollander GA, McMichael A. Evolution of the immune system in humans from infancy to old age. Proc Biol Sci 2015; 282:20143085.
577
2.
Bieber T. Atopic dermatitis. Ann Dermatol 2010; 22:125-37.
578
3.
Zhou L, Leonard A, Pavel AB, Malik K, Raja A, Glickman J, et al. Age-
579
specific changes in the molecular phenotype of patients with moderate-to-
580
severe atopic dermatitis. J Allergy Clin Immunol 2019; 144:144-156.
581
4.
Davidson WF, Leung DYM, Beck LA, Berin CM, Boguniewicz M, Busse
582
WW, et al. Report from the National Institute of Allergy and Infectious
583
Diseases Workshop on "Atopic Dermatitis and the Atopic March:
584
Mechanisms and Interventions". J Allergy Clin Immunol 2019; 143:894-
585
913.
586
5.
Sandmand M, Bruunsgaard H, Kemp K, Andersen-Ranberg K, Pedersen
587
AN, Skinhoj P, et al. Is ageing associated with a shift in the balance
588
between Type 1 and Type 2 cytokines in humans? Clin Exp Immunol
589
2002; 127:107-14.
590
6.
Kawamoto N, Kaneko H, Takemura M, Seishima M, Sakurai S, Fukao T,
591
et al. Age-related changes in intracellular cytokine profiles and Th2
592
dominance in allergic children. Pediatr Allergy Immunol 2006; 17:125-33.
593
7.
Chipeta J, Komada Y, Zhang XL, Deguchi T, Sugiyama K, Azuma E, et al.
594
CD4+ and CD8+ cell cytokine profiles in neonates, older children, and
595
adults: increasing T helper type 1 and T cytotoxic type 1 cell populations
596
with age. Cell Immunol 1998; 183:149-56.
28 597
8.
598 599
secretion in newborns. Biol Neonate 2004; 85:55-60. 9.
600 601
Kotiranta-Ainamo A, Rautonen J, Rautonen N. Imbalanced cytokine
Marodi L. Down-regulation of Th1 responses in human neonates. Clin Exp Immunol 2002; 128:1-2.
10.
Gasparoni A, Ciardelli L, Avanzini A, Castellazzi AM, Carini R, Rondini G,
602
et al. Age-related changes in intracellular TH1/TH2 cytokine production,
603
immunoproliferative T lymphocyte response and natural killer cell activity
604
in newborns, children and adults. Biol Neonate 2003; 84:297-303.
605
11.
606 607
Debock I, Flamand V. Unbalanced Neonatal CD4(+) T-Cell Immunity. Front Immunol 2014; 5:393.
12.
van der Velden VH, Laan MP, Baert MR, de Waal Malefyt R, Neijens HJ,
608
Savelkoul HF. Selective development of a strong Th2 cytokine profile in
609
high-risk children who develop atopy: risk factors and regulatory role of
610
IFN-gamma, IL-4 and IL-10. Clin Exp Allergy 2001; 31:997-1006.
611
13.
Herberth G, Heinrich J, Roder S, Figl A, Weiss M, Diez U, et al. Reduced
612
IFN-gamma- and enhanced IL-4-producing CD4+ cord blood T cells are
613
associated with a higher risk for atopic dermatitis during the first 2 yr of
614
life. Pediatr Allergy Immunol 2010; 21:5-13.
615
14.
616 617
Tang ML, Kemp AS, Thorburn J, Hill DJ. Reduced interferon-gamma secretion in neonates and subsequent atopy. Lancet 1994; 344:983-5.
15.
Kaminishi K, Soma Y, Kawa Y, Mizoguchi M. Flow cytometric analysis of
618
IL-4, IL-13 and IFN-gamma expression in peripheral blood mononuclear
619
cells and detection of circulating IL-13 in patients with atopic dermatitis
29 620
provide evidence for the involvement of type 2 cytokines in the disease. J
621
Dermatol Sci 2002; 29:19-25.
622
16.
Katsunuma T, Kawahara H, Yuki K, Akasawa A, Saito H. Impaired
623
interferon-gamma production in a subset population of severe atopic
624
dermatitis. Int Arch Allergy Immunol 2004; 134:240-7.
625
17.
Jung T, Lack G, Schauer U, Uberuck W, Renz H, Gelfand EW, et al.
626
Decreased frequency of interferon-gamma- and interleukin-2-producing
627
cells in patients with atopic diseases measured at the single cell level. J
628
Allergy Clin Immunol 1995; 96:515-27.
629
18.
Glines KR, Stiff KM, Freeze M, Cline A, Strowd LC, Feldman SR. An
630
update on the topical and oral therapy options for treating pediatric atopic
631
dermatitis. Expert Opin Pharmacother 2019:1-9.
632
19.
Siegfried EC, Igelman S, Jaworsk JC, Antaya RJ, Cordoro KM, Eichenfield
633
LF, et al. Use of dupilimab in pediatric atopic dermatitis: Access, dosing,
634
and implications for managing severe atopic dermatitis. Pediatr Dermatol
635
2019; 36:172-6.
636
20.
Carel K, Bratton DL, Miyazawa N, Gyorkos E, Kelsay K, Bender B, et al.
637
The Atopic Dermatitis Quickscore (ADQ): validation of a new parent-
638
administered atopic dermatitis scoring tool. Ann Allergy Asthma Immunol
639
2008; 101:500-7.
640 641
21.
Laudanska H, Reduta T, Szmitkowska D. Evaluation of skin barrier function in allergic contact dermatitis and atopic dermatitis using method
30 642
of the continuous TEWL measurement. Rocz Akad Med Bialymst 2003;
643
48:123-7.
644
22.
Czarnowicki T, Gonzalez J, Shemer A, Malajian D, Xu H, Zheng X, et al.
645
Severe atopic dermatitis is characterized by selective expansion of
646
circulating TH2/TC2 and TH22/TC22, but not TH17/TC17, cells within the
647
skin-homing T-cell population. J Allergy Clin Immunol 2015; 136:104-15.
648
23.
Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector
649
memory T cell subsets: function, generation, and maintenance. Annu Rev
650
Immunol 2004; 22:745-63.
651
24.
Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets of
652
memory T lymphocytes with distinct homing potentials and effector
653
functions. Nature 1999; 401:708-12.
654
25.
Ferenczi K, Burack L, Pope M, Krueger JG, Austin LM. CD69, HLA-DR
655
and the IL-2R identify persistently activated T cells in psoriasis vulgaris
656
lesional skin: Blood and skin comparisons by flow cytometry. J Autoimmun
657
2000; 14:63-78.
658
26.
Czarnowicki T, He HY, Wen HC, Hashim PW, Nia JK, Malik K, et al.
659
Alopecia areata is characterized by expansion of circulating
660
Th2/Tc2/Th22, within the skin-homing and systemic T-cell populations.
661
Allergy 2018; 73:713-23.
662 663
27.
Czarnowicki T, He H, Leonard A, Kim HJ, Kameyama N, Pavel AB, et al. Blood endotyping distinguishes the profile of vitiligo from that of other
31 664
inflammatory and autoimmune skin diseases. J Allergy Clin Immunol
665
2019; 143:2095-2107.
666
28.
Gorski K TJ, Gavin M, Cottrell S, Cox C, Vincent M, Ferbas J. CCR4
667
improves phenotypic identification of T-regulatory cells; Validation and
668
implementation of clinical test. Journal Immunol 2010; 184:26.
669
29.
Ferran M, Romeu ER, Rincon C, Sagrista M, Arnau AMG, Celada A, et al.
670
Circulating CLA+ T lymphocytes as peripheral cell biomarkers in T-cell-
671
mediated skin diseases. Exp Dermatol 2013; 22:439-42.
672
30.
Simpson E PA, Siegfried EC, Boguniewicz M, Pariser D, Blauvelt A,
673
Hultsch T, Staudinger H, Zhang R, Kamal MA, Davis JD, Ruddy M,
674
Graham NMH, Bansal A. Dupilumab Efficacy and Safety in Adolescents
675
with Moderate-to-Severe Atopic Dermatitis: Results from a Multicenter,
676
Randomized, Placebo-Controlled, Double-Blind, Parallel-Group, Phase 3
677
Study. Paris, France 2018; 09/15.
678
31.
Simpson EL, Bieber T, Guttman-Yassky E, Beck LA, Blauvelt A, Cork MJ,
679
et al. Two Phase 3 Trials of Dupilumab versus Placebo in Atopic
680
Dermatitis. N Engl J Med 2016; 375:2335-48.
681
32.
Saule P, Trauet J, Dutriez V, Lekeux V, Dessaint JP, Labalette M.
682
Accumulation of memory T cells from childhood to old age: central and
683
effector memory cells in CD4(+) versus effector memory and terminally
684
differentiated memory cells in CD8(+) compartment. Mech Ageing Dev
685
2006; 127:274-81.
32 686
33.
Vieira PL, Wassink L, Smith LM, Nam S, Kingsbury GA, Gutierrez-Ramos
687
JC, et al. ICOS-mediated signaling regulates cytokine production by
688
human T cells and provides a unique signal to selectively control the
689
clonal expansion of Th2 helper cells. Eur J Immunol 2004; 34:1282-90.
690
34.
Paulos CM, Carpenito C, Plesa G, Suhoski MM, Varela-Rohena A,
691
Golovina TN, et al. The Inducible Costimulator (ICOS) Is Critical for the
692
Development of Human T(H)17 Cells. Sci Trans Med 2010; 2.
693
35.
Wikenheiser DJ, Ghosh D, Kennedy B, Stumhofer JS. The Costimulatory
694
Molecule ICOS Regulates Host Th1 and Follicular Th Cell Differentiation
695
in Response to Plasmodium chabaudi chabaudi AS Infection. J Immunol
696
2016; 196:778-91.
697
36.
Vocanson M, Rozieres A, Hennino A, Poyet G, Gaillard V, Renaudineau
698
S, et al. Inducible costimulator (ICOS) is a marker for highly suppressive
699
antigen-specific T cells sharing features of TH17/TH1 and regulatory T
700
cells. J Allergy Clin Immunol 2010; 126:280-9.
701
37.
Kobayashi T, Iijima K, Dent AL, Kita H. Follicular helper T cells mediate
702
IgE antibody response to airborne allergens. J Allergy Clin Immunol 2017;
703
139:300-13.
704
38.
705 706
Wikenheiser DJ, Stumhofer JS. ICOS Co-Stimulation: Friend or Foe? Front Immunol 2016; 7:304.
39.
Simpson TR, Quezada SA, Allison JP. Regulation of CD4 T cell activation
707
and effector function by inducible costimulator (ICOS). Curr Opin Immunol
708
2010; 22:326-32.
33 709
40.
Coyle AJ, Gutierrez-Ramos JC. The role of ICOS and other costimulatory
710
molecules in allergy and asthma. Springer Semin Immunopathol 2004;
711
25:349-59.
712
41.
713 714
Maazi H, Akbari O. ICOS regulates ILC2s in asthma. Oncotarget 2015; 6:24584-5.
42.
Czarnowicki T, Esaki H, Gonzalez J, Malajian D, Shemer A, Noda S, et al.
715
Early pediatric atopic dermatitis shows only a cutaneous lymphocyte
716
antigen (CLA)(+) TH2/TH1 cell imbalance, whereas adults acquire CLA(+)
717
TH22/TC22 cell subsets. J Allergy Clin Immunol 2015; 136:941-51.
718
43.
Esaki H, Czarnowicki T, Gonzalez J, Oliva M, Talasila S, Haugh I, et al.
719
Accelerated T-cell activation and differentiation of polar subsets
720
characterizes early atopic dermatitis development. J Allergy Clin Immunol
721
2016; 138:1473-7.
722
44.
Uwadiae FI, Pyle CJ, Walker SA, Lloyd CM, Harker JA. Targeting the
723
ICOS/ICOS-L pathway in a mouse model of established allergic asthma
724
disrupts T follicular helper cell responses and ameliorates disease. Allergy
725
2019; 74:650-62.
726
45.
Czarnowicki T, Krueger JG, Guttman-Yassky E. Novel concepts of
727
prevention and treatment of atopic dermatitis through barrier and immune
728
manipulations with implications for the atopic march. J Allergy Clin
729
Immunol 2017; 139:1723-34.
730 731
46.
Ferenczi K, Burack L, Pope M, Krueger JG, Austin LM. CD69, HLA-DR and the IL-2R identify persistently activated T cells in psoriasis vulgaris
34 732
lesional skin: blood and skin comparisons by flow cytometry. J Autoimmun
733
2000; 14:63-78.
734
47.
Hulstaert F, Hannet I, Deneys V, Munhyeshuli V, Reichert T, De Bruyere
735
M, et al. Age-related changes in human blood lymphocyte subpopulations.
736
II. Varying kinetics of percentage and absolute count measurements. Clin
737
Immunol Immunopathol 1994; 70:152-8.
738
48.
Erkeller-Yuksel FM, Deneys V, Yuksel B, Hannet I, Hulstaert F, Hamilton
739
C, et al. Age-related changes in human blood lymphocyte subpopulations.
740
J Pediatr 1992; 120:216-22.
741
49.
Protonotariou E, Malamitsi-Puchner A, Rizos D, Papagianni B, Moira E,
742
Sarandakou A, et al. Age-related differentiations of Th1/Th2 cytokines in
743
newborn infants. Mediators Inflamm 2004; 13:89-92.
744
50.
745 746
Adkins B. Development of neonatal Th1/Th2 function. Int Rev Immunol 2000; 19:157-71.
51.
Brunner PM, Israel A, Leonard A, Pavel AB, Kim HJ, Zhang N, et al.
747
Distinct transcriptomic profiles of early-onset atopic dermatitis in blood and
748
skin of pediatric patients. Ann Allergy Asthma Immunol 2019; 122:318-30.
749
52.
Schlapbach C, Gehad A, Yang C, Watanabe R, Guenova E, Teague JE,
750
et al. Human TH9 cells are skin-tropic and have autocrine and paracrine
751
proinflammatory capacity. Sci Transl Med 2014; 6:219ra8.
752
53.
Wong MT, Ong DE, Lim FS, Teng KW, McGovern N, Narayanan S, et al.
753
A High-Dimensional Atlas of Human T Cell Diversity Reveals Tissue-
754
Specific Trafficking and Cytokine Signatures. Immunity 2016; 45:442-56.
35 755
54.
Ciprandi G, De Amici M, Giunta V, Marseglia A, Marseglia G. Serum
756
interleukin-9 levels are associated with clinical severity in children with
757
atopic dermatitis. Pediatr Dermatol 2013; 30:222-5.
758
55.
Esaki H, Brunner PM, Renert-Yuval Y, Czarnowicki T, Huynh T, Tran G, et
759
al. Early-onset pediatric atopic dermatitis is TH2 but also TH17 polarized
760
in skin. J Allergy Clin Immunol 2016; 138:1639-51.
761
56.
Ma L, Xue HB, Guan XH, Shu CM, Zhang JH, Yu J. Possible pathogenic
762
role of T helper type 9 cells and interleukin (IL)-9 in atopic dermatitis. Clin
763
Exp Immunol 2014; 175:25-31.
764
57.
765 766
Immunopathol 2017; 39:47-54. 58.
767 768
Clark RA, Schlapbach C. TH9 cells in skin disorders. Semin
Fujisawa T, Katsumata H, Kato Y. House dust mite extract induces interleukin-9 expression in human eosinophils. Allergol Int 2008; 57:141-6.
59.
Brough HA, Cousins DJ, Munteanu A, Wong YF, Sudra A, Makinson K, et
769
al. IL-9 is a key component of memory TH cell peanut-specific responses
770
from children with peanut allergy. J Allergy Clin Immunol 2014; 134:1329-
771
38.
772
60.
Xie J, Lotoski LC, Chooniedass R, Su RC, Simons FE, Liem J, et al.
773
Elevated antigen-driven IL-9 responses are prominent in peanut allergic
774
humans. PLoS One 2012; 7:e45377.
775 776
61.
Walton S, Nayagam AT, Keczkes K. Age and sex incidence of allergic contact dermatitis. Contact Dermatitis 1986; 15:136-9.
36 777
62.
Statescu L, Branisteanu D, Dobre C, Solovastru LG, Vasilca A, Petrescu
778
Z, et al. Contact dermatitis - epidemiological study. Maedica (Buchar)
779
2011; 6:277-81.
780
63.
Thijs JL, Strickland I, Bruijnzeel-Koomen C, Nierkens S, Giovannone B,
781
Knol EF, et al. Serum biomarker profiles suggest that atopic dermatitis is a
782
systemic disease. J Allergy Clin Immunol 2018; 141:1523-6.
783
64.
Vekaria AS, Brunner PM, Aleisa AI, Bonomo L, Lebwohl MG, Israel A, et
784
al. Moderate-to-severe atopic dermatitis patients show increases in serum
785
C-reactive protein levels, correlating with skin disease activity. F1000Res
786
2017; 6:1712.
787
65.
Brunner PM, Suarez-Farinas M, He H, Malik K, Wen HC, Gonzalez J, et
788
al. The atopic dermatitis blood signature is characterized by increases in
789
inflammatory and cardiovascular risk proteins. Sci Rep 2017; 7:8707.
790
66.
Brunner PM, Silverberg JI, Guttman-Yassky E, Paller AS, Kabashima K,
791
Amagai M, et al. Increasing Comorbidities Suggest that Atopic Dermatitis
792
Is a Systemic Disorder. J Invest Dermatol 2017; 137:18-25.
793
67.
Black A, Bhaumik S, Kirkman RL, Weaver CT, Randolph DA.
794
Developmental regulation of Th17-cell capacity in human neonates. Eur J
795
Immunol 2012; 42:311-9.
796
68.
Lynch JP, Ferreira MA, Phipps S. Th2/Th17 reciprocal regulation: twists
797
and turns in the complexity of asthma phenotypes. Ann Transl Med 2016;
798
4:S59.
37 799
69.
Choy DF, Hart KM, Borthwick LA, Shikotra A, Nagarkar DR, Siddiqui S, et
800
al. TH2 and TH17 inflammatory pathways are reciprocally regulated in
801
asthma. Sci Transl Med 2015; 7:301ra129.
802
70.
Nograles KE, Zaba LC, Shemer A, Fuentes-Duculan J, Cardinale I,
803
Kikuchi T, et al. IL-22-producing "T22" T cells account for upregulated IL-
804
22 in atopic dermatitis despite reduced IL-17-producing TH17 T cells. J
805
Allergy Clin Immunol 2009; 123:1244-52.
806
71.
Weerkamp F, de Haas EF, Naber BA, Comans-Bitter WM, Bogers AJ, van
807
Dongen JJ, et al. Age-related changes in the cellular composition of the
808
thymus in children. J Allergy Clin Immunol 2005; 115:834-40.
809
72.
Pennino D, Bhavsar PK, Effner R, Avitabile S, Venn P, Quaranta M, et al.
810
IL-22 suppresses IFN-gamma-mediated lung inflammation in asthmatic
811
patients. J Allergy Clin Immunol 2013; 131:562-70.
812
73.
Lou H, Lu J, Choi EB, Oh MH, Jeong M, Barmettler S, et al. Expression of
813
IL-22 in the Skin Causes Th2-Biased Immunity, Epidermal Barrier
814
Dysfunction, and Pruritus via Stimulating Epithelial Th2 Cytokines and the
815
GRP Pathway. J Immunol 2017; 198:2543-55.
816
74.
817 818
Fujita H. The role of IL-22 and Th22 cells in human skin diseases. J Dermatol Sci 2013; 72:3-8.
75.
Teraki Y, Sakurai A, Izaki S. IL-13/IL-22-coproducing T cells, a novel
819
subset, are increased in atopic dermatitis. J Allergy Clin Immunol 2013;
820
132:971-4.
38 821
76.
Liang SC, Tan XY, Luxenberg DP, Karim R, Dunussi-Joannopoulos K,
822
Collins M, et al. Interleukin (IL)-22 and IL-17 are coexpressed by Th17
823
cells and cooperatively enhance expression of antimicrobial peptides. J
824
Exp Med 2006; 203:2271-9.
825
77.
Gittler JK, Shemer A, Suarez-Farinas M, Fuentes-Duculan J, Gulewicz KJ,
826
Wang CQ, et al. Progressive activation of T(H)2/T(H)22 cytokines and
827
selective epidermal proteins characterizes acute and chronic atopic
828
dermatitis. J Allergy Clin Immunol 2012; 130:1344-54.
829
78.
Hulshof L, Overbeek SA, Wyllie AL, Chu M, Bogaert D, de Jager W, et al.
830
Exploring Immune Development in Infants With Moderate to Severe Atopic
831
Dermatitis. Front Immunol 2018; 9:630.
832
79.
833 834
Long SA, Buckner JH. CD4+FOXP3+ T regulatory cells in human autoimmunity: more than a numbers game. J Immunol 2011; 187:2061-6.
80.
McHugh RS, Shevach EM. The role of suppressor T cells in regulation of
835
immune responses. Journal of Allergy and Clinical Immunology 2002;
836
110:693-702.
837
81.
Hall BM. T Cells: Soldiers and Spies--The Surveillance and Control of
838
Effector T Cells by Regulatory T Cells. Clin J Am Soc Nephrol 2015;
839
10:2050-64.
840
82.
Burr ML, Dunstan FD, Hand S, Ingram JR, Jones KP. The natural history
841
of eczema from birth to adult life: a cohort study. Br J Dermatol 2013;
842
168:1339-42.
39 843
83.
Carlsten C, Dimich-Ward H, Ferguson A, Watson W, Rousseau R,
844
Dybuncio A, et al. Atopic dermatitis in a high-risk cohort: natural history,
845
associated allergic outcomes, and risk factors. Ann Allergy Asthma
846
Immunol 2013; 110:24-8.
847
84.
Guttman-Yassky E, Krueger JG. Atopic dermatitis and psoriasis: two
848
different immune diseases or one spectrum? Curr Opin Immunol 2017;
849
48:68-73.
850
85.
Czarnowicki T, He H, Krueger JG, Guttman-Yassky E. Atopic dermatitis
851
endotypes and implications for targeted therapeutics. J Allergy Clin
852
Immunol 2019; 143:1-11.
853
86.
Leung DY, Guttman-Yassky E. Deciphering the complexities of atopic
854
dermatitis: shifting paradigms in treatment approaches. J Allergy Clin
855
Immunol 2014; 134:769-79.
856
87.
Guttman-Yassky E, Nograles KE, Krueger JG. Contrasting pathogenesis
857
of atopic dermatitis and psoriasis--part I: clinical and pathologic concepts.
858
J Allergy Clin Immunol 2011; 127:1110-8.
859
88.
Burr ML, Dunstan FD, Hand S, Ingram JR, Jones KP. The natural history
860
of eczema from birth to adult life: a cohort study. Br J Dermatol 2013;
861
168:1339-42.
862
89.
863 864 865
Margolis JS, Abuabara K, Bilker W, Hoffstad O, Margolis DJ. Persistence of mild to moderate atopic dermatitis. JAMA Dermatol 2014; 150:593-600.
90.
Abuabara K, Margolis DJ. Do children really outgrow their eczema, or is there more than one eczema? J Allergy Clin Immunol 2013; 132:1139-40.
40 866
Figure legends
867
Figure 1. A-D) The frequency of CLA+ Tem (CD45RO+CCR7-) and Tcm
868
(CD45RO+CCR7+) in CD4+/CD8+ T cells and (E-L) ICOS+ activation in CLA+/-
869
CD4+/CD8+ Tcm/Tem cells in healthy controls and AD patients across ages
870
(ICOS+CLA+CD45RO+CCR7+/-). Bar plots represent means (black)/medians
871
(red) ± SEMs. AD, Atopic dermatitis; CLA, Cutaneous lymphocyte antigen; SEM,
872
Standard error of the mean/median; Tcm, central memory T-cells; Tem, effector
873
memory T-cells. P-values are designated as ***<0.001, **<0.01, *<0.05, and
874
+
875
Figure 2. A-D) IFN-γ+, (E-H) IL-13+, and (I-L) IFN-γ+/IL-13+ cytokine frequencies
876
in CLA+, CLA-, CD4+ and CD8+ T-cells in healthy controls and AD patients across
877
ages. The ratio between the % of IFN-γ+CLA+CD4+ (or CD8+) T cells and the %
878
of their IL-13+ counterpart was calculated for each sample but was not multiplied
879
by 100 and is therefore unitless. Bar plots represent means (black)/medians (red)
880
± SEMs. AD, Atopic dermatitis; CLA, Cutaneous lymphocyte antigen; SEM,
881
Standard error of the mean/median. P-values are designated as ***<0.001,
882
**<0.01, *<0.05, and +<0.1.
883
Figure 3. A-D) IL-9+ and (E-H) IL-17+ and (I-L) IL-22+ frequencies in CLA+, CLA-,
884
CD4+ and CD8+ T-cells in healthy controls and AD patients across ages. Bar
885
plots represent means (black)/medians (red) ± SEMs. AD, Atopic dermatitis;
886
CLA, Cutaneous lymphocyte antigen; SEM, Standard error of the mean/median.
887
P-values are designated as ***<0.001, **<0.01, *<0.05, and +<0.1.
<0.1.
41 888
Figure 4. Unsupervised hierarchical clustering heatmap displaying polarized T-
889
cell subsets for control and AD across age groups (red: positive/increase; blue:
890
negative/decrease). Fold-changes (FCHs) of the mean frequencies of AD vs.
891
controls for each age group are listed on the right (stars and plus signs display
892
significance). The green cluster includes subsets that were relatively low and
893
stable among controls, but incrementally increased with age in AD. The pink box
894
shows increased IL-9 frequencies in childhood, which decline in adulthood,
895
particularly in AD. The yellow cluster shows markers that increased in both
896
controls and AD.
897
Figure 5. Unsupervised hierarchical clustering of polarized cytokine subset
898
frequencies (%) with AD clinical measures using Spearman correlation as a
899
similarity metric. CD4+/CD8+ IL-13+, IL-17+ and IL-22+ subsets clustered together
900
(turquoise box). IFN-γ+-producing T-cell subsets grouped together with age and
901
disease duration (yellow box). IL-9+ and some IL-13+ and IL-22+ T-cells (purple
902
box) clustered adjacent to AD clinical measures (green box). The heatmap
903
shows the positive (red) or negative (blue) correlations of all parameters, with
904
color intensity reflecting the strength of the correlation (-1 to +1). Dendrograms
905
are shown as a tree, representing the distance between variables. CLA,
906
Cutaneous lymphocyte antigen. P-values are designated as ***<0.01, **<0.01,
907
*<0.05, and +<0.1.
908
Figure 6. Spearman correlation scatter plots (linear regression
909
[red=AD/blue=control line] with their CI [in gray]) for (A-E) SCORAD and (F-L)
910
age versus clinical measures and Tem/Tcm subset frequencies (%). Dots colors
42 911
in plots A-D, F designate different AD patient ages from infancy to adulthood, as
912
shown in Figure 1. CI, Confidence interval; CLA, Cutaneous lymphocyte antigen;
913
EASI, Eczema area and severity index; LS, Lesional; NL, Non-lesional;
914
SCORAD, SCORing of Atopic Dermatitis; Tcm, Central memory T-cells; Tem,
915
Effector memory T-cells; TEWL, Transepidermal water loss.
916
Figure 7. Spearman correlation scatter plots (linear regression
917
[red=AD/blue=control line] with their CI [in gray]) for CD4+/CD8+, CLA+/CLA- (A-
918
D) IFN-γ+/IL-13+ producing T-cell ratio, (E-H) IL-13+, and (I-L) IL-22+ producing T-
919
cell frequencies (%) versus age in AD patients (red dots/line) and controls (blue
920
dots/line). CI, Confidence interval; CLA, Cutaneous lymphocyte antigen.
921
Figure 8. Unsupervised clustering of AD patients and healthy controls across all
922
principal components of the blood flow cytometry marker frequencies (%) by k-
923
means. In AD, the frequencies of different markers defined three meaningful age-
924
clusters, aligning along a spectrum. While infants (pink ellipse) clustered on the
925
far left, and adults (green ellipse) on the right, children and adolescents (blue
926
ellipse) clustered together between the other age cohorts. The markers that best
927
distinguished between clusters appear in the boxes between two cohorts (colors
928
of markers parallel the colors of the relative age group). Arrows designate
929
elevated frequencies of a given marker among the specific age group. In healthy
930
controls, clusters did not clearly align patients along an age spectrum. CLA,
931
Cutaneous lymphocyte antigen; ICOS, Inducible co-stimulator molecule; Tregs,
932
T-regulatory cells.
933
Figure 1
A
100 100
**
** **
75
Frequency (%)
B
CLA CD4 Tcm cells
CD4 CM CLA+
150
** **
75
** **
100
50
50
25
0
40
+*
40 40
0
lts du ents
A
sc
e ol
0
0
+
+ +
+*
0
en
lts
sc
le
du
do
A
A
n re ld s nt
hi
fa
ts
AD
ICOS CLA CD8 Tem cells CD8 EM ICOS+/CLA
30 30
**
** **
C
s
L
In
lts
nt
ce
**
**
es
*** + *** *
100
du
**
ol
fa
ts
Normal
CLA CD8 Tem cells
CD8 EM ICOS+/CLA+
A
d n A re ld hi C ts n
In
lts
AD
100
20
du
n
**
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
en
**
K ICOS
sc
le
s
+
A
do
lts
nt
ce
es
A re ld hi s nt fa
C
In
ol
du
fa
+*
A
d n A re ld hi s nt
C
lts
ts
+
In
en
sc
le
+ *
40
30
Normal
CLA CD8 Tcm cells
CD8 CM ICOS+/CLA
30
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
AD
40 *** ***
**
30
du
do
A
J ICOS
A
hi
fa
s
n re ld s nt
C
In
lts
nt
ce
es
du
**
ol
* **
A
d n A re ld hi C ts n
fa
lts
ts
+
n d A re ld hi C ts n fa In s lt du
90
0
In
en
sc
le
* * +
+ +
+*
** **
50
Normal
+ *
CLA CD4 Tem cells
CD4 EM ICOS+/CLA
90
50
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
du
do
A
A
hi
fa
s
n re ld s nt
C
In
lts
nt
ce
es
fa
ol
du
A
d n A re ld hi s nt
C
In
CD8 CM ICOS+/CLA+
120
AD
30
AD
ICOS CLA CD8 Tcm cells
ts en
*
** *** *** **
120
60
0
Normal
sc
*** ***
H ICOS
60
20 0
e ol
CLA CD4 Tem cells
CD4 EM ICOS+/CLA+
100
*** **
+
A
Normal
20
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
d n A re ld hi C ts n fa In
lts du
ts en
sc
e ol
ts en
sc
e ol
G ICOS
A
n d A re ld hi C ts n fa In s lt du
A
d n A re ld hi C ts n fa In
lts du
ts en
sc
e ol
*** ***
**
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
AD
100
**
+
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
+*
+*
**
20
A
n d A re ld hi C ts n fa In s lt du
** ** ** ***
40
0
100
+
Normal
** **
**
40
25
100
**
** ** + * *** ***
** **
*** ***
20
0
CLA CD4 Tcm cells
60
* **
** ***
AD
CD4 CM ICOS+/CLA
60
25 0
ts en
**
sc
**
** **
e ol
75
A
75
** *
**
80
60
20
d n A re ld hi C ts n fa In
*** ***
+
50
lts du
100
50
ts en
sc
sc
le
e ol
100
F ICOS 80
*** ***
**
CLA CD8 Tem cells
CD8 EM CLA+
60
20
Normal
CLA CD4 Tcm cells
** **
0
AD
CD4 CM ICOS+/CLA+
** **
40
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
A
n do A re ld hi C ts n fa In s lt du ents
A
d n A re ld hi C ts n fa In
E ICOS Frequency (%)
60
**
D
CLA CD8 Tcm cells
CD8 CM CLA+
60
*** *** ** *
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
Normal
I
** ** *
50
25 0
Frequency (%)
**
100
50
0
+ *
** **
**
**
C
CLA CD4 Tem cells
CD4 EM CLA+ 150
+ *
+
20 20
20
50 50
50 50
10 10
10 10
0
0
0
0
0
ts en
AD
ts
sc
le
l du
sc
e ol
Normal
A
A
n do A re ld hi C ts n fa In s lt du ents
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
d n A re ld hi C ts n fa In
ts en
AD
ts
sc
le
l du
sc
e ol
Normal
A
A
n do A re ld hi C ts n fa In s lt du ents
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
d n A re ld hi C ts n fa In
ts en
AD
ts
sc
le
l du
sc
e ol
Normal
A
n do A re ld hi C ts n fa In s lt du ents
A
ts en
ts
sc
le
l du
sc
e ol
AD
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
d n A re ld hi C ts n fa In
A
n do A re ld hi C ts n fa In s lt du ents
A
d n A re ld hi C ts n fa In
Normal
0
0
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
Figure 2
Frequency (%)
A
CD4 T cells
CD4 IFNg+CLA+
60
60
40
** **
**
IFN-
*** ***
+
+*
**
B
CLA
100
*** ***
75
+
75
40
*** *** *** ***
50 50
**
C
CLA
**
IFN-
CLA
CD8 IFNg+CLA+
* **
*** *** *** *** *** ***+ + + + ** **
** *
+ +
75 75
D
+
CD8 IFNg+CLA
IFN-
**
*** *** ** *** *** ***
100 100
CLA *** *** ** ** *** + ** * **
50
+
50
50 50
20
lts du
ts en
le
ts
en
lts
sc
AD
CLA /IL-13 CLA
CD8 IFNg+CLA /IL13+CLA
1000
+
**
**
1000
**
du
s
IFN-
do
nt
n re ld s nt
hi
lts
s
*** *
A
A
C
fa
du
In
nt
ce
es
ol
lts
s
+
A
du
ce
es
ol
fa
lts
nt
L
CLA /IL-13 CLA
CD8 IFNg+CLA+/IL13+CLA+
75
Infants.Normal Children.Normal Adolescents.Normal Adults.Normal Infants.AD Children.AD Adolescents.ADAdults.AD
d n A re ld hi C ts n fa In
A
n d A re ld hi s nt C
du
In
A
K IFN75 50
** **
A
sc
*
Normal
50
*
500
500
25 25
0
0
0
0 Adults.AD
lts
du ts
n
ts
AD
en
sc
le
do
A
A e r ld hi s nt fa
Children.AD Adolescents.AD
C
In
en
n Normal
Infants.AD
lts
du
sc
le
do
A
A
nt
re ld s
fa
hi
C
Infants.Normal Children.Normal Adolescents.NormalAdults.Normal
In
ts
en
lts
n
ts
AD
du
sc
le
do
lts
du
C
Adults.AD
A
Children.AD Adolescents.AD
A re ld hi s nt fa
Infants.AD
In
n
ts
Normal
en
sc
le
do
A
A
nt
re ld s
fa
hi
C
lts
du
n
en
sc
le
do
ts
en
AD
Infants.Normal Children.NormalAdolescents.NormalAdults.Normal
In
Adults.AD
A
C
In
A re ld hi s nt fa
Children.AD Adolescents.AD
lts
n
ts
Normal
Infants.AD
du
A
sc
le
do
A
nt
re ld s
fa
hi
C
Infants.Normal Children.NormalAdolescents.NormalAdults.Normal
In
lts
du
n
ts
AD
en
sc
le
do
A
A re ld hi s nt fa
C
lts
du
In
n
en
sc
le
do
A
A
nt
re ld s
fa
hi
C
In
Normal
ce
*** * *** * ** +*
0
AD
*
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
es
*** ***
ol
*** ***
+*
*** *** ** **
d n A re ld hi C ts n fa In
ts
0
en
0
lts
sc
10
le
20
**
**
+ *
*** *** *** ***
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
du
20
40
do
** *
40
+*
0
Normal
CLA /IL-13 CLA
+
*** *** * *** * * *** *
1
AD
CD4 IFNg+CLA /IL13+CLA
60
e ol
10
1
2
A
n re ld s nt
fa
hi
60
A
C
s
*
nt
20
lts
J IFN-
40
*
du
*
In
*** *
ce
80
es
s
30
ol
nt
s
*** *
*** **
* +
120
A
lts
nt
ce
es
du
ol
fa
lts
Normal
CLA /IL-13 CLA
CD4 IFNg+CLA+/IL13+CLA+
40
d n A re ld hi C ts n fa In
A
n d A re ld hi s nt C
du
In
A
ce
es
ol
d n A re ld hi C ts n fa In
IFN-
AD
n d A re ld hi C ts n fa In s lt du
3
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
ts en
3 2
+ *
+ *
4
20
AD
IL-13 CLA
CD8 IL13+CLA
4
10
0
sc
30
0 Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
e ol
5
*** *** ** ** *** ***
30
A
5
*** ***
+
40
5
d n A re ld hi C ts n fa In
lts du
ts en
sc
H
IL-13 CLA
40 +
Normal
20
Normal
0
e ol
*
**
A
50
CD8 IL13+CLA+
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
AD
50
10
0
I
n d A re ld hi C ts n fa In s lt du
10
ts en
**
A
G
5
0
sc
15
e ol
Normal
** **
** *
** **
15
d n A re ld hi C ts n fa In
lts du
ts en
sc
le
sc
*** + *** +
AD
IL-13 CLA
CD4 IL13+CLA
0
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
A
n do A re ld hi C ts n fa In s lt du ents
A
e ol
F
*
**
d n A re ld hi C ts n fa In
Normal
IL-13 CLA
40
* **
lts du
AD
*** ***
40
ts en
sc
e ol
ts en
CD4 IL13+CLA+
0 0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
A
n d A re ld hi C ts n fa In s lt du
A
sc
e ol
d n A re ld hi C ts n fa In
Normal
20
0 0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
20
25
25
0
E
25
25
20
0
Frequency (%)
IFN-
CD4 IFNg+CLA
100
*** *** *** ***
**
CD8 T cells
Figure 3
A
CD4 T cells ** *
** *
Frequency (%)
B
IL-9 CLA
CD4 IL9+CLA+
*** **
40
+*
90
*** *
0
ts en
sc
le
lts du
A
n do A re ld hi C ts n fa In s lt du ents
A
lts
0
ts
0
en
sc
2
le
2
du
**
0
*** *** *** * ***
*** *** *** *** ** *** * **
4
+*
AD
IL-22 CLA
CD8 IL22+CLA
4
do
**
A
s
n re ld s nt
hi
fa
nt
ce
lts
L
Adults.AD
A
C
In
Children.AD Adolescents.AD
Normal
*** + *** *** *
Infants.AD
du
**
A
lts
ts
en
sc
es
ol
le
du
do
d n A re ld hi C ts n fa In
Infants.Normal Children.Normal Adolescents.NormalAdults.Normal
A
s
n re ld s nt
hi
fa
nt
**
25
0
sc
le
lts du ents
Normal
A
sc
n re
s
n do A re ld hi C ts n fa In s lt du ents
A
le
do
A
ld
nt
fa
hi
C
AD
Infants.Normal Children.Normal Adolescents.Normal Adults.Normal Infants.AD Children.AD Adolescents.ADAdults.AD
In
sc
le
lts du ents
Normal
A
n do A re ld hi C ts n fa In s lt du ents
A
n re
sc
le
do
A
s
nt
ld
fa
hi
C
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
In
sc
le
sc
le
lts du ents
A
n do A re ld hi C ts n fa In s lt du ents
do
A
A
s
n re
ld
nt
fa
hi
C
AD
sc
0.5
Adults.AD
50
Normal
e ol
ts en
C
lts
ce
es
du
In
A
Children.AD Adolescents.AD
50
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
d n A re ld hi C ts n fa In
lts du
A
sc
0.5
AD
+
25
In
sc
le
lts du ents
A
n do A re ld hi C ts n fa In s lt du ents
A
s
n re
sc
le
do
A
ld
nt
fa
hi
C
In
AD
ol
0
Infants.AD
*
0
Normal
**
IL-22 CLA
75
10
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
CD8 IL22+CLA+
75
5
0
A
ts
en
lts
sc
*** *** *** *** *** *** ** *** ** **
*** *** * *** *** *** **
5
0
1.0
0.0
d n A re ld hi C ts n fa In
le
du
10
do
10
A
s
n re ld s nt
fa
hi
lts
nt
*
15
10
1
**
0 Infants.Normal Children.Normal Adolescents.NormalAdults.Normal
K
+
*** *** *** * **
1.5
Normal
IL-22 CLA
CD4 IL22+CLA
+*
A
C
In
du
ce
es
ol
20
1.5
+ +
AD
15
+ +
20
A
lts
ts
en
sc
+
d n A re ld hi C ts n fa In
le
J
*** * *** ** * **
2.0
0.0
Infants.Normal Children.Normal Adolescents.Normal Adults.Normal Infants.AD Children.AD Adolescents.ADAdults.AD
du
do
s
30
+ +
le
0
0
A
nt
n re ld s nt
hi
fa
lts
40
+
AD
IL-17 CLA
CD8 IL17+CLA
2
2.5
Normal
IL-22 CLA
*** *** *** ** +* *** ***
**
2.5
AD
40 30
A
C
du
In
A
ce
es
ol
CD4 IL22+CLA+
H
+
5
0
d n A re ld hi C ts n fa In
50
n do A re ld hi C ts n fa In s lt du ents
7.5
2
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
50
+ +
7.5
2
0
I
sc
10
Normal
IL-17 CLA
CD8 IL17+CLA+
10.0
5
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
AD
5.0
5
Normal
e ol
** **
A
G
10
0
d n A re ld hi C ts n fa In
4
lts du ents
sc
sc
le
e ol
**
4
**
Normal
*** *** + *** ** ** *
6
10
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
AD
IL-17 CLA
CD4 IL17+CLA
+*
0
0
A
n do A re ld hi C ts n fa In s lt du ents
A
d n A re ld hi C ts n fa In
ts en
sc
le
sc
lts du
A
n do A re ld hi C ts n fa In s lt du ents
A
e ol
d n A re ld hi C ts n fa In
F **
**
4
0
Infants.Normal Children.Normal Adolescents.Normal Adults.Normal Infants.AD Children.AD Adolescents.ADAdults.AD
6
15
8
4
Normal
+
15
8
*
30
AD
IL-17 CLA
20
+
*** *** ** * *** ***
30
0
Infants.Normal Children.Normal Adolescents.Normal Adults.NormalInfants.AD Children.AD Adolescents.ADAdults.AD
20
+
12
+ +
+
IL-9 CLA
CD8 IL9+CLA
12
60
2
CD4 IL17+CLA+
+
** *
90
2
Normal
Frequency (%)
**
+
60
0
Frequency (%)
+
**
120
D
IL-9 CLA
CD8 IL9+CLA+
120
4
20
E
+
** *
4
20
0
+*
+ +
6
*** ***
** **
C
IL-9 CLA
CD4 IL9+CLA
6
*** ***
*** **
40
CD8 T cells
AD
Figure 4 Color Key 1.5
0.5 0.5 1.5 Row Z Score
Normal
AD
AD/Normal
AdolesInfants Children Adolescents Adults Infants Children cents Adults CD4⁺ Tem ICOS⁺/CLA⁺ CD8⁺ Tcm HLA-DR⁺/CLA⁻ CD8⁺ Tem HLA-DR⁺/CLA⁺ CD4⁺ IL22⁺CLA⁺ CD8⁺ IL22⁺CLA⁺ CD8⁺ IL13⁺ CD8⁺ IL13⁺CLA⁺ CD4⁺ IL13⁺CLA⁺ CD8⁺ Effector CD4⁺ Effector CD8⁺ Tem ICOS⁺/CLA⁺ CD4⁺ Tem HLA-DR⁺/CLA⁺ CD8⁺ Tcm HLA-DR⁺/CLA⁺ CD4⁺ Tcm ICOS⁺/CLA⁺ CD8⁺ Naïve CD8⁺ Tcm ICOS⁺/CLA⁺ CD8⁺ Tcm ICOS⁺/CLA ⁻ Tregs CCR4⁺CLA⁺ CD4⁺ IL13⁺ CD4⁺ IL13⁺CLA⁻ CD4⁺ Tcm HLA-DR⁺/CLA⁺ CD4⁺ Tcm HLA-DR⁺/CLA⁻ CD4⁺ Tem HLA-DR⁺/CLA⁻ T Regs (CD25⁺CD127⁻) CD4⁺ IL9⁺ CD4⁺ IL9⁺CLA⁻ CD4⁺ IL9⁺CLA⁺ CD4⁺ Tcm ICOS⁺/CLA⁻ CD8⁺ IL9⁺CLA⁻ CD4⁺ Tem CD8⁺ IL17⁺CLA⁺ CD8⁺ IL17⁺ CD8⁺ IL17⁺CLA⁻ CD4⁺ Naïve CD8⁺ Effector CLA⁺ CD8⁺ Tem CLA⁺ CD8⁺ Tcm CLA⁺ CD8⁺ Tem ICOS⁺/CLA⁻ CD4⁺ Tem CLA⁺ CD4⁺ Tcm CLA⁺ CD8⁺ IL9⁺CLA⁺ CD8⁺ IL9⁺ CD4⁺ Tem ICOS⁺/CLA⁻ CD8⁺ IFN-γ⁺CLA⁻ CD8⁺ IFN-γ⁺ CD4⁺ IFN-γ⁺CLA⁻ CD4⁺ IFN-γ⁺ CD4⁺ IFN-γ⁺CLA⁺ CD4⁺ Tcm CD4⁺ IFN-γ⁺CLA⁻/IL13⁺CLA⁻ CD8⁺ IFN-γ⁺CLA⁺ CD4⁺ IL22⁺ CD4⁺ IL22⁺CLA⁻ CD4⁺ IL17⁺ CD4⁺ IL17⁺CLA⁻ CD8⁺ IL13⁺CLA⁻ CD8⁺ IL22⁺ CD8⁺ IL22⁺CLA⁻ CD8⁺ Tem HLA-DR⁺/CLA⁻ CD4⁺ IL17⁺CLA⁺ CD4⁺ Effector CLA⁺ CD4⁺ CLA⁺ CD8⁺ IFN-γ⁺CLA⁻/IL13⁺CLA⁻ CD8⁺ Tcm CD8⁺ Tem CD8⁺ IFN-γ⁺CLA⁺/IL13⁺CLA⁺ CD4⁺ IFN-γ⁺CLA⁺/IL13⁺CLA⁺
: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :
Infants Children Adolescents Adults 1.48⁺ 1.52 1.14 1.16 2.51 1.67 1.21 3.69** 1.09 1.16 1.12 1.62⁺ 1.08 1.45⁺ 1.23* 1.24 1.10 1.38 1.21 1.04 2.04⁺ 1.98 1.40 1.12 1.05 1.03 1.17 1.91* 1.40 1.18 1.53 1.24 1.32 1.02 1.22 1.12 1.11 1.57 1.43** 1.51** 1.20 1.39 2.03** 1.11 1.10 1.02 1.04 1.54* 1.07 1.73⁺ 1.16 1.00 1.17 1.35 1.38 1.13 2.12⁺ 1.86 1.08 1.41⁺ 1.10 1.56* 1.27 1.65* 1.39⁺ 1.65 5.97*
1.03 1.01 1.55 1.62⁺ 2.79* 2.33⁺ 2.02⁺ 1.38 1.24 1.33 1.81* 1.34 1.09 1.02 1.06 1.71⁺ 2.27* 2.88** 4.04** 4.20** 1.87* 2.52⁺ 1.64 1.74** 2.10⁺ 2.22⁺ 1.05 1.13 1.33 1.29 1.60 1.01 1.47 1.08 1.13 1.92** 1.89** 1.61 1.46* 1.66* 1.60 1.19 1.13 1.43* 1.42* 1.46* 1.46* 1.40⁺ 1.08 3.28** 1.16 2.28* 2.34* 1.19 1.22 2.54* 1.90 1.76 1.94⁺ 1.39 1.35 1.57 2.58* 1.50** 1.15 1.25 3.31*
1.40 1.98* 1.66 2.21** 1.88 1.44 1.13 1.64⁺ 1.15 1.14 1.73* 1.95 3.24* 1.26 1.33** 1.08 1.52 1.40 1.71* 1.78* 1.82 1.72 1.72 1.01 1.38 1.48 1.08 1.24 1.46 1.80* 4.65* 8.49 8.88 1.30** 1.11 1.08 1.10 1.24 1.28 1.05 1.37 1.24 1.11 1.16 1.17 1.14 1.14 1.36⁺ 1.41⁺ 1.61 1.31 1.16 1.15 1.25 1.29 1.66 1.10 1.11 1.12 1.00 1.38 1.21 1.55 1.79** 2.40** 1.41 1.76⁺
1.99** 1.89** 3.18** 2.04** 3.59** 1.90** 2.85** 2.43** 1.57** 2.03⁺ 1.95** 2.13** 2.84** 2.42** 1.09 1.47* 1.03 1.28⁺ 2.31** 1.85** 3.01** 2.12⁺ 1.02 1.39** 1.86* 1.38 1.08 1.65** 1.23 1.23 1.51⁺ 1.03 1.08 1.02 1.52* 1.02 1.14 1.22 1.16 1.00 1.01 1.45⁺ 1.44⁺ 1.04 1.03 1.05 1.07 1.14 1.23⁺ 1.76 1.07 1.16 1.31⁺ 1.25 1.24 1.50* 1.16 1.04 1.66** 1.13 1.23 1.72** 1.43⁺ 1.05 1.01 2.24* 3.49**
CD8−CLA− CD8⁺CLA⁺
**
**
**
TEWL−LS TEWL LS
+
**
***
*
+
*
**
*
**
**
*
*
***
**
*
*
***
***
*
*
+
*
*
***
***
***
***
***
***
**
+
+
***
***
***
***
***
***
**
**
***
*
***
***
**
***
***
***
***
***
***
***
***
***
*
***
***
***
***
***
***
***
**
***
***
***
***
***
CD8−IL13−CLA−−1 CD8⁺IL13⁺CLA⁻
***
***
***
***
***
CD8−IL−17−CLA− CD8⁺IL17⁺CLA⁺
***
***
CD8−IL13−CLA− CD8⁺IL13⁺CLA⁺
***
CD8−IL22−CLA− CD8⁺IL22⁺CLA⁺
*
***
***
***
***
***
***
***
***
X−CD8−IL22− CD8⁺IL22⁺ CD8−IL22−CLA−−1 CD8⁺IL22⁺CLA⁻ X−CD8−IL13− CD8⁺IL13⁺
X−CD8−IL−17− CD8⁺IL17⁺
***
CD8−IL−17−CLA−−1 CD8⁺IL17⁺CLA⁻
***
*
*
**
**
**
*
+
**
**
+
*
***
**
*
***
***
***
***
***
***
***
*
+
** +
+
*
CD8⁺IL9⁺CLA⁻
CD8⁺IL9⁺
CD8⁺IL9⁺CLA⁺
CD4⁺IL9⁺CLA⁻
+
***
*
+
+
*
*
*
*
***
**
**
*
+
***
***
***
***
*
***
***
***
***
***
**
*
***
***
***
***
*
*
+
***
***
***
***
***
***
**
**
*
*
CD4⁺IL9⁺ *
** *
+
CD4⁺IL9⁺CLA⁺
CD4⁺IL13⁺CLA⁻
CD4⁺IL13⁺
CD4⁺IL13⁺CLA⁺
CD4⁺IL22⁺CLA⁺
+
+ *
*
**
+
TEWL−NL TEWL NL CD4−IL−17−CLA− CD4⁺IL17⁺CLA⁺
Total ADQ
Pruritus ADQ
Eosinophils CD8⁺IFNγ⁺CLA⁻
CD8⁺IFNγ⁺
CD8⁺IFNγ⁺CLA⁺
CD4⁺IFNγ⁺CLA⁻
CD4⁺IFNγ⁺
CD4⁺IFNγ⁺CLA⁺
Age
CD4⁺IL17⁺CLA⁻
CD4⁺IL17⁺
CD4⁺IL22⁺CLA⁻
CD4⁺IL22⁺
*
EASI
***
SCORAD
CD4−CLA− CD4⁺CLA⁺
CD8⁺IL17⁺CLA⁻
CD8⁺IL17⁺
CD8⁺IL17⁺CLA⁺
CD8⁺IL13⁺CLA⁻
CD8⁺IL13⁺
CD8⁺IL22⁺CLA⁻
Distance: spearman / average
Duration of Disease
−1.0
CD8⁺IL22⁺
−0.5
CD8⁺IL22⁺CLA⁺
0.0
CD8⁺IL13⁺CLA⁺
TEWL NL
0.5
CD8⁺CLA⁺
CD4⁺CLA⁺
1.0
TEWL LS
cor
CD4⁺IL17⁺CLA⁺
Figure 5
+
+
+ +
+
*
+
*
+
***
***
*
+
*
*
**
***
**
**
* +
**
***
* **
**
***
***
***
+
***
***
*
*
***
***
***
***
***
*
***
***
*
**
***
***
***
***
***
**
***
***
**
**
+
+
**
***
***
***
***
***
***
***
***
***
***
+
*
**
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
X−CD4−IL22− CD4⁺IL22⁺ CD4−IL22−CLA−−1 CD4⁺IL22⁺CLA⁻ X−CD4−IL−17− CD4⁺IL17⁺ CD4−IL−17−CLA−−1 CD4⁺IL17⁺CLA⁻ Age Age Duration−of−disease Duration of Disease CD4−IFNg−CLA− CD4⁺IFNγ⁺CLA⁺
+ *
+
***
***
***
***
***
***
***
***
***
***
**
**
**
***
***
***
***
***
***
***
***
***
***
***
***
***
***
*
***
***
+
***
***
***
***
+
***
*
***
***
***
***
***
***
+
***
***
**
**
+
***
***
**
**
***
***
+
*
+
*
***
*
+
**
+
*
***
*
+
**
CD4⁺IFNγ⁺ CD4−IFNg− CD4−IFNg−CLA−−1 CD4⁺IFNγ⁺CLA⁻ CD8−IFNg−CLA− CD8⁺IFNγ⁺CLA⁺ CD8−IFNg− CD8⁺IFNγ⁺
+
+
* +
**
*
CD8−IFNg−CLA−−1 CD8⁺IFNγ⁺CLA⁻
Eosinophils Eosinophils
**
Pruritus−ADQ Pruritus ADQ
*
*
**
***
***
***
***
***
Total−ADQ Total ADQ SCORAD SCORAD
***
EASI EASI
+ +
*
** *
+
*
+
+
*
***
**
***
*
+
**
**
**
**
**
**
** **
*
*
***
*
* *
*
*
+
CD4−IL22−CLA− CD4⁺IL22⁺CLA⁺
** ***
CD4−IL13−CLA− CD4⁺IL13⁺CLA⁺
***
***
+
***
+
*
*
**
*
**
+
*
*
+
+
*
***
**
***
***
*
***
***
+
*
***
***
***
***
*
**
***
***
***
*
**
***
***
***
**
**
***
***
***
***
***
***
***
***
***
X−CD4−IL13− CD4⁺IL13⁺ CD4−IL13−CLA−−1 CD4⁺IL13⁺CLA⁻ CD4−IL9−CLA− CD4⁺IL9⁺CLA⁺ X−CD4−IL9− CD4⁺IL9⁺ CD4−IL9−CLA−−1 CD4⁺IL9⁺CLA⁻ CD8−IL9−CLA− CD8⁺IL9⁺CLA⁺ X−CD8−IL9− CD8⁺IL9⁺
***
***(p<0.001) **(p<0.01) *(p<0.05) +(p<0.1)
CD8⁺IL9⁺CLA⁻ CD8−IL9−CLA−−1
CD8⁺IL9⁺ X−CD8−IL9−
CD8⁺IL9⁺CLA⁺ CD8−IL9−CLA−
CD4⁺IL9⁺CLA⁻ CD4−IL9−CLA−−1
CD4⁺IL9⁺ X−CD4−IL9−
CD4⁺IL9⁺CLA⁺ CD4−IL9−CLA−
CD4⁺IL13⁺CLA⁻ CD4−IL13−CLA−−1
CD4⁺IL13⁺ X−CD4−IL13−
CD4⁺IL13⁺CLA⁺ CD4−IL13−CLA−
CD4⁺IL22⁺CLA⁺ CD4−IL22−CLA−
EASI EASI
SCORAD SCORAD
Total ADQ Total−ADQ
Pruritus ADQ Pruritus−ADQ
Eosinophils Eosinophils
CD8⁺IFNγ⁺CLA⁻ CD8−IFNg−CLA−−1
CD8⁺IFNγ⁺ CD8−IFNg−
CD8⁺IFNγ⁺CLA⁺ CD8−IFNg−CLA−
CD4⁺IFNγ⁺CLA⁻ CD4−IFNg−CLA−−1
CD4⁺IFNγ⁺ CD4−IFNg−
CD4⁺IFNγ⁺CLA⁺ CD4−IFNg−CLA−
Age
Age
CD4⁺IL17⁺CLA⁻ CD4−IL−17−CLA−−1
CD4⁺IL17⁺ X−CD4−IL−17−
CD4⁺IL22⁺CLA⁻ CD4−IL22−CLA−−1
CD4⁺IL22⁺ X−CD4−IL22−
CD8⁺IL17⁺CLA⁻
CD8−IL−17−CLA−−1
CD8⁺IL17⁺ X−CD8−IL−17−
CD8⁺IL17⁺CLA⁺ CD8−IL−17−CLA−
CD8⁺IL13⁺CLA⁻ CD8−IL13−CLA−−1
CD8⁺IL13⁺ X−CD8−IL13−
CD8⁺IL22⁺CLA⁻ CD8−IL22−CLA−−1
Duration of Disease Duration−of−disease
***p<0.001 **p<0.01 *p<0.05 +p<0.1
CD8⁺IL22⁺ X−CD8−IL22−
CD8⁺IL22⁺CLA⁺ CD8−IL22−CLA−
CD8⁺IL13⁺CLA⁺ CD8−IL13−CLA−
CD4⁺IL17⁺CLA⁺ CD4−IL−17−CLA−
TEWL NL TEWL−NL
TEWL LS
TEWL−LS
CD8⁺CLA⁺ CD8−CLA−
CD4⁺CLA⁺ CD4−CLA−
CD8−IL9−CLA−−1 CD8⁺IL9⁺CLA⁻
Figure 6
20
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Age Age
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p=0.88
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r=−0.37 (CI: −0.52−−0.2) (CI: -0.52–-0.2) ● 100 r=-0.37 p=5.2e−05
15
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Age
80
p=0.095
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75 ●
50
25
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r=-0.17 (CI: -0.53–-019)
r=−0.17 (CI: −0.53−−0.19) p=0.095
p=5.2e-05
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0
Age
10
L Age vs. CD4 EM CLA+
r=-0.015 (CI: -0.51–-0.17)
●
● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ●● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ●● ●● ● ● ●● ● ● ● ●● ● ●●● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●● ●● ● ● ●● ● ●●● ●● ●● ● ● ●● ● ● ● ● ●● ● ●● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ●● ●● ● ●● ● ● ●● ● ●●●● ● ● ● ●
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Age Age
r=−0.015 (CI: −0.51−−0.17) p=0.88
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Age Age
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p=0.00013
0
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40
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80
SCORAD SCORAD
r=0.27 (CI: 0.028−0.47) r=0.27 (CI: 0.028–0.47) p=0.029 p=0.029
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20
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r=−0.35 (CI:(CI: −0.5−−0.18) r=-0.35 -0.5–-0.18) ● p=0.00013
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H Age vs. Eosinophils ●
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0
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40
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40
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20
80
K Age vs. CD4 CM CLA+
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40
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5
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15
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r=0.081 (CI: −0.12−0.27) r=0.081 (CI: -0.12–0.27) ● p=0.42 p=0.42
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J Age vs. TEWL.NL
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Age
TEWL NL TEWL.NL
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60
●
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150
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Age
r=0.32 (CI: 0.14−0.49) r=0.32 (CI: 0.14–0.49) ● p=0.00097 p=0.00097
50
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10
0 0
80
Age vs. TEWL.LS
TEWL.LS TEWL LS
● ●
●
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Age Age
●
●●
r=0.19 (CI: 0.0017−0.36) r=0.19 (CI: 0.0017–0.36) ● ●● p=0.048 p=0.048 ● ● ●
●
●
20
100
●
●
20
0
0
0
●
G Age vs. Pruritus.ADQ
●
●
●
SCORAD
●
●
●
● ●
SCORAD
● ●
EASI EASI
SCORAD SCORAD
● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ●●● ● ●●● ●● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ●●● ● ● ● ● ● ● ● ● ● ● ● ●
● ●
● ●
● ●
● ●
●
●
●
0
● ● ●
● ● ●
●
●
●
● ●
● ●
●
60
●
I
20
●
●
●
● ● ●
● ● ●● ● ●
100
●
20 ● ● ● ● ● ●● ●
●●
●
80
r=0.34 (CI: 0.17−0.49) r=0.34 p=0.00015 (CI: 0.17–0.49) ● p=0.00015
●
●
●
●
●
●
● ● ●
20
●
● ●
F Age vs. EASI
p=0.0016 ●●
●
●
SCORAD
r=0.25 (CI: 0.096−0.39) r=0.25 (CI: 0.096–0.39) ● p=0.0016
●
●
SCORAD
EAge vs. SCORAD
40
●
40
EASI
60
●
● ● ● ● ● ● ●● ●●● ● ● ● ●● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ●● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ●●
EASI
80
●
0
0
100
●
●
●
● ●
●
●
20
●●
●
●
●
●●
●
5
●
●
●
Pruritus ADQ Pruritus.ADQ
60
●
●
15
r=0.25 (CI: 0.01−0.47) r=0.25 (CI: 0.01–0.47) p=0.0011 p=0.0011
● ● ● ●
30
CLA⁺CD4⁺ CD4 CMTcm CLA+ cells
SCORAD SCORAD
●
p=0.82e-14
Pruritus ADQ Pruritus.ADQ
●● ●
r=0.54 (CI: 0.39–0.66)
●
●
●
● ●
r=0.24 (CI: 0.064–0.41) p=0.0016
r=0.54 (CI: 0.39−0.66) ● p=8.2e−14
Eosinophils Eosinophils
r=0.74 (CI: 0.64–0.81) p=1.4e-21
CD4⁺CLA⁺ CD4 CLA+
100
D SCORAD vs. Eosinophils
C SCORAD vs. Pruritus.ADQ
r=0.24 (CI: 0.064−0.41) p=0.0016
Eosinophils Eosinophils
B SCORAD vs. CD4 CLA+
r=0.74 (CI: 0.64−0.81) p=1.4e−21
CD4 EM Tem CLA+ cells CLA⁺CD4⁺
A EASI vs. SCORAD
●
● ●
●
● ● ●●
●
● ● ●● ● ● ● ● ● ● ● ● ● ●
● ● ●
● ● ● ●
● ● ● ● ● ● ●●● ●
●
●
● ●
● ●●
● ● ● ●●
● ●
● ●
●
20
40
Age Age
60
80
CD4⁺ T cells r=0.11 (CI: 0.01−0.38) ● p=0.28
●
● ●
● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ●● ● ● ●●● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ●● ● ●●●● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ●● ●● ●● ● ●● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ●●●● ●●●● ● ● ● ● ● ●●●●● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●● ● ●● ● ●● ● ● ● ●● ● ●●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●●● ● ● ●
0
20
40
●
●
60
0
80
12
p=0.51
20
40
●
60
●
●
●
●
●
●
●
● ● ●
0
●
●
●
●
● ● ● ● ● ● ● ● ● ●● ● ●● ●● ● ● ● ● ●● ● ● ●● ●●● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ●●●●●● ●●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ●● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ●●●
0
80
●
● ●● ● ● ●● ● ● ● ●● ●● ● ● ●●●● ● ● ● ● ● ●● ● ● ● ● ●●●●● ● ● ●●● ●● ●● ●● ● ●● ● ● ● ● ● ● ● ●
20
40
●
60
p=0.53
30
p=0.63
p=0.38
●
2000 ●
●
● ● ● ●
1000 ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ●● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ●● ● ●● ● ● ● ● ● ● ●● ● ●●● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ●●●● ● ●● ● ● ● ● ●● ●● ●● ●● ● ● ● ●● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●●●● ● ●●● ● ●● ● ●● ● ● ● ●● ●● ● ● ● ●●●● ● ●●●●● ● ● ● ●●● ● ●
0
●
80
G Age vs. CD8 IL13+CLA+
Age vs. CD8 IFNg+CLA−/IL13+CLA− r=−0.058 (CI: −0.23−0.12) (CI: −0.25−0.14) r=-0.058 (CI: -0.23–0.12)r=−0.089 r=0.089 (CI: -0.25–0.14) p=0.53 ● p=0.38
0
Age Age
r=0.072 (CI: −0.11−0.25) (CI: −0.13−0.26) r=0.072 (CI: -0.11–0.25) r=−0.048 r=0.048 (CI: -0.13-0.26) p=0.44● p=0.63
p=0.44
20
40
80
H Age vs. CD8 IL13+CLA−
r=0.36 (CI: 0.19−0.51) r=0.14 (CI: 0.17−0.52) r=0.36 (CI: 0.19–0.51) ●r=0.14 (CI: 0.17-0.52) p=6.5e−05 p=0.16 ● p=6.5e-05 p=0.16
3
r=0.65 (CI: 0.53−0.74) r=0.45 (CI: 0.52−0.75) r=0.65 0.53–0.74) r=0.45 (CI: 0.52-0.75) ● p=3.7e−15 (CI: p=1.9e−06 p=3.7e-15 p=1.96e-06
● ●
● ● ● ● ●
20
● ●
●
●
●
●
● ●
● ● ● ● ●
● ● ●
● ●
●
●●
●● ● ● ●
●
●
● ●
● ● ● ● ● ● ●● ● ●● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ●● ● ● ●●● ●● ● ● ●● ●●● ● ● ●● ● ● ● ●● ● ●● ● ● ● ● ● ●●●● ● ● ●●● ●● ● ●● ●●●● ●●●●● ● ● ● ● ● ● ● ●● ● ● ●
0
●● ●●
●
● ●
●● ● ● ●
● ●
●
●● ●
20
●
●
●
●
●
●
0
●● ● ● ●
40
60
●
●
●
● ● ● ● ●
●
6
●
●
●● ●
●
● ●
● ●
● ●
●●
●
3
●
●
●
● ●●
●
80
●
●
9
●
●
●
● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ●● ● ● ●● ● ● ● ●● ● ● ●●● ● ● ● ● ●● ● ●● ● ● ●●● ● ●● ● ● ● ●●● ● ●● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ●●● ● ●●● ●● ● ● ●●● ●● ● ● ● ●● ● ●● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ●●●● ●●●● ● ● ● ● ●● ● ●●● ● ● ● ● ●● ● ● ●● ● ● ● ●●● ● ● ● ● ●● ● ● ● ●●● ●
20
0
20
40
●
●
●
● ● ●
10
●
●
0
60
●
●
● ●
Age
●
●
80
●
● ●
●
● ●
● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ●● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ●● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ●●● ● ● ●● ●● ●● ●● ● ●● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ● ● ● ● ● ● ●● ●● ● ●● ● ●● ●● ● ● ●●●● ● ● ●●●●● ● ●●●● ● ●● ● ● ●●
0
20
40
Age
●
CD8 IL13+CLA−
●
IL-13⁺CLA⁻
●
2
●
●
1
●
●
● ●
●
● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ●●● ● ● ●● ●● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●●● ●● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●●●● ● ● ●● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ●
●
0
60
0
80
20
40
60
Age
Age
Age
I Age vs. CD4 IL22+CLA+
J Age vs. CD4 IL22+CLA−
K Age vs. CD8 IL22+CLA+
L Age vs. CD8 IL22+CLA−
r=0.31 (CI: 0.14–0.47) r=0.22 (CI: 0.12-0.48) ● p=0.00052 p=0.03 r=0.31 (CI: 0.14−0.47) p=0.00052
r=0.22 (CI: 0.12−0.48) ● p=0.03
10.0
r=0.66 (CI: 0.48−0.73) r=0.66 (CI: 0.48-0.73) p=1e−13 p=1e-13
r=0.62 (CI: 0.5−0.72) r=0.62 p=3.9e−14●(CI: 0.5–0.72) p=3.9e-14
●
●
80
Age
Age
Age 30
60
Age Age
● ●
● ●
IL-13⁺CLA⁻ CD4 IL13+CLA−
IL-13⁺CLA⁺ CD4 IL13+CLA+
● ●
F Age vs. CD4 IL13+CLA−
●
0
●
p=0.38
Age Age
r=0.16 (CI: −0.018−0.33) (CI: −0.034−0.35) r=0.16 (CI: ●-0.018–0.33) r=0.067 r=0.067 (CI: -0.034–0.35) p=0.077 p=0.51
10
●
● ●●
● ● ●
● ●● ● ● ● ● ●● ●● ● ● ● ●●● ● ● ●● ● ● ●● ● ●● ● ● ● ●● ● ●● ● ● ● ● ●● ●● ● ● ●● ● ● ● ● ●● ●●● ●● ●● ●●● ●● ●● ● ● ●● ●● ● ● ● ● ● ● ● ● ●●● ● ● ● ●●●● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●
0
EAge vs. CD4 IL13+CLA+ 30
●
●
● ● ●
●
r=0.088 (CI: −0.21−0.17) p=0.38
p=0.81
100
● ●
Age Age
p=0.077
r=-0.022 (CI: -0.2–0.16) r=0.088 (CI: -0.21–0.17) r=−0.022 (CI: −0.2−0.16) ● p=0.81
200
200
●
0
●
400
●
40
20
r=0.54 (CI: 0.4−0.67) p=3.5e−09
600
●
60
D
C Age vs. CD8 IFNg+CLA+/IL13+CLA+
r=0.55 (CI: 0.41–0.66) r=0.54 (CI: 0.4–0.67) ● p=5.9e-11 p=3.5e-09 r=0.55 (CI: 0.41−0.66) p=5.9e−11
CD8 IFNg+CLA+/IL13+CLA+ IFN-γ⁺CLA⁺/IL-13⁺CLA⁺
B Age vs. CD4 IFNg+CLA−/IL13+CLA−
r=0.2 (CI: 0.026–0.37) r=0.11 (CI: 0.01–0.38) p=0.025 p=0.028 r=0.2 (CI: 0.026−0.37) ● p=0.025
IL-13⁺CLA⁺ CD8 IL13+CLA+
IFN-γ⁺CLA⁺/IL-13⁺CLA⁺ CD4 IFNg+CLA+/IL13+CLA+
80
CD4 IFNg+CLA−/IL13+CLA− IFN-γ⁺CLA⁻/IL-13⁺CLA⁻
A Age vs. CD4 IFNg+CLA+/IL13+CLA+
CD8⁺ T cells IFN-γ⁺CLA⁻/IL-13⁺CLA⁻ CD8 IFNg+CLA−/IL13+CLA−
Figure 7
r=0.43 (CI: 0.27−0.56) r=0.3 (CI: 0.25−0.57) r=0.43 (CI: 0.27–0.56) r=0.3 (CI: 0.25-0.57) ● p=1.1e−06 p=0.0023 p=1.1e-06 p=0.0023
4
r=0.55 (CI: 0.41−0.66) r=0.55 (CI: 0.39−0.67) r=0.55 (CI: 0.41–0.66) r=0.55 (CI: 0.39-0.67) p=1.4e−10 ● p=2.6e−09 p=1.4e-10 p=0.2.6e-09
●
●
●
●
● ●
● ● ●
10
0
● ●
●
● ●
●
●
●
●
●
● ● ● ● ● ● ●● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●●● ● ● ● ●● ● ●● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ●●●● ● ● ● ● ●●●● ● ● ● ● ● ●● ●● ●● ● ● ●● ● ● ●●● ● ●● ● ● ● ●● ● ● ●● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ●●
0
20
40
Age Age
60
7.5
●
●
●
●
●
●
5.0
●● ● ●
● ●
0.0
●
●
●
●
● ● ●●●
● ●●● ●●●●● ● ● ●●● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ●● ● ●● ●● ●● ●● ● ● ●● ● ●● ● ●● ● ●● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ●●●●●●●●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ●● ● ● ●● ● ● ● ● ● ●
0
●
●
●
●
2.5
80
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20
●
●
●
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●
●
● ● ●●
●
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40
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20
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60
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80
0
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Age Age
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40
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●
20
●
●
40
Age Age
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60
●
● ● ●
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●
● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ●● ● ●● ●● ● ● ●● ● ● ● ● ●● ● ● ●● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●●●●●●●●●●● ● ● ● ● ●● ● ● ● ● ●● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ●● ● ●●● ● ● ● ● ● ● ● ● ●● ●●● ● ● ● ● ● ●
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0 80
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2
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3
1
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● ●
● ● ● ●●●●● ● ● ● ●● ●● ●●● ● ● ● ● ● ●● ● ● ●● ● ● ●●●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ●● ● ● ● ●● ● ●●● ●● ● ●●●● ● ● ●● ●● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ●● ● ● ●● ● ●● ● ● ● ●●● ● ●● ● ● ● ● ● ●●● ● ● ● ● ● ●● ●● ● ● ● ●● ● ● ● ● ●
0
CD8 IL22+CLA−
●
IL-22⁺CLA⁻
●
20
●
●
IL-22⁺CLA⁺ CD8 IL22+CLA+
CD4 IL22+CLA− IL-22⁺CLA⁻
IL-22⁺CLA⁺ CD4 IL22+CLA+
● ●
●
0
20
40
Age Age
60
80
Figure 8 cluster3
Figure E8. Healthy controls cluster3
AD patients
Normal patients ●
CD4+/CD8+ IFN-γ+ CD4+/CD8+ IL-22+ CD4+ IL-9+CLA+ Total Tregs Tcm HLA-DR+CLA+ Tem ICOS+CLA+ CD4+/CD8+ Tem/Tcm CLA+
10
CD4+/CD8+ IFN-γ+ CD8+ Tem HLA-DR+ CD8+ IL-13+CLA+ Th1/Th2 ratio CD8+ Tem cells CD4+ IL-9+CLA+ Total Tregs
5
●
labels ●
1
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2
●
3
●
Children and Adolescents PC2
cluster3
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Infants
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0
●
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●
cum.df$AgeGrp
●
● ● ●
●
Adults
●
labels ●
1
●
2
PC2
−5
0
CD4+/CD8+ IFN-γ+ CD4/+CD8+ IL-22+ Th1/Th2 ratio CD8+ Tem HLA-DR+ CD4+ Tem/Tcm CLA+ CD4+ Tem ICOS+CLA-
●
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●
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3
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●
●
● ● ● ● ●● ● ●
●
●
● ●
● ●
●
● ●
●
●
cum.df$AgeGrp ●
●
●
Adolescents Adults
●
Children ●
●
Infants
●
−5
● ●
● ●
● ●
●
● ●
0
●
●
●
Infants
●
●
Adolescents
Children
●
●
●
Adults
● 5
●
5
PC2
●
●
0
5
PC1
−5 10
−5
0
5
PC1
10
Table E1. Patient demographic and clinical severity scores Characteristic
Age
Parameter
Normal Infants (n=24)
Normal Children (n=27)
Normal Adolescents (n=28)
Normal Adults (n=30)
AD Infants (n=40)
AD Children (n=26)
AD Adolescents (n=23)
AD Adults (n=43)
Mean ± SD
2.45 ± 1.44 2.00 (0.564.93)
9.16 ± 2.12 9.01 (5.0511.87)
14.36 ± 1.69
1.93 ± 1.44
7.54 ± 1.94
15.06 ± 1.90
14.30 (12.1317.46)
39.27 ± 11.97 37 (1966)
1.56 (0.414.96)
7.47 (5.0811.51)
14.29 (12.1917.99)
43.30 ± 15.09 43 (1881)
Male
13
7
11
18
22
13
13
24
Female
11
20
17
12
18
13
10
19
White
18
18
16
24
19
9
16
34
Hispanic
3
4
10
1
3
6
2
0
Black
3
3
1
2
8
6
2
6
Median (range) Sex
Race
Asian
0
2
1
3
10
5
3
3
4.04 ± 4.05 2.15 (0.012.6)
2.25 ± 1.95 1.9 (0.710.0)
3.19 ± 2.38
NA
6.42 ± 3.44
6.71 ± 3.85
9.72 ± 5.90
NA
2.4 (0.5-8.9)
NA
6.4 (2.2-15)
5.6 (0.7-15.6)
8.0 (1.923.7)
NA
Mean ± SD
NA
NA
NA
NA
52.48 ± 13.15
58.43 ± 14.91
Median (range)
NA
NA
NA
NA
52.5 (20.883.6)
57.3 (25.489.0)
58.95 ± 21.78 53.5 (21.391.2)
EASI
Mean ± SD
NA
NA
NA
NA
14.04 ± 8.64
17.76 ± 10.21
64.90 ± 15.64 65 (32.597) NA
NA
NA
NA
NA
TEWL LS
Median (range) Mean ± SD
NA
NA
NA
NA
13.0 (2.8-38.6) 36.24 ± 31.34
16.6 (3.7-51.4) 47.13 ± 24.78
NA
NA
NA
NA
TEWL NL
Median (range) Mean ± SD
NA
NA
NA
NA
28.6 (8.3-176.8) 19.08 ± 15.27
42.7 (16.6-103.4) 19.02 ± 11.00
NA
NA
NA
NA
14.5 (2.9-76.7)
16.5 (5.9-49.2)
Eosinophils (%)
Mean ± SD
Median (range) Disease Severity Scores SCORAD
Median (range)
27.93 ± 20.56 18.0 (4.6-68.4) 58.30 ± 29.68 57.6 (13.5-96.6) 22.07 ± 13.66 18.8 (5.4-53.2)
NA NA NA NA NA
P-Value b/w Normal and AD Infants 0.1656
P-Value b/w Normal and AD Children 0.005724
P-Value b/w Normal and AD Adolescents 0.1699
P-Value b/w Normal and AD Adults 0.2263
1
0.1275
0.3446
0.9082
0.03131
0.1289
0.1007
0.4775
0.09849
1.03E-05
4.70E-05
Pruritus ADQ
Mean ± SD
NA
NA
NA
NA
16.95 ± 7.51
21.96 ± 8.11
20.82 ± 8.73
NA
NA
NA
NA
NA
16.0 (1-32)
20.5 (7-35)
22.0 (5-35)
NA
Total ADQ
Median (range) Mean ± SD
NA
NA
NA
NA
33.65 ± 13.62
43.38 ± 13.36
NA
Median (range) Mean ± SD
NA
NA
NA
NA
34.0 (5-61)
46.5 (18-70)
42.75 ± 13.67 44.4 (19-65)
NA
NA
NA
NA
1.61 ± 1.38
6.33 ± 2.45
14.23 ± 2.45
NA
NA
NA
NA
NA
1.2 (0.2-4.7)
5.1 (2.0-10.5)
NA
0
1
5
0
18
16
13.7 (9.617.7) 13
0
0
0
0
1
3
3
2
0
0
0
0
1
6
4
10
Disease Duration
Median (range) History of other atopic disorder (allergic rhinitis,
NA
7
asthma, allergic conjunctivitis, environmental)
Food Allergies (FA) FA + other atopic disorder
AD: Atopic dermatitis; SCORAD: Scoring of Atopic Dermatitis; SD: Standard deviation; NA: not available; EASI: Eczema Area and Severity Index; TEWL LS: Transepidermal Water Loss Lesional Skin; TEWL NL: Transepidermal Water Loss Non-Lesional Skin; Pruritus ADQ: Pruritus Atopic Dermatitis Quickscore; Total ADQ: Total Atopic Dermatitis Quickscore
Table E2A: Sensitivity analysis with race-matched infants CD4+IL13+CLA+
N Infant (mean) 2.575
AD Infant (mean) 8.551
N Infant (median) 2.040
AD Infant (median) 6.094
P-value (means)Infant AD vs. N 0.00284
P-value (median)Infant AD vs. N 0.00174
CD4+EM+CLA+
26.698
40.595
25.365
38.290
0.00296
0.00398
CD4+Tem ICOS+CLA-
8.604
18.119
7.009
15.642
0.00413
0.000722
CD4+CM+CLA+
13.4755
21.267
12.420
20.200
0.0163
0.0177
CD4+IFNγ+CLA-/IL13+CLA-
15.278
7.346
10.116
4.707
0.0187
0.03243
CD4+Tcm ICOS+CLA-
3.909
6.841
3.151
4.548
0.0222
0.0348
CD4+IFNγ+CLA+/IL13+CLA+
9.386
1.518
6.051
1.156
0.0239
0.000335
CD4+Tem HLADR+CLA+
10.207
5.309
8.512
4.078
0.0303
0.0225
CD4+Tcm HLADR+CLA+
11.102
4.954
6.438
4.326
0.0638
0.0868
CD4+IFNγ+CLA+
11.945
8.274
10.730
8.046
0.0952
0.146
Table E2B: Sensitivity analysis with age-matched children CD4+IFNγ+CLA-/IL13+CLA-
N Child (mean) 32.881
AD Child (mean) 9.934
N Child (median) 35.858
AD Child (median) 6.515
P-value (mean)Child AD vs. N 1.11E-05
P-value (median)Child AD vs. N 1.51E-05
Tregs (CCR4+CLA+)
7.598
21.365
5.030
18.20
0.00232
0.00113
CD8+Tem ICOS+CLA+
11.481
28.929
4.974
23.208
0.00572
0.00850
CD4+IL13+CLA-
0.647
2.925
0.672
2.105
0.00698
0.0105
CD8+Tem CLA+
4.826
9.588
5.066
7.690
0.00753
0.0163
Tregs (CD25+CD127-)
6.656
11.630
6.470
9.530
0.0121
0.0368
CD8+Tcm ICOS+CLA-
3.285
7.970
2.958
7.313
0.0144
0.0236
CD8+Tcm CLA+
5.812
11.146
4.025
8.480
0.0182
0.0169
CD4+Tcm HLADR+CLA+
9.344
17.661
7.158
16.245
0.0263
0.0458
CD4+IL22+CLA-
0.775
2.395
0.729
1.682
0.0353
0.0253
Table E2C: Sensitivity analysis using only adolescent controls with no atopic history CD4+IL22+CLA+
N Adol (mean) 4.137
AD Adol (mean) 8.021
N Adol (median) 4.441
AD Adol (median) 6.400
P-value (mean)Adol AD vs. N 0.00518
P-value (median)Adol AD vs. N 0.00203
CD8+Tem ICOS+CLA+
12.689
29.184
10.301
26.667
0.00580
0.0137
CD4+IFNγ+CLA+
20.417
13.482
18.937
12.608
0.0101
0.0136
CD4+IL13+CLA-
1.065
1.660
0.938
1.403
0.0424
0.0504
CD8+Tcm HLADR+CLA+
7.686
23.516
4.006
19.950
0.0428
0.0677
CD4+Tem ICOS+CLA+
19.775
31.052
18.330
29.024
0.0502
0.103
CD4+IFNγ+CLA+/IL13+CLA+
5.738
3.104
3.663
2.616
0.0634
0.0173
CD8+IFNγ+CLA-
42.573
32.263
42.495
33.569
0.0687
0.0736
CD4+Tem ICOS+CLA-
9.488
14.020
8.163
12.741
0.0688
0.138
CD4+Tem HLADR+CLA+
6.108
12.230
6.259
6.503
0.0978
0.557
Sensitivity analyses for demographic parameters that were significantly different between AD and N within an age-group, with means, medians, and respective Pvalues for the top 10 most significant comparisons. These analyses were done for race-matched infants (A), age-matched children (B), and adolescent AD versus adolescent controls without atopic history (C). Abbreviations AD: Atopic dermatitis; Adol: Adolescent; Child: Children; CLA: Cutaneous lymphocyte antigen; HLADR: Human leukocyte antigen-DR; ICOS: Inducible co-stimulator; IFNγ: Interferon-gamma; IL: Interleukin; N: Normal; Tcm: Central memory T; Tem: Effector memory T; Treg: Regulatory T
Table E3A. Mean values for normal and atopic dermatitis infants, children, adolescents, and adult patients and respective p-values P- Values
CD4+IFNγ+ CD4+CLA+ CD4+IFNγ+CLA+ CD4+IFNγ+CLACD4+IL13+
N Infants
AD Infants
N Child
AD Child
N Adol
AD Adol
N Adults
AD Adults
Infants AD vs. N Infants
7.648
7.365
20.242
13.973
22.037
18.974
25.870
24.200
0.871
2.383 11.945 7.517 1.168
3.726 7.769 7.380 1.411
2.066 19.511 20.273 0.757
3.398 13.690 14.006 3.067
2.035 18.326 22.132 1.024
1.610 13.734 19.072 1.804
2.934 19.588 26.068 0.831
5.053 17.228 24.780
0.044 0.047 0.938
Child AD vs. N Child
Adol AD vs. N Adol
Adults AD vs. N Adults
N Infants vs. N Child
N Infants vs. N Adol
N Infants vs. N Adults
N Child vs. N Adol
N Child vs. N Adults
N Adol vs. N Adults
AD Infants vs. AD Child
AD Infants vs. AD Adol
AD Infants vs. AD Adults
AD Child vs. AD Adol
AD Child vs. AD Adults
0.040
0.371
0.501
8.17E06
2.51E05
1.12E12
0.604
0.037
0.223
0.010
3.30E05
4.59E10
0.093
0.001
0.071
0.076
1.58E04
0.107 0.058 0.040 0.004
0.469 0.075
0.009
0.641
0.010
0.010
0.378
0.765
0.478
0.164
0.666
0.367
0.036
0.215
0.130
0.106
0.142
0.750
0.009
0.010
0.033
0.481
0.203
0.479
0.858
0.659
0.409
0.780
0.300
0.215
0.341
0.692
0.365
0.141
0.920
0.211
0.022
0.026
0.375
0.139
0.041
0.132
1.916
0.625
0.173
0.109
6.99E06
0.01
0.007
0.006
0.084
0.010
0.841
0.255
0.311
0.276
0.197
0.021
0.244
0.095
0.056
0.501
0.250
0.564
0.010
0.001
0.689
0.007
0.006
0.456
0.155
0.012
0.249
0.015
0.056
0.104
0.071
0.044
0.160
0.006
0.121
0.061
0.685
0.437
0.085
0.047
0.678
2.850
0.934
1.719
0.693
1.283
0.922
9.386
1.572
8.546
2.457
5.470
3.206
14.262
4.088
0.025
0.032
0.141
0.137
1.48E04
0.474
0.036
0.591
CD4+IFNγ+CLAIL13+CLA-
15.278
8.853
34.346
10.760
24.806
12.470
39.339
26.164
0.053
1.94E06
CD4+IL9+
0.491
0.514
0.406
0.881
0.906
1.194
0.250
0.464
0.877
0.054
CD4+IL9+CLA+
3.824
3.256
6.701
7.052
9.708
10.259
2.162
2.332
0.676
0.862
0.845
0.788
0.154
CD4+IL9+CLA-
0.399
0.409
0.333
0.766
0.743
1.051
0.214
0.295
0.930
0.049
0.372
0.188
0.615
CD4+IL22+CLA-
0.467
0.399
0.870
2.063
2.313
7.910 2.595
4.746 2.239
0.051
0.217
1.077
3.627
0.090
0.034
1.124
6.750
0.099
0.594
CD4+IL13+CLA-
4.139
0.236 3.96E04
0.609
CD4+IFNγ+CLA+ IL13+CLA+
5.261
0.987
2.93E10
4.95E04
11.055
4.552
0.030
0.376
4.548
CD4+IL22+CLA+
0.028
3.37E05
8.483
2.314
0.127 7.63E06
0.001
5.338
2.654
0.005 3.94E04
8.77E13
9.114
2.340
0.608
0.679
0.029
6.428
2.169
0.970
0.057
2.30E05
9.492
0.935
0.675
0.189
0.004
2.575
0.594
0.960
6.83E06
CD4+IL13+CLA+
0.593
0.311
0.242
9.20E05
CD4+IL22+
0.488
AD Adol vs. AD Adults
2.675 9.663 2.943
0.995 0.595 0.562
0.031 0.073 0.037
0.024
0.217
0.271
0.118
0.073
8.11E05
1.28E07
0.594
0.437
0.049
0.002
3.78E05
0.753
0.452
0.874
0.006
3.72E05
3.28E08
0.635
0.085
0.010
0.002
0.010
0.064
0.243
0.013
0.087
0.001
6.13E06
0.952
0.008
0.510
0.433
0.068
0.304
0.001
5.10E06
0.005
0.865
0.182
0.147
0.001
2.53E04
0.037
0.172
0.405
0.024
0.015
2.57E04
5.45E06
0.491
0.445
0.972
0.052
0.001
0.496
0.077
0.253
1.49E04
1.08E09
0.453
0.164
0.544
7.54E07
0.719
0.145
0.254
0.001
CD4+IL17+
0.510
0.377
0.805
0.973
0.883
1.087
1.120
1.403
0.270
0.529
0.452
0.241
0.083
0.067
0.003
0.712
0.137
0.317
0.020
0.004
CD4+IL17+CLA+
2.906
2.062
3.012
2.166
2.633
2.608
2.605
2.953
0.083
0.265
0.964
0.441
0.892
0.657
0.585
0.632
0.585
0.960
0.804
0.193
0.013
0.392
0.096
0.455
0.002
1.11E06
0.693
0.192
0.342
0.720
0.001
0.002
CD4+IL17+CLACD8+IFNγ+
0.442 18.328
0.320 20.179
0.765 38.883
0.946 28.485
0.846 37.309
1.072 30.301
1.076 44.737
1.336 46.063
0.261 0.647
0.504 0.052
0.401 0.217
0.284
0.048
0.038
0.002
0.782
6.51E05
0.001
2.00E07
0.693
0.139
0.323
0.017
0.785
0.245
0.186
0.081
0.029
1.37E07
3.39E01
0.415
N/A
0.758
N/A
N/A
0.113
0.165
N/A
0.965
N/A
N/A
0.631
0.174
0.062
0.933
0.233
0.152
0.259
0.124
0.008
6.71E08
0.684
0.001
0.002
0.822
0.060
0.031
0.008
3.78E04
CD8+CLA+
2.744
3.770
1.684
1.939
1.924
1.979
N/A
N/A
0.452
0.752
0.950
N/A
CD8+IFNγ+CLA+
21.484
24.946
27.724
24.546
25.570
19.225
33.830
31.589
0.441
0.494
0.166
0.605
0.156
0.337
0.005
0.696
4.44E05
0.001
1.51E07
0.781
0.245
0.187
0.073
0.023
0.948
0.290
0.009
0.207
0.001
0.179
0.549
0.675
0.028
0.765
3.97E04
CD8+IFNγ+CLA-
18.014
19.931
39.176
28.670
37.558
30.772
45.066
46.953
0.642
0.051
0.240
CD8+IL13+
0.225
0.376
0.219
0.528
0.345
0.480
0.485
0.920
0.488
0.067
0.425
0.005
0.839
7.15E05
CD8+IL13+CLA+
2.936
3.544
2.091
4.429
2.989
3.223
3.094
8.822
0.627
0.041
0.452
0.968
0.892
0.310
0.111
0.911
0.475
0.364
CD8+IL13+CLA-
0.114
0.101
0.179
0.467
0.287
0.461
0.419
0.630
0.694
0.046
0.291
0.028
0.151
0.032
4.73E06
0.175
2.34E04
0.139
0.012
0.023
6.47E08
0.975
0.297
0.312
CD8+IFNγ+CLA+ IL13+CLA+
13.535
10.690
19.937
7.252
10.347
10.440
18.629
8.298
0.449
0.766
0.488
0.010
0.718
0.500
0.630
0.579
0.825
0.666
0.865
0.479
0.219
0.549
0.400
0.610
CD8+IFNγ+CLAIL13+CLA-
44.484
31.193
54.077
45.658
61.714
47.565
73.607
49.528
0.559
0.038
0.227
0.097
0.183
0.817
0.181
0.107
0.011
0.184
0.739
0.254
0.020
0.577
0.168
0.186
CD8+IL9+
0.732
1.020
0.626
0.624
0.895
0.849
0.363
0.528
0.324
0.996
0.906
0.075
0.743
0.669
0.069
0.524
0.334
0.120
0.183
0.573
0.041
0.433
0.650
0.159
0.092
0.004
0.002
CD8+IL9+CLA+
13.578
16.237
6.775
8.695
8.720
14.186
3.847
3.881
0.580
0.430
0.141
0.972
0.133
0.296
CD8+IL9+CLA-
0.297
0.414
0.410
0.426
0.800
0.673
0.289
0.355
0.413
0.945
0.730
0.225
0.580
CD8+IL22+ CD8+IL22+CLA+
0.160 1.434
0.341 3.598
0.206 1.564
0.395 4.147
0.670 2.375
0.593 4.692
0.649 2.398
0.753 8.611
0.096 0.190
0.152 0.023
0.170
0.053
0.015
0.599
5.47E05
0.306
0.526
0.133
0.947
0.209
0.635
0.248
0.610
0.078
0.006
3.25E05
0.901
0.725
0.283
0.002
0.416
0.017
0.481
0.024
0.522
0.147
0.936 3.75E06
0.764
0.372
0.472
0.002
0.066
4.16E05
0.824
0.214
0.112
0.227
0.088
0.972
0.768
0.565
0.017
0.714
0.010
0.025
0.004
2.45E05
0.916
0.400
0.136
0.001
0.321
0.017
0.686
0.295
0.491
0.038
0.531
CD8+IL22+CLA-
0.120
0.224
0.186
0.329
0.620
0.545
0.604
0.630
0.244
0.172
0.749
0.814
0.213
0.001
1.18E06
CD8+IL17+
0.237
0.294
0.219
0.229
2.883
0.325
0.422
0.410
0.636
0.940
0.326
0.884
0.893
0.309
0.063
0.307
0.128
0.344
0.581
0.839
CD8+IL17+CLA+
2.382
1.552
1.579
0.974
4.694
1.023
1.383
2.094
0.301
0.353
0.046
0.058
0.376
0.223
0.180
0.096
0.737
0.067
0.233
0.274
0.270
0.914
0.019
0.024
0.510
0.047
0.472
CD8+IL17+CLATregs (CD25+CD127-)
0.180 6.403
0.137 5.697
0.146 7.037
0.222 12.212
2.925 11.527
0.315 11.642
0.384 5.177
Tregs (CCR4+CLA+)
8.118
11.242
8.272
23.786
9.468
13.293
13.998
CD4+CM+CLA+
13.475
20.412
11.350
18.839
11.218
11.750
12.870
0.416
0.481
0.392
0.336
0.729
0.650
0.005
0.333
0.105
0.004
0.456
12.824
0.007
0.011
0.863
0.979
0.309
0.366
0.749
0.957
0.410
0.472
0.615
0.007
0.002
0.041
0.033
0.691
0.608
0.330
0.843
0.317
0.013
0.012
0.026
0.907
0.586
0.641
0.022
0.025
3.66E04
0.365
0.462
0.322
0.439
0.322
0.945
0.077
0.140
0.954
0.045
0.357
0.097
0.363
0.021
0.260
1.57E06
0.014
0.011
0.496
0.741
0.003
0.003
0.338
0.024
0.020
0.167
0.281
0.556
0.701
3.57E04
0.002
0.357
0.087
0.352
0.518
0.103
0.013 0.392
5.538
7.478
5.662
4.117
8.248
10.135
0.682
0.350
3.738
0.162
0.033
7.607
11.385
0.018
0.006
6.932
9.197
0.010
0.595
0.318
9.625
0.003
0.014
0.030
8.496
0.002
0.592
0.002
7.457
0.770
0.950
23.511
3.909
0.007
0.083
20.343
8.953
0.033
0.158
20.683
23.955
0.004
0.230
19.273
18.971
0.165 1.96E04
17.906
27.768
18.018
0.266 1.75E04
0.950
18.972
17.700
0.349 5.88E05
0.003
38.082
14.789
0.005
0.306
4.88E04
26.698
10.221
0.019
0.573
7.197
CD4+Effector cells CLA+
CD4+Tcm ICOS+CLA-
0.312
4.43E04
CD4+EM+CLA+
CD4+Tcm ICOS+CLA+
0.656
1.50E04
21.656 6.174
0.072 0.013
CD4+Tem ICOS+CLA+
18.372
27.272
19.461
18.874
22.129
31.052
18.027
35.898
0.052
CD4+Tem ICOS+CLA-
8.604
17.477
11.149
9.824
12.679
14.020
8.851
12.783
0.003
0.931 0.618
0.360
0.003
0.860
0.111
1.91E06
0.001
0.002
0.835
0.779
0.356
0.924
0.438
0.622
0.194
0.040
0.516
0.048
0.031
2.65E05
0.516
0.617
0.062
0.139
0.052
0.886
0.489
0.234
0.091
0.016
0.305
0.133
0.105
0.179
0.630
0.451
0.361
0.015
0.800
0.003
0.009
0.001
0.030
0.026
0.739
0.015
0.156
CD4+Tcm HLADR+CLA+
11.102
5.452
8.450
15.817
7.852
14.303
2.862
8.602
0.088
0.020
0.125
3.52E05
CD4+Tcm HLADR+CLA-
6.051
3.063
2.844
7.171
2.753
4.730
1.181
2.502
0.305
0.066
0.247
0.058
0.251
0.237
0.084
0.907
0.015
0.018
0.099
0.383
0.642
0.370
0.049
0.198
CD4+Tem HLADR+CLA+
10.207
6.294
8.139
10.876
6.277
12.230
5.419
11.540
0.072
0.455
0.103
0.001
0.517
0.066
0.023
0.500
0.319
0.467
0.107
0.106
0.005
0.751
0.824
0.853
CD4+Tem HLADR+CLA-
4.400
3.133
2.537
4.169
2.607
4.494
2.019
1.980
0.435
0.122
0.241
0.949
0.206
0.236
0.087
0.951
0.576
0.554
0.411
0.426
0.288
0.832
0.008
0.082
0.557
9.13E05
3.78E04
0.657
0.037
0.670
0.115
0.943
0.072
0.019
0.053
0.008
0.195
0.387
0.392
0.069
0.831
0.078
0.481
0.001
0.602
0.125
0.185
CD8+Tcm CLA+
12.795
11.574
5.653
10.673
6.408
7.069
12.344
10.814
0.654
0.008
0.724
0.386
0.009
0.017
0.868
CD8+Tem CLA+
15.463
13.867
4.904
9.409
7.466
8.036
11.186
10.925
0.623
0.003
0.683
0.877
0.001
0.009
0.147
0.040
2.13E05
CD8+Effector cells CLA+
9.152
7.482
4.253
4.797
5.333
5.898
4.992
3.278
0.469
0.635
0.812
0.035
0.025
0.121
0.055
0.497
0.461
CD8+Tcm ICOS+CLA+
10.895
8.773
13.133
22.439
18.967
20.423
12.525
18.407
0.613
0.093
0.776
0.020
0.669
0.115
0.689
0.222
0.869
0.068
0.005
0.012
0.001
0.727
0.396
0.659
CD8+Tcm ICOS+CLA-
3.927
4.334
3.127
7.089
5.013
7.632
4.366
4.476
0.737
0.013
0.114
0.899
0.427
0.421
0.655
0.102
0.058
0.560
0.100
0.036
0.900
0.776
0.110
0.039
CD8+Tem ICOS+CLA+
17.315
15.412
14.538
26.308
16.905
29.184
13.409
26.175
0.711
0.033
0.039
1.55E04
0.626
0.943
0.431
0.618
0.759
0.360
0.028
0.012
0.003
0.649
0.979
0.577
CD8+Tem ICOS+CLA-
5.846
3.734
2.508
4.036
2.920
3.632
3.248
3.950
0.146
0.140
0.414
0.298
0.019
0.041
0.066
0.553
0.266
0.650
0.788
0.915
0.785
0.725
0.933
0.701
0.034
5.64E08
0.055
1.09E04
0.128
0.052
0.574
0.046
1.94E04
1.18E06
0.061
0.245
3.50E08
CD8+Tcm HLADR+CLA+ CD8+Tcm HLADR+CLACD8+Tem HLADR+CLA+ CD8+Tem HLADR+CLACD4+Naïve cells CD4+Tcm
9.883 4.954 7.410 2.979 76.014 11.930
9.115 3.978 8.462 3.216 77.562 11.100
10.513 4.440 8.486 4.474 66.520 18.075
11.498 4.381 13.154 2.306 61.472 19.438
7.252 3.830 7.839 3.474 52.718 24.907
18.053 7.602 13.029 3.904 68.480 17.675
6.880 5.921 5.729 4.577 60.613 26.410
19.539 11.174 18.233 7.613 59.677 21.455
0.786 0.508 0.691 0.819 0.666 0.715
0.823 0.958 0.304 0.055 0.255 0.601
0.780 0.003 0.059
0.004 0.803 0.068
0.849 0.726 0.738
0.388 0.471 0.889
0.212 0.539 0.423
0.225
0.615
0.141
0.032
5.47E05
2.59E04
0.001
1.60E06
0.016
0.331 0.602 0.853 0.363 0.012 0.060
0.194 0.217 0.307 0.930 0.148 0.002
0.877 0.112 0.402
0.558 0.406 0.262
0.235
0.310
0.115
7.36E05
0.683
0.001
0.988
0.344
0.080
0.275
2.06E04
0.981
0.223
0.213
0.663
3.26E05
0.309
6.73E07
0.029
0.007
1.81E06
0.085
0.665
0.022
0.012
2.39E05
0.540
0.455
0.176
0.051
0.546
0.048
0.372
0.574
0.264
0.041
0.003
3.26E07 4.34E05
CD4+Tem
6.514
5.500
9.947
12.796
15.585
8.660
9.414
11.536
0.457
0.253
0.011
0.163
0.101
0.002
0.077
0.057
0.793
0.023
0.001
0.012
6.77E06
CD4+Effector cells
5.041
5.854
4.729
6.299
5.947
5.202
3.623
7.360
0.392
0.184
0.531
0.075
0.772
0.381
0.304
0.286
0.447
0.105
0.672
0.560
0.41
CD8+Naïve cells
51.973
64.082
60.900
64.812
55.577
73.750
56.350
51.734
0.031
0.394
0.001
0.189
0.106
0.568
0.386
0.338
0.257
0.880
0.874
0.025
0.003
CD8+Tcm
25.817
15.653
16.197
10.792
17.599
9.835
19.878
18.906
0.026
0.010
0.006
0.626
0.024
0.068
0.146
0.612
0.078
0.391
0.070
0.035
0.237
0.616
1.04E04
CD8+Tem
11.849
8.531
10.927
9.493
15.175
6.317
13.680
13.604
0.088
0.457
0.002
0.965
0.646
0.245
0.352
0.123
0.122
0.577
0.599
0.194
0.003
0.101
0.034
1.36E04
CD8+Effector cells
10.340
11.280
11.979
14.896
11.626
10.095
10.036
15.749
0.637
0.141
0.329
0.008
0.325
0.475
0.863
0.825
0.214
0.352
0.111
0.507
0.056
0.016
0.724
0.006
Table E3B. Median values for normal and atopic dermatitis infants, children, adolescents, and adult patients and respective p-values
P- Values
CD4+IFNγ+ CD4+CLA+
N Infants
AD Infants
N Child
AD Child
N Adol
AD Adol
N Adults
AD Adults
Infants AD vs. N Infants
5.663
3.992
17.861
12.174
21.903
17.790
26.391
23.214
0.388
1.581
2.927
1.500
2.190
1.484
0.718
2.302
3.870
0.078
Child AD vs. N Child
Adol AD vs. N Adol
Adults AD vs. N Adults
N Infants vs. N Child
N Infants vs. N Adol
N Infants vs. N Adults
N Child vs. N Adol
N Child vs. N Adults
N Adol vs. N Adults
AD Infants vs. AD Child
AD Infants vs. AD Adol
AD Infants vs. AD Adults
AD Child vs. AD Adol
AD Child vs. AD Adults
0.052
0.302
0.364
6.40E06
6.41E05
2.57E10
0.500
0.029
0.103
0.003
3.38E06
9.57E11
0.088
0.001
0.097
0.628
1.66E04
0.127
4.12E06
0.022
0.085
0.042
0.283
0.570
0.265
0.528
0.013
0.086
0.214
0.006
AD Adol vs. AD Adults
CD4+IFNγ+CLA+
10.730
6.255
19.172
12.946
18.538
12.720
19.349
15.540
0.042
0.011
0.113
0.136
0.002
0.026
8.30E05
0.500
0.891
0.448
0.017
2.09E04
2.63E05
0.461
0.154
0.433
CD4+IFNγ+CLA-
5.645
3.714
18.088
12.198
21.919
17.845
26.398
23.216
0.379
0.054
0.324
0.400
4.28E06
4.58E05
2.05E10
0.538
0.028
0.096
0.003
3.75E06
5.58E11
0.093
4.73E04
0.066
CD4+IL13+
0.521
1.086
0.779
1.803
0.847
1.582
0.529
1.369
0.123
0.007
0.009
0.001
0.810
0.381
1.000
0.365
0.769
0.342
0.097
0.127
0.241
0.767
0.443
0.634
CD4+IL13+CLA+
2.040
6.758
4.588
6.114
4.115
6.701
3.455
9.400
0.001
0.123
0.108
2.39E05
0.022
0.017
0.185
0.893
0.360
0.551
0.749
0.939
0.148
0.786
0.293
0.090
CD4+IL13+CLA-
0.464
0.891
0.673
1.459
0.777
1.504
0.437
1.023
0.289
0.013
0.005
0.009
0.810
0.418
0.769
0.345
0.497
0.180
0.058
0.014
0.399
1.000
0.161
0.060
1.497
5.73E05
4.08E04
0.056
6.41E06
0.901
0.433
0.467
0.644
0.263
0.107
0.039
0.007
0.043
0.415
0.994
0.350
0.001
0.001
3.09E04
0.006
1.58E06
0.213
0.006
0.008
2.40E07
0.260
8.06E05
0.006
0.564
0.363
0.393
0.115
0.185
0.020
0.115
0.561
0.022
0.002 3.73E07
CD4+IFNγ+CLA+ IL13+CLA+
6.051
0.843
4.175
1.304
3.389
2.624
5.464
CD4+IFNγ+CLAIL13+CLA-
10.116
4.970
35.306
7.101
22.904
11.877
34.706
24.109
0.043
1.37E06
CD4+IL9+
0.267
0.351
0.214
0.523
0.481
0.507
0.208
0.211
0.435
0.046
0.001
0.129
0.518
0.001
0.214
0.071
0.005
1.23E05
0.140
0.228
1.70E04
CD4+IL9+CLA+
1.961
1.865
2.652
5.394
5.230
8.935
1.101
1.656
0.652
0.346
0.315
0.485
0.118
0.008
0.367
0.247
0.003
6.44E05
CD4+IL9+CLA-
0.253
0.296
0.164
0.394
0.410
0.472
0.167
0.147
0.582
0.039
0.291
0.597
0.314
0.211
0.090
0.018
0.609
0.002
0.226
0.022
0.055
0.401
0.011
1.66E04
CD4+IL22+
0.435
0.444
0.807
1.512
2.260
2.116
1.840
1.989
0.980
0.085
0.812
0.375
0.012
7.99E05
6.31E09
0.002
4.71E06
0.847
4.39E04
5.27E10
4.14E08
0.165
0.378
0.351
0.035
2.46E04
8.19E05
0.662
0.651
0.062
0.939
0.145
0.089
0.304
0.054
0.001
0.345
0.043
0.383
0.003
1.57E05
4.00E10
0.002
1.53E06
0.913
1.26E04
5.31E12
3.73E14
0.165
0.034
0.477
0.007
0.939
0.286
0.317
0.011
8.70E05
4.30E08
0.282
0.045
0.312
0.537
0.869
0.894
0.819
0.889
0.160
0.023
0.211
0.042
0.701
0.005
2.16E05
2.25E09
0.271
0.055
0.593
0.670
0.003
0.016
CD4+IL22+CLA+ CD4+IL22+CLA-
3.442 0.377
5.174 0.263
3.170 0.763
5.982 1.450
3.606 2.218
6.283 2.099
4.395 1.727
8.241 2.307
0.137 0.362
0.141
0.919
0.281
CD4+IL17+
0.385
0.271
0.651
0.582
0.780
0.762
0.996
0.936
0.426
0.844
0.434
0.349
0.049
8.96E02
CD4+IL17+CLA+
2.638
1.825
2.091
1.865
2.235
2.711
2.141
2.628
0.140
0.351
0.591
0.230
0.675
0.440
CD4+IL17+CLACD8+IFNγ+
0.355 15.667
0.220 16.053
0.605 45.807
0.512 26.526
0.731 40.483
0.751 29.900
0.884 46.182
0.811 41.604
0.260 0.967
0.776 0.060
0.462 0.251
0.318
0.021
0.037
0.008
0.907
1.36E04
0.003
1.72E06
0.773
0.425
0.229
0.060
0.025
4.76E07
1.13E01
0.406
N/A
0.271
N/A
N/A
0.034
0.002
N/A
0.560
N/A
N/A
0.434
0.012
0.764
0.154
0.052
0.640
0.514
0.116
0.181
0.161
0.006
0.002
1.34E06
0.021
2.00E07
0.633
0.002
0.010
0.303
4.63E04
0.034
6.71E12
0.928
0.001
0.001
CD8+CLA+
0.855
2.059
0.719
0.927
1.417
0.753
N/A
N/A
0.097
0.166
0.833
N/A
CD8+IFNγ+CLA+
23.358
19.786
25.432
19.330
21.156
13.050
34.618
26.705
0.791
0.526
0.081
0.506
0.229
0.960
7.16E05
CD8+IFNγ+CLACD8+IL13+ CD8+IL13+CLA+ CD8+IL13+CLA-
15.342 0.103 1.310 0.060
16.881 0.141 2.247 0.078
45.703 0.140 1.438 0.122
26.485 0.208 3.121 0.184
40.481 0.183 1.638 0.158
31.094 0.270 2.421 0.264
46.282 0.467 2.460 0.419
44.073 0.692 6.702 0.536
0.915 0.226 0.237
0.060 0.044 0.164
0.261 0.261
0.007
0.449
4.44E05
0.431 0.843
0.923
0.334
0.818
0.415
0.525
4.67E04
0.351
0.224 0.030
0.056 0.042
0.774
0.123
0.712
0.114
0.134
0.655
0.824
1.16E04
0.583
0.010
3.90E04
0.577
3.51E04
0.023
2.14E04
4.82E05
2.48E15
0.867
0.025
0.024
0.169
0.096
0.070
0.074
4.46E06
0.037
0.982
9.33E05
0.363
0.395
0.129
0.055
0.729
0.007
0.786
0.700
0.802
1.000
0.954
0.924
0.512
0.021
0.224
0.113
0.132
0.804
0.685
0.147
0.017
0.240
0.745
0.743
0.212
0.908
0.607
0.474
0.388
0.054
0.777
0.419
0.085
0.431
0.682
0.003
0.054
0.115
0.312
0.953
0.202
0.271
0.994
0.357
0.173
0.002
2.10E05
0.181
0.492
0.252
0.426
CD8+IFNγ+CLA+ IL13+CLA+
7.190
5.842
13.043
4.218
4.559
3.723
12.751
4.242
0.465
CD8+IFNγ+CLAIL13+CLA-
38.959
29.056
56.026
41.155
59.395
45.281
77.830
48.033
CD8+IL9+
0.297
0.451
0.111
0.306
0.471
0.534
0.360
0.369
0.057
CD8+IL9+CLA+
7.202
9.416
2.744
7.499
5.078
12.186
2.299
1.883
0.233
0.118
0.036
0.578
0.074
0.346
0.004
0.347
0.664
0.052
0.185
0.859
4.98E06
CD8+IL9+CLA-
0.216
0.181
0.091
0.297
0.361
0.394
0.291
0.278
0.640
0.129
0.743
0.467
0.245
0.094
0.282
0.002
0.028
0.240
0.928
0.150
0.301
CD8+IL22+
0.100
0.192
0.128
0.184
0.357
0.332
0.580
0.668
0.123
0.067
0.937
0.406
0.444
0.005
8.05E07
0.013
6.32E06
0.360
0.370
0.005
1.77E07
0.104
1.83E04
0.015
CD8+IL22+CLA+
0.674
1.438
0.835
2.976
0.982
2.916
1.732
5.576
0.061
0.039
0.039
1.59E05
0.459
0.216
0.003
0.425
0.043
0.261
0.227
0.029
4.70E06
0.548
0.026
0.067
CD8+IL22+CLA-
0.068
0.140
0.114
0.160
0.345
0.318
0.536
0.457
0.224
0.166
0.830
0.905
0.219
0.001
1.35E07
0.013
7.66E06
0.486
0.162
2.79E04
1.44E08
0.093
0.001
0.085
CD8+IL17+
0.107
0.089
0.081
0.114
0.264
0.083
0.297
0.301
0.987
0.081
0.162
0.914
0.431
0.054
0.003
0.006
1.34E05
0.455
0.475
0.899
5.81E05
0.651
0.001
0.003
CD8+IL17+CLA+
0.799
0.674
0.538
0.364
1.661
0.606
1.251
1.204
0.508
0.481
0.033
0.962
0.369
0.290
0.454
0.057
0.099
0.522
0.233
0.786
0.093
0.431
0.009
0.041
0.006
1.02E05
0.351
0.274
0.355
8.64E07
0.887
0.001
0.003
7.92E05
1.13E05
0.004
0.918
0.010
0.002
0.008
0.077
0.476
0.117
CD8+IL17+CLATregs (CD25+CD127-)
0.103 5.150
0.078 5.420
0.072 6.630
0.084 9.530
0.211 10.900
0.079 11.200
0.272 5.260
Tregs (CCR4+CLA+)
5.820
7.630
6.230
18.200
7.925
11.900
11.850
CD4+CM+CLA+
12.420
20.090
9.514
16.810
10.289
8.830
12.297
0.245
0.967
0.166
0.191
0.135
15.300
0.156
0.094
0.144
0.912
0.193
0.001
0.367
0.002
0.002
0.004
0.287
9.650
0.007
0.023
0.990
0.779
0.325
0.103
0.876
0.759
0.256
0.181
0.479
0.001
0.002
0.019
0.042
0.342
0.335
0.283
0.910
0.850
0.031
0.003
0.038
0.776
0.621
0.666
0.018
0.002
1.78E04
0.647
0.318
0.116
0.102
0.629
0.974
0.033
0.021
0.928
0.124
0.466
0.188
0.368
0.030
0.261
1.68E07
0.037
0.033
0.392
0.775
0.005
0.013
0.248
0.026
0.003
0.145
0.164
0.440
0.992
1.95E04
0.001
0.268
0.018
0.637
0.258
0.283
0.015 0.340
19.290
18.250
19.660
18.330
0.005
0.092
0.852
7.651
5.440
4.320
4.045
3.202
3.450
7.387
4.999
0.378
0.497
3.151
4.463
8.001
7.264
19.187 8.328
22.861 8.355
6.401 2.454
19.259 5.857
0.169 0.039
CD4+Tem ICOS+CLA+
16.725
24.570
17.600
18.115
25.072
29.024
15.782
32.419
0.042
CD4+Tem ICOS+CLA-
7.010
14.018
11.264
8.993
10.428
12.741
7.049
10.065
0.003
CD4+Tcm HLADR+CLA+ CD4+Tcm HLADR+CLA-
6.438 2.061
4.326 0.911
6.473 2.262
15.789 4.716
6.279 1.996
9.549 2.209
1.862 0.520
6.813 1.190
0.086 0.250
1.000 0.788
0.358
0.002
0.974
0.189
1.38E06
0.463
0.164
0.302
0.669
0.024
0.194
0.264
3.98E09
0.881
1.09E04
0.709
0.769
0.148 0.285
0.058 0.071
CD4+Tem HLADR+CLA+
8.512
4.757
3.968
7.464
6.259
6.503
3.799
9.215
0.059
0.243
0.579
1.23E04
CD4+Tem HLADR+CLA-
1.529
1.468
1.360
2.354
1.271
2.606
0.944
1.510
0.507
0.038
0.360
0.102
CD8+Tcm CLA+
11.109
9.430
4.050
8.480
6.160
5.735
12.660
0.015
0.001
24.100
15.758
0.005
0.782
16.490
18.023
0.818
8.76E07
0.017
36.470
10.720
0.001
3.99E04
25.365
8.761
0.025
0.561
6.970
CD4+Effector cells CLA+
CD4+Tcm ICOS+CLA-
0.810
2.45E04
CD4+EM+CLA+
CD4+Tcm ICOS+CLA+
0.898
8.890
0.942
0.009
0.659
0.002
1.000
0.005
0.550
0.613
0.111
0.109
0.420
0.020
0.042
9.36E05
0.536
0.087
0.044
0.017
0.679
0.149
0.107
0.182
0.429
0.724
2.13E04
0.029
0.003
0.152
0.019
0.461
0.044
0.538
0.034
0.841
4.24E05
1.43E05
0.001
0.303
0.223
0.242
0.005
0.873
0.157
0.060
0.657
0.945
0.408
0.162
0.330
0.001
0.991
0.352
0.365
0.565
0.162
0.716
0.965
0.265
0.036
0.278
0.824
0.530
0.028
0.256
0.296
1.33E04
0.001
0.653
0.016
0.700
0.071
0.863
0.012
0.019
0.159
0.115
0.605
0.602
0.610
0.256
0.804
0.283
0.094
0.666
0.620
1.67E05
0.324
0.010
0.025
0.526
0.009
0.419
0.079
5.54E05
CD8+Tem CLA+
11.382
9.080
5.102
7.690
6.023
6.645
10.313
9.412
0.531
0.009
0.560
0.554
5.98E05
CD8+Effector cells CLA+
6.460
5.543
4.110
3.606
4.030
3.030
3.621
3.260
0.566
0.846
0.762
0.183
0.023
0.023
0.041
0.831
0.435
0.713
0.225
0.214
0.021
0.599
0.376
0.924
CD8+Tcm ICOS+CLA+
3.735
7.295
5.822
19.633
16.667
24.721
12.136
15.659
0.558
0.091
0.921
0.088
0.628
0.014
0.023
0.080
0.219
0.255
0.013
0.029
0.001
0.782
0.751
0.686
CD8+Tcm ICOS+CLA-
1.961
3.105
2.459
5.675
3.218
6.940
4.356
3.548
1.000
0.055
0.068
0.484
0.965
0.174
0.161
0.090
0.043
0.876
0.145
0.027
0.474
0.575
0.242
0.029
3.46E04
0.805
0.664
0.624
0.456
0.584
0.831
0.049
0.019
0.003
0.667
0.818
0.544
0.036
0.087
0.148
0.529
0.298
0.748
0.869
0.806
0.250
0.943
0.337
0.786
0.049
1.39E05
0.111
0.001
0.958
0.032
1.96E06
0.089
4.47E06
0.017
CD8+Tem ICOS+CLA+
9.841
9.695
13.601
21.448
16.689
26.667
11.837
21.531
0.597
0.040
0.052
CD8+Tem ICOS+CLA-
4.818
3.143
2.042
2.679
2.364
3.850
2.686
3.106
0.141
0.435
0.496
0.140
0.042
2.29E09
0.048
1.39E04
CD8+Tcm HLADR+CLA+ CD8+Tcm HLADR+CLA-
9.502 2.673
6.457 2.016
6.354 3.448
7.056 2.393
4.270 2.903
18.549 5.044
5.924 4.121
18.648 11.376
0.749 0.664
0.972 0.691
0.938 0.462
0.432 0.953
0.663 0.205
0.560 0.332
0.827 0.396
0.388 0.101
0.865 0.352
CD8+Tem HLADR+CLA+
4.361
6.255
4.416
4.751
5.331
9.719
4.596
15.959
0.728
0.438
0.361
3.06E10
0.990
0.981
0.650
0.990
0.649
0.802
0.597
0.430
2.39E05
0.989
0.016
0.024
CD8+Tem HLADR+CLA-
1.786
2.302
3.203
1.638
2.874
1.806
3.300
6.633
0.842
0.046
0.383
0.001
0.139
0.194
0.027
0.741
0.642
0.382
0.376
0.826
2.31E06
0.559
7.00E07
0.002
CD4+Naïve cells
80.750
79.750
66.200
63.000
58.100
69.250
61.750
63.700
0.706
0.240
0.005
1.000
0.038
3.37E05
1.49E04
0.015
0.144
0.176
5.04E05
0.006
7.57E06
0.150
0.719
0.056
CD4+Tcm
9.370
8.510
17.400
15.700
22.950
16.550
25.500
17.900
0.460
0.869
0.072
0.049
0.007
2.41E04
3.65E06
0.049
0.004
0.442
3.95E04
0.006
3.84E05
0.723
0.349
0.248
0.159
3.44E04
0.027
0.767
0.013
2.39E04
0.007
2.27E05
0.205
0.958
0.121
0.286
3.90E04
0.008
2.93E04
0.597
0.494
0.041
0.309
0.058
0.217
CD4+Tem CD4+Effector cells
4.560 4.400
4.290 4.718
8.950 2.940
10.100 4.970
12.350 5.260
8.345 4.600
7.860 1.195
10.500 2.490
0.633 0.500
0.162 0.125
0.016 0.365
0.141 0.014
0.314
0.030 0.150
CD8+Naïve cells
53.350
69.800
61.500
65.200
61.250
78.500
57.850
53.300
0.026
0.497
0.003
0.317
0.130
0.496
0.550
0.624
0.257
0.511
0.824
0.058
0.002
0.044
0.005
9.57E06
CD8+Tcm
22.600
10.200
15.800
10.600
15.200
10.030
19.700
17.200
0.026
0.015
0.007
0.532
0.110
0.213
0.566
0.915
0.086
0.090
0.200
0.144
0.013
0.689
0.001
3.02E04
CD8+Tem
9.875
6.160
9.460
7.660
11.900
4.655
13.400
11.400
0.046
0.237
0.002
0.818
0.806
0.330
0.265
0.220
0.106
0.846
0.640
0.197
0.002
0.098
0.015
1.10E04
CD8+Effector cells
9.095
8.285
11.800
15.100
11.250
11.050
7.325
14.100
0.801
0.181
0.450
0.018
0.130
0.311
0.585
0.765
0.056
0.185
0.046
0.410
0.034
0.024
0.952
0.056
Mean expression of all T-cell subset frequencies in healthy and AD individuals, as well as P-values (measured by Welch’s t-test) for all AD versus healthy comparisons and comparisons within the healthy group and within the AD group (A). Median expression of all T-cell subset frequencies in healthy and AD individuals, as well as P-values (measured by Wilcoxon-Mann-Whitney test) for all AD versus healthy comparisons and comparisons within the healthy group and within the AD group (B). Abbreviations AD: Atopic dermatitis; Adol: Adolescent; Child: Children; CLA: Cutaneous lymphocyte antigen; HLADR: Human leukocyte antigen-DR; ICOS: Inducible co-stimulator; IFNγ: Interferon-gamma; IL: Interleukin; N: Normal; Tcm: Central memory T; Tem: Effector memory T; Treg: Regulatory T
Table E4. Selected Spearman correlations and their P-values in atopic dermatitis patients AD Age Marker Duration of Disease CD4+IFNγ+CLACD8+IL13+CLACD4+IFNγ+ CD8+IL13+ CD8+IL22+CLACD8+IL22+ CD8+IL17+CLACD4+IFNγ+CLA+ CD8+IL22+CLA+ CD8+IL9+CLA+ CD8+IL17+ CD8+IL13+CLA+ EASI CD4+IL22+CLA+ CD8+CLA+ SCORAD Total ADQ Eosinophils CD4+IL13+CLA+ Pruritus ADQ CD8+IFNγ+CLA+ CD4+IL9+CLACD8+IL17+CLA+ CD4+IL9+ CD8+IL9+CLACD4+IL13+ CD8+IL9+ CD4+IL9+CLA+ CD4+IL13+CLACD4+CLA+ CD8+IFNγ+CLA-IL13+CLACD8+IFNγ+CLA+IL13+CLA+
EASI r 0.980 0.647 0.646 0.638 0.581 0.546 0.501 0.462 0.439 0.428 -0.405 0.372 0.358 0.340 0.312 -0.370 0.250 0.237 0.238 0.163 0.155 0.150 -0.145 0.143 -0.122 0.111 0.098 -0.093 -0.088 0.072 0.061 -0.058 -0.022
P-value 1.12E-56 1.39E-15 3.67E-15 4.76E-15 6.66E-12 1.36E-10 8.84E-09 1.06E-07 5.45E-07 1.11E-06 5.03E-06 3.15E-05 6.46E-05 1.52E-04 5.16E-04 9.35E-04 1.38E-03 1.14E-02 3.48E-02 7.74E-02 1.02E-01 1.03E-01 1.15E-01 1.21E-01 1.85E-01 2.30E-01 2.86E-01 3.17E-01 3.41E-01 4.37E-01 5.07E-01 5.26E-01 8.09E-01
Marker r P-value CD4+IL9+CLA+ 0.331 3.44E-03 CD8+IL9+CLA0.316 5.45E-03 CD8+IL9+CLA+ 0.265 2.05E-02 CD8+IL9+ 0.260 2.35E-02 CD4+IL9+CLA0.245 3.30E-02 CD4+IL22+CLA+ 0.224 5.18E-02 CD4+IL9+ 0.221 5.52E-02 CD4+CLA+ -0.213 6.50E-02 CD8+CLA+ -0.209 6.94E-02 CD8+IFNγ+CLA-IL13+CLA0.112 3.24E-01 CD4+IL13+CLA+ 0.093 4.24E-01 CD4+IL13+ 0.081 4.88E-01 CD4+IL13+CLA0.071 5.41E-01 CD8+IFNγ+CLA+IL13+CLA+ 0.019 8.67E-01 CD8+IFNγ+CLA+ -0.002 9.86E-01 SCORAD Marker
r EASI 0.738 CD8+IL9+CLA0.271 CD4+CLA+ 0.246 CD8+IL9+ 0.203 CD4+IL22+CLA+ 0.201 CD4+IL13+CLA+ 0.200 CD4+IL13+ 0.126 CD4+IL9+CLA-0.071 CD8+IL9+CLA+ 0.070 CD8+CLA+ -0.063 CD4+IL9+CLA+ -0.050 CD4+IL9+ -0.046 CD8+IFNγ+CLA+ 0.044 CD4+IL13+CLA0.029 CD8+IFNγ+CLA+IL13+CLA+ -0.017 CD8+IFNγ+CLA-IL13+CLA0.013
P-value 1.42E-21 3.32E-03 7.53E-03 2.89E-02 2.95E-02 3.17E-02 1.75E-01 4.45E-01 4.56E-01 5.93E-01 5.97E-01 6.19E-01 6.41E-01 7.54E-01 8.58E-01 8.86E-01
Total ADQ Marker
r SCORAD 0.576 EASI 0.532 CD8+IL9+CLA0.261 CD4+IL9+CLA+ 0.240 CD8+CLA+ -0.192 CD8+IL9+ 0.192 CD4+IL13+CLA+ 0.182 CD4+IL13+ 0.162 CD4+IL9+ 0.136 CD4+IL9+CLA0.121 CD4+IL22+CLA+ 0.120 CD4+IL13+CLA0.113 CD4+CLA+ -0.107 CD8+IFNγ+CLA+IL13+CLA+ -0.095 CD8+IL9+CLA+ 0.088 CD8+IFNγ+CLA+ 0.070 CD8+IFNγ+CLA-IL13+CLA- -0.039
P-value 2.54E-11 1.59E-09 2.68E-02 4.26E-02 1.05E-01 1.07E-01 1.26E-01 1.74E-01 2.56E-01 3.12E-01 3.15E-01 3.44E-01 3.69E-01 4.20E-01 4.64E-01 5.59E-01 7.39E-01
Eosinophil Count Marker r Pruritus ADQ 0.350 EASI 0.335 Total ADQ 0.288 SCORAD 0.267 CD4+CLA+ -0.133 CD4+IL9+CLA0.086 CD4+IL13+CLA+ 0.080 CD4+IL9+ 0.070 CD8+CLA+ 0.063 CD8+IFNγ+CLA+ 0.048 CD4+IL22+CLA+ -0.048 CD8+IFNγ+CLA+IL13+CLA+ 0.046 CD8+IL9+ 0.044 CD4+IL13+ 0.036 CD8+IL9+CLA0.032 CD4+IL13+CLA0.030 CD8+IL9+CLA+ 0.025 CD8+IFNγ+CLA-IL13+CLA- 0.020 CD4+IL9+CLA+ -0.012
P-value 2.42E-03 2.76E-03 1.35E-02 1.88E-02 3.46E-01 5.46E-01 5.74E-01 6.21E-01 6.56E-01 7.36E-01 7.37E-01 7.43E-01 7.56E-01 8.00E-01 8.22E-01 8.30E-01 8.62E-01 8.85E-01 9.33E-01
Pruritus ADQ Marker r Total ADQ 0.945 SCORAD 0.547 EASI 0.456 CD8+IL9+CLA0.185 CD8+CLA+ -0.185 CD4+IL13+CLA+ 0.170 CD4+IL9+CLA+ 0.151 CD4+CLA+ -0.138 CD4+IL13+ 0.133 CD8+IL9+ 0.121 CD8+IFNγ+CLA+IL13+CLA+ -0.108 CD4+IL13+CLA0.087 CD8+IFNγ+CLA+ 0.062 CD4+IL22+CLA+ 0.044 CD4+IL9+ 0.032 CD8+IL9+CLA+ 0.031 CD4+IL9+CLA0.021 CD8+IFNγ+CLA-IL13+CLA- -0.002
TEWL LS
TEWL NL Marker EASI
P-value 9.72E-56 3.80E-10 4.45E-07 1.20E-01 1.21E-01 1.54E-01 2.06E-01 2.49E-01 2.65E-01 3.11E-01 3.62E-01 4.68E-01 6.07E-01 7.17E-01 7.88E-01 7.97E-01 8.62E-01 9.89E-01
r 0.294
P-value 2.71E-03
Marker Age
r 0.333
P-value 5.89E-04
Significant Spearman correlations (P<0.05), with accompanying correlation coefficient (r) and P-value between various T-cell subsets and age or clinical scores (e.g. EASI, SCORAD, pruritus ADQ, TEWL NL, TEWL LS, eosinophil count). Abbreviations ADQ: Atopic dermatitis Quickscore; Adol: Adolescent; Child: Children; CLA: Cutaneous lymphocyte antigen; EASI: Eczema Area and Severity Index; HLADR: Human leukocyte antigen-DR; IFNγ: Interferon-gamma; IL: Interleukin; LS: Lesional; N: Normal; NL: Non-lesional; SCORAD: SCORing Atopic Dermatitis; Tcm: Central memory T; Tem: Effector memory T; TEWL: Transepidermal water loss; Treg: Regulatory T
Duration of Disease Marker r CD4+IFNγ+ 0.584 CD4+IFNγ+CLA0.583 CD4+IL9+CLA+ 0.570 CD8+IL13+CLA0.507 CD4+IFNγ+CLA+ 0.406 CD8+IL22+CLA0.392 CD4+IL9+CLA0.381 EASI 0.376 CD4+IL9+ 0.350 CD8+IL22+ 0.317 CD4+CLA+ -0.304 CD8+IL13+ 0.299 CD8+CLA+ -0.299 CD4+IL13+CLA0.289 CD8+IL22+CLA+ 0.281 Total ADQ 0.284 CD4+IL22+CLA+ 0.276 CD8+IL9+CLA0.235 Pruritus ADQ 0.222 CD4+IL13+ 0.217 Eosinophils 0.234 CD8+IL17+CLA0.155 SCORAD 0.153 CD4+IL13+CLA+ 0.149 CD8+IFNγ+CLA-IL13+CLA0.143 CD8+IL17+CLA+ -0.093 CD8+IL9+ 0.055 CD8+IL17+ 0.049 CD8+IFNγ+CLA+IL13+CLA+ 0.039 CD8+IL9+CLA+ 0.028 CD8+IL13+CLA+ 0.016 CD8+IFNγ+CLA+ -0.006
P-value 2.45E-08 2.74E-08 6.39E-08 2.50E-06 2.51E-04 4.28E-04 6.34E-04 6.46E-04 1.81E-03 4.96E-03 7.22E-03 8.15E-03 8.34E-03 1.09E-02 1.35E-02 1.41E-02 1.50E-02 3.95E-02 5.68E-02 5.84E-02 9.18E-02 1.77E-01 1.80E-01 1.96E-01 2.05E-01 4.26E-01 6.35E-01 6.75E-01 7.30E-01 8.07E-01 8.89E-01 9.61E-01
Table E5: Pairwise comparisons between age group associated clusters P-values Adults vs. Infants
Children and Adolescents vs. Infants
Children and Adolescents vs. Adults
Adults vs. Infants
Children and Adolescents vs. Infants
Children and Adolescents vs. Adults
P-value ANOVA
FDR ANOVA
CD4+IFNγ+
23.985
11.339
-12.646
2.82E-14
1.03E-08
6.47E-08
2.56E-19
1.74E-17
CD4+IFNγ+CLA-
24.326
11.726
-12.600
2.88E-14
7.95E-09
1.44E-07
4.46E-19
2.99E-17
CD4+IFNγ+CLA+
16.491
5.763
-10.728
1.30E-13
1.00E-03
1.85E-07
2.49E-13
1.64E-11
CD8+IFNγ+CLA-
37.265
13.395
-23.870
2.22E-13
8.51E-04
4.17E-07
5.10E-13
3.31E-11
CD8+IFNγ+
36.183
12.373
-23.810
3.67E-13
1.86E-03
2.93E-07
9.04E-13
5.78E-11
CD4+IL22+CLA-
2.477
2.543
0.067
3.26E-07
3.52E-10
0.987
5.75E-11
3.62E-09
CD4+IL22+
1.010
2.831
1.821
7.52E-02
3.82E-11
2.49E-04
6.00E-11
3.72E-09
CD4+IL9+CLA+
-0.975
5.543
6.518
6.81E-01
2.10E-07
2.57E-07
2.51E-09
1.53E-07
CD8+IL13+CLA-
0.701
0.334
-0.367
5.93E-08
1.44E-03
4.61E-03
8.43E-08
5.06E-06
CD4+Naïve cells
-22.633
-8.536
14.097
5.04E-08
1.05E-02
3.47E-04
1.12E-07
6.60E-06
CD8+IL22+CLA-
0.780
0.311
-0.469
7.63E-08
1.11E-02
1.01E-03
1.63E-07
9.45E-06
CD4+IL17+CLA-
1.167
0.843
-0.324
2.34E-06
3.36E-05
0.300
3.66E-07
2.09E-05
CD4+IL17+
1.174
0.816
-0.358
1.75E-06
5.30E-05
0.228
3.79E-07
2.12E-05
CD8+Tem HLADR+CLA-
5.887
0.329
-5.558
1.62E-06
9.26E-01
2.96E-06
5.29E-07
2.91E-05
CD4+IL13+
0.068
1.791
1.723
0.987
6.20E-06
2.73E-04
1.50E-06
8.11E-05
CD4+IL9+
-0.135
0.734
0.869
0.782
6.58E-05
6.80E-05
3.48E-06
1.84E-04
CD4+IL9+CLA-
-0.092
0.603
0.695
0.848
7.50E-05
1.24E-04
5.57E-06
2.90E-04
CD4+Tcm
12.321
7.113
-5.208
1.07E-05
1.25E-03
8.09E-02
7.32E-06
3.73E-04
CD4+IL13+CLA-
0.141
1.539
1.398
0.934
1.85E-05
1.47E-03
7.95E-06
3.97E-04
CD8+IL22+
0.767
0.292
-0.474
4.99E-06
0.047
5.16E-03
9.67E-06
4.74E-04
-21.405
-12.280
9.124
2.02E-05
0.002
0.090
1.47E-05
0.001
CD4+Tem
8.138
2.779
-5.358
8.69E-06
0.081
0.003
1.70E-05
0.001
CD4+IFNγ+CLA+IL13+CLA-
73.154
5.044
-68.110
6.60E-05
0.924
1.73E-04
3.91E-05
0.002
CD4+IL22+CLA+
2.017
4.986
2.969
0.295
4.58E-05
0.065
7.44E-05
0.003
Tregs CD25+CD127+
1.378
4.627
3.249
0.561
7.94E-05
0.028
7.69E-05
0.003
CD8+Tcm ICOS+CLA+
6.025
13.250
7.225
0.257
7.12E-05
0.123
1.20E-04
0.005
CD8+IL17+CLA-
0.395
0.152
-0.243
7.12E-05
0.102
0.018
1.28E-04
0.005
CD8+Naïve cells
-14.301
3.940
18.241
4.73E-03
0.490
9.87E-05
1.67E-04
0.007
CD4+Tem CLA+
CD4+IFNγ+CLA+IL13+CLA+
5.002
0.293
-4.709
3.34E-04
0.955
6.48E-04
2.05E-04
0.008
CD4+Tcm CLA+
-10.615
-6.947
3.668
5.60E-04
0.004
0.345
2.68E-04
0.010
CD8+IFNγ+CLA+
13.375
-4.151
-17.526
0.010
0.496
3.61E-04
5.62E-04
0.021
CD8+Tem CLA+
-4.363
-6.058
-1.696
0.105
0.001
0.683
0.002
0.059
CD4+Tcm HLADR+CLA+
2.583
7.579
4.996
0.629
0.002
0.148
0.003
0.101
CD8+IL13+CLA+
5.232
1.302
-3.930
0.002
0.546
0.026
0.003
0.118
CD8+Effector cells
6.634
-0.497
-7.131
0.012
0.958
0.004
0.004
0.135
CD8+Tem ICOS+CLA+
5.485
12.108
6.622
0.453
0.003
0.280
0.004
0.143
CD8+Tcm HLADR+CLA+
10.335
10.257
-0.079
0.033
0.007
1.000
0.005
0.145
CD8+Tem
5.247
-0.345
-5.592
0.017
0.970
0.006
0.006
0.198
CD4+IL17+CLA+
1.288
0.507
-0.781
0.004
0.272
0.113
0.007
0.198
CD8+Effector cells CLA+
-4.570
-2.774
1.796
0.010
0.055
0.440
0.008
0.226
CD4+IL13+CLA+
-0.035
4.409
4.444
1.000
0.015
0.049
0.008
0.229
CD8+IL17+CLA+
0.731
-0.664
-1.395
0.286
0.220
0.010
0.012
0.321
CD8+IL9+CLA+
-8.800
-2.116
6.684
0.009
0.649
0.055
0.012
0.321
CD8+IL13+
0.709
0.244
-0.464
0.009
0.405
0.115
0.013
0.328
CD4+Tem ICOS+CLA-
-8.238
-4.029
4.209
0.019
0.201
0.308
0.023
0.542
CD8+Tcm CLA+
-2.498
-4.210
-1.713
0.420
0.019
0.638
0.026
0.597
CD8+Tem HLADR+CLA+
7.009
7.170
0.161
0.123
0.031
0.999
0.027
0.597
CD4+Tcm ICOS+CLA-
-1.975
2.717
4.693
0.571
0.177
0.034
0.028
0.597
CD4+Tcm ICOS+CLA+
4.248
7.190
2.942
0.434
0.023
0.639
0.031
0.617
CD8+Tcm ICOS+CLA-
-1.177
2.186
3.363
0.702
0.136
0.047
0.031
0.617
CD4+Tem HLADR+CLA+
1.993
5.482
3.489
0.753
0.034
0.387
0.040
0.716
CD8+IL22+CLA+
4.546
1.976
-2.571
0.062
0.450
0.383
0.076
1.00
CD8+Tcm
2.806
-2.710
-5.517
0.558
0.407
0.087
0.090
1.00
CD4+Effector cells
2.203
-1.360
-3.563
0.406
0.564
0.077
0.092
1.00
CD8+IL9+CLA-
0.036
0.240
0.203
0.965
0.107
0.313
0.101
1.00
CD8+IL17+
0.226
0.031
-0.194
0.123
0.938
0.194
0.125
1.00
CD4+Tem HLADR+CLA-
-1.824
0.474
2.298
0.297
0.873
0.123
0.144
1.00
-101.376
-49.219
52.157
0.167
0.526
0.609
0.184
1.00
-0.238
0.178
0.415
0.592
0.640
0.190
0.213
1.00
CD8+CLA+
3.071
-1.244
-4.315
0.519
0.494
0.283
0.227
1.00
CD8+IFNγ+CLA+IL13+CLA+
-8.738
-7.932
0.807
0.386
0.310
0.992
0.257
1.00
CD8+Tcm HLADR+CLA+
3.841
0.516
-0.885
0.943
-4.726
0.335
0.364
1.00
Tregs CCR4+CLA+
3.026
0.678
3.157
0.502
0.131
0.999
0.499
1.00
CD4+Effector cells CLA+
-2.420
0.647
-0.254
0.992
2.167
0.683
0.642
1.00
CD4+Tcm HLADR+CLA+
-0.908
0.887
-0.975
0.797
-0.067
0.999
0.798
1.00
CD4+CLA+
-0.589
0.801
-0.168
0.974
0.421
0.887
0.816
1.00
CD4+Tem ICOS+CLA+
2.145
0.902
0.733
0.980
-1.412
0.952
0.910
1.00
CD8+Tem ICOS+CLA-
0.128
0.990
-0.225
0.951
-0.353
0.922
0.914
1.00
CD8+IFNγ+CLA+IL13+CLACD8+IL9+
P-values, as measured by Welch’s t-test, that compare the three clusters that were obtained by k-means unsupervised clustering of all AD patients, as well as both raw and FDR-adjusted ANOVA score among all clusters. Abbreviations ANOVA: Analysis of variance; CLA: Cutaneous lymphocyte antigen; FDR: False discovery rate; HLADR: Human leukocyte antigen-DR; ICOS: Inducible co-stimulator; IFNγ: Interferon-gamma; IL: Interleukin; Tcm: Central memory T; Tem: Effector memory T; Treg: Regulatory T
Table E6. Evolution of pathologic T-cell subsets in atopic dermatitis from infancy to adulthood Healthy controls
0-5y/o IFN-γ CLA+>CLACD4+>CD8+
IL-13 CLA+>CLACD4+>CD8+
IL-9
6-11y/o
12-17y/o
Atopic Dermatitis
≥ 18y/o
0-5y/o
6-11y/o
12-17y/o
CLA->CLA+
CLA->CLA+
≥ 18y/o
---
-----
IL-17 ---
CLA-
CLA-
CLA-
---
CLA-
CLA-
CLA-
IL-22 ---
---
---
---
---
CLA-
---
CLA-
---
CLA+
CLA+
Tregs (CLA+)
---
---
---
Arrows show developmental changes in healthy controls and AD across ages, as well as AD vs. control changes in the AD groups. CLA, Cutaneus Lymphocyte Antigen; Tregs, T-regulatory cells
Figure E1
A
B 10
C
5
5
10
5
10 4
10
4
10
3
10
10 4
76.6
12.6
65.2
10
0
0
50K
FSC-A
Frequency (%)
D
100K
150K
CLA CD4 Tcm cells
CD4 CM CLA+
** **
100 100
** **
** **
200K
50
0
CD8
200
**
**
10 2
10 3
CLA CD4 Tem cells
CD4 EM CLA+ 200
** *
**
150 150
100 100
** **
**
*** ***
F
AD N
Infants
H
AD N
AD
N
0
AD
Children Adolescents
Adults
HLA-DR CLA CD4 Tcm cells CD4 CM HLDR+/CLA+
** ***
100
100
+ +
*** * + *
50
50
0
*
**
100
**
AD N
AD N AD
Children Adolescents
CD4 CM HLADR+/CLA
N
0
AD
Adults
** *
*** +
+* + +
50
50
*** ***
N
AD N
AD N
AD
N
AD
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
Infants
Children Adolescents
Adults
+*
*** *
** ***
AD N
40
AD N
AD
N
AD
AD N
AD N
AD
N
AD
HLA-DR CLA CD4 Tem cells CD4 EM HLADR+/CLA+
50
0
Infants
Children Adolescents
Adults
0
Adults
**
** **
N
AD N
AD N
Infants
K HLA-DR
Children Adolescents
N
AD
Adults
CLA CD4 Tem cells
CD4 EM HLADR+/CLA
* **
60
*
+ *
AD
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
60 +
+
40 40
*** ***
+ +
20
*
20
0 0
0
N
+ *
40
Children Adolescents
+
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
*** ***
** **
+
*** ***
0
N
*** * *** +
**
+
60
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
100
25
CLA CD8 Tem cells
20
100
25
5
CD8 EM CLA+
20
Infants
J
G
10
60
** **
50
0
+ *** ***
**
10
4
80
*** **
0
0
**
10
3
80
0
N
75
* **
10
20
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
75 *** **
** **
40
HLA-DR CLA CD4 Tcm cells
100
0
20
Infants
I
4.84 2
CLA CD8 Tcm cells
40
0
N
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
5.95
CD45RO
CD8 CM CLA+
60
+ *
0
10 5
60
50
0
10 2
10 4
**
** *
50 0
Frequency (%)
41.3
0
250K
E
**
50
51.9
10 2
CD4
CD3
10 2
3
CCR7
10
3
N
AD N
AD N
AD
N
AD
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
Infants
Children Adolescents
Adults
N
AD N
AD N
AD
N
AD
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
Infants
Children Adolescents
Adults
Figure E2
CD4⁺ T cells
A
B
Infants Normal
Normal
CD4 Infants
Normal
CD4 Children
Normal
CD8 Infants
CD8 Children
Naive 56%
Naive 62%
Naive 56%
Naive 62%
Naive 64%
Naive 68%
AD
CD8 Children
Naive 64%
Naive 50%
Naive 55%
Children
AD
CD8 Infants
Naive 50%
Naive 55%
Naive 68%
D
Infants
AD
CD4 Children
Naive 78%
Naive 78%
C
Children
AD
CD4 Infants
Naive 78%
Naive 78%
Tcm 12%
m Tc
13
12 %
%
%
%
%
15
%
m Tc
10
17
9%
28
%
H
Adults Normal
AD
CD8 Adults
CD8 Adults
Naive 56%
Naive 54% Naive 54%
Naive 56%
Naive 77%
Naive 70%
Tcm 15%
m Te
% 11
m Tc
m Te
m Tc
19 Naive 60%
or ct fe Ef 20%
Tem 10%
CD8 Adolescents
Naive 60%
Naive 60%
Effector 20%
Tem 13%
r to
Tcm 17%
CD8 Adolescents
Naive 60%
Effector 13%
Adolescents Normal AD
AD
Naive 61%
c fe Ef
%
m Tc
% 11
%
% 11
G
Normal
Tem 9%
Tcm 28%
CD4 Adults
Naive 61%
Effector 11%
m Te or ct fe Ef 13%
or ct fe Ef 10%
12
m Te
Tcm 19%
CD4 Adults
Naive 70%
Naive 57%
Effector 10%
Tem 12%
Adults
CD4 Adolescents
Naive 57%
16
m Te
F
Adolescents Normal AD CD4 Adolescents
Tem 17%
m Tc
6%
Tem 11%
Effector 8%
m Te
Tcm 16%
5%
6%
% 11
m Te
% 11
m Te
Tem 5%
m Tc
5%
Tem 6%
r to
c fe Ef
r to
m Tc
Tcm 11%
Effector 8%
Effector 5%
Effector 6%
c fe Ef
Tcm 11%
or ct fe Ef 5%
Effector 5%
E
CD8⁺ T cells
Naive 77%
0
20
40
60
80
Age Age
M
r=0.42 (CI: 0.26–0.56)
r=0.56 (CI: 0.25-0.57)
p=2.7e-06
p=2.4e-09
r=0.42 (CI: 0.26−0.56) ● p=2.7e−06
0
●
●
25
●
p=0.11
● ●
●
●●●
●
●
●
● ● ● ● ●
●●
●
●
●
●●
●
● ●
●
●
●
●
●
●
●
●
●
●● ●
● ●
●
Effector
● ●
●
●
●
●
● ●
●●
●
●
●
● ●
●
●
●
40
●
●
●
● ●
●
60
80
0
0
20
40
r=0.24 (CI: 0.21-0.54)
p=2e-05
p=0.018
r=0.24 (CI: 0.21−0.54) p=0.018
60
Age Age
OAge vs. CD8 CM
r=0.39 (CI: 0.22–0.53) r=0.39 (CI: 0.22−0.53) ● p=2e−05
20
10
●
P
r=0.008 (CI: 0.022-0.4)
p=0.019
p=0.94
r=0.008 (CI: 0.022−0.4) p=0.94
40
40 ●
60
●
20
0
20
●
● ● ●
●
●
●
● ●
● ●
● ●●
●● ●●
●●
●● ● ● ● ● ●
● ● ●
●
●
●
●
●● ● ●● ● ● ●
● ● ● ●
●
10
●
●
0 40
Age Age
60
●● ●●
80
0
● ●
● ●
● ● ●
●
● ●
●
● ●
●
● ● ●● ● ● ●●● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ●● ● ● ● ● ●●●● ● ● ●● ● ● ● ● ● ● ● ●●● ●● ● ● ● ● ● ● ●● ●● ● ● ●● ● ● ● ● ●● ●● ● ● ● ● ● ● ●●● ● ●● ●● ● ●● ● ● ● ● ●● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●●
●
●
● ●
20
●
●
● ●● ●●● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ●● ● ●● ●●●● ●● ● ● ● ●● ● ●● ● ● ● ●● ●● ●● ● ●●● ●●● ● ● ● ● ● ● ● ●●●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ●● ● ●●● ● ● ● ● ● ●●● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●
● ● ● ●
●
●
●●
● ●
● ● ●
●
● ●
●
● ●
● ●
● ●● ● ●● ●
20
● ●● ●
● ●●● ● ● ●● ● ●● ● ● ● ● ● ●
40
60
80
Age Age
Age vs. CD8 EM r=0.16 (CI: 0.056−0.43) r=0.16 (CI:p=0.11 0.056-0.43)
r=0.25 (CI: 0.07−0.42) r=0.25 (CI: 0.07–0.42) ● p=0.0071
p=0.11
● p=0.0071 ●
●
●
●
●
20
●
●
● ● ● ● ●
● ●
● ●
● ●
● ● ●● ●
● ● ●
●
● ●●
●
●
20
●
● ● ● ● ● ● ● ●● ● ● ● ● ●
0 40
Age Age
60
80
● ● ● ● ● ● ●
●
●● ●
40
● ●
●
●
● ● ●
● ●
● ●●
●
● ● ●
● ●
●
●
● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ●●●●● ● ● ● ● ●● ●● ● ●●● ● ● ●● ● ● ●● ● ●● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●●● ●●● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ●● ● ●● ● ●● ● ●● ●● ● ●● ● ●● ● ● ● ●
0
20
● ●● ● ● ●
● ● ● ● ● ● ●
●
●● ●
● ● ●
● ●
● ● ●
●● ● ●
● ●
●●
●●
●●
●
20
● ●
●
●
●●
●
● ● ●●
CD8 EM
●
●
Tem
●
● ● ● ● ● ● ● ●● ●● ●● ●● ● ●● ● ● ● ● ● ● ● ●● ● ● ●●●● ●● ● ● ● ●● ● ●● ● ● ●● ●● ●● ● ●● ● ● ●●● ●●● ● ● ●● ● ●● ● ●● ●● ●●● ● ●● ● ●● ● ● ● ●● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ●●● ● ● ● ●● ● ●● ● ● ●● ● ● ● ● ●● ● ● ●● ●●
0
●
●
●
●
30
● ●
Tcm CD8 CM
●● ●
● ●
●
●
●
Tem CD4 EM
Tcm CD4 CM
40
● ●
●
●
●
30
● ●
●● ●
●
● ●
●
● ●
●
●
60
●
●
0
80
r=0.22 (CI: 0.036–0.39) r=0.22 (CI: 0.036−0.39) ● p=0.019
● ●
●
●
● ●● ● ● ● ● ● ● ● ●
● ● ●
●
20
30
●
● ● ● ●●● ● ● ● ● ● ●
p=0.32
40 ●
●
Age vs. CD4 EM
r=0.56 (CI: 0.25−0.57) p=2.4e−09
r=0.15 (CI: −0.033−0.33) (CI: −0.047−0.34) r=0.15 (CI: -0.033–0.33) r=−0.1 r=-0.1 (CI:p=0.32 -0.047-0.34)● p=0.11
p=0.85
CD8 Effector
Naive ●
●
● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ●● ●●● ● ● ● ● ● ● ●● ● ●
Age Age
N
Age vs. CD4 CM
CD8 Naïve
Effector
Naive
CD4 Naïve
CD4 Effector
0
● ●
%
● ●
●
18
●
●● ● ● ● ● ● ● ●● ● ●● ● ● ●● ● ●● ● ● ●● ●● ● ●●●● ● ● ● ●● ●● ●● ● ●● ●●● ●● ●●● ● ● ●● ● ●● ● ● ● ●●● ● ● ●● ●● ● ● ● ●●● ●● ●● ● ●●● ● ● ●● ●● ● ● ● ● ●●● ●●●● ●● ● ● ● ● ● ● ● ●●● ●● ● ●● ●● ●● ●● ● ● ● ● ● ●
or ct fe Ef 15%
●
25
●
50
m Tc
●
● ●
●
●
●
● ●
10
●
● ● ●
%
●
●
●
13
● ●
●
●
●
Tcm 18%
%
● ●●
●
●
20
●
●
m Te or ct fe Ef 10%
●
● ●●
●
20
m Tc
●
●
●
●
%
●
●●
14
●
75
●
m Te
●
% 11
●
●
r to
●
8% m Tc 5%
● ●
●
●
c fe Ef
● ● ●● ●
●
●
● ●
m Te
●
●
Tcm 20%
Age vs. CD8 Effector
r=−0.24 (CI: −0.4−−0.056) (CI: −0.42−−0.042) r=0.019 (CI: -0.42--0.042) r=-0.24 (CI: -0.4–-0.056)r=0.019 p=0.011 ● p=0.85 ● ● ● ●● ● ● ●● ●●● ●● ● ● ● ● ● ● ●● ● ● ●● ●● ● ●●● ●● ●● ●●● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ●● ● ●● ● ●●● ● ● ● ● ● ●●● ●● ●●● ● ● ● ●●● ● ●● ● ● ●● ● ●● ● ●● ● ●● ● ● ● ●● ● ●● ● ● ● ● ● ●● ● ● ●
Effector 15%
Tem 13%
Tem 14%
L
p=0.011 ●
p=0.0021
●
●
● ●
%
●●
●
●
Tem 5%
15
● ●
●
●●
● ● ● ●
●
●
●● ● ● ● ● ●
100
r=−0.22 (CI: −0.39−−0.039) (CI: −0.4−−0.025) r=p=0.018 -0.22 (CI: -0.39–-0.039) r=−0.31 r=-0.31 (CI: -0.4--0.025) ● p=0.0021
p=0.018
m Tc
%
50
●
%
21
%
%
%
75
●● ●●● ● ● ● ● ●● ● ●● ●● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ●● ● ● ● ●● ● ● ●● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ●●● ● ●● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●●● ● ●● ● ● ● ● ●●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●
30
p=1.6e-05
Tcm 15%
Tcm 8%
K Age vs. CD8 Naïve
Age vs. CD4 Effector
r=−0.41 (CI: −0.55−−0.24) (CI: −0.56−−0.23) r=-0.41 (CI: -0.55–-0.24) r=−0.42 r=-0.42 (CI: -0.56--0.23) ●● p=7.1e−06 p=1.6e−05 ●● ●● ● ● ● p=7.1e-06 ● ● ●● ●
Tem 12%
12
m Tc
12
26
10
J
Age vs. CD4 Naïve
or ct fe Ef 13%
Tcm 21%
Effector 10%
Effector 11%
Effector 13%
m Te
m Te
m Tc
%
9%
15
m Te
%
%
20
16
I
Effector 7%
or ct fe Ef 4%
Effector 7%
Tem 12%
Tcm 26%
m Te
Tem 10%
Tem 9%
5%
m Tc
7%
m Tc
Tcm 20%
Tcm 15%
r to
c fe Ef
r to
c fe Ef
m Te
Tem 16%
Effector 4%
Effector 5%
Effector 7%
● ● ●
10 ●●
●
●
●
● ●
0 40
Age Age
60
80
● ●
●
● ● ●
●
●● ●
●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ●● ● ● ● ●●● ● ●● ●●● ● ●● ● ●● ● ●● ● ● ● ●● ●●● ● ● ● ●● ●●● ● ● ● ● ●● ● ● ● ●● ● ● ●● ● ●● ● ● ● ● ●● ● ● ● ●● ●●● ● ● ● ● ● ● ● ● ● ●●●● ●●● ● ● ● ●● ● ● ● ● ●
0
●
●
20
● ●
● ● ● ●
●
●
● ●●●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ●
●
●●
●
●
●
40
Age Age
60
●
80
Figure E3 Non-Activated T-cells 10 5
0.0128
0
10 5
10 4
10 3
10 3
10 2
10 2
0
0
IFNγ
10 4
99.1 0
10
5
0.924 10 2
10 3
10 4
0.0257
5
10 4
10 3
10 3
0
0
IL-13
10 4
0.977
10
5
10 2
10 3
10 4
0.0129
10 5
10 4
10 3
10 3
0
0
CLA
0.926 10 2
10 3
10 4
3.16
10 5
10 2
10 3
10 4
10.8
10 5 0.267
3.91
0
10 4
0
78.4
85
10 5
6.43e-3
99.1
0.322
0
6.43e-3
99
18.1
10 5
10
0
IL-22
Activated T-cells
10 2
10 3
10 4
10 5
3.47
0.187
93.3
3.02
0
10 2
10 3
10 4
10 5
Figure E4
A
CD4 IFNg+CLA+
100 100
Frequency (%)
CD4 T cells IFN*** ***
**
75 75
50 50
**
** **
*** ***
+
*** ***
**
B
CLA
CD4 IFNg+CLA
100
+*
IFN-
+
50 50
C
CLA *** ***
*** *** + + ** ** *** *** ** *** *** *** *** **
100
25
IFN-
+
*** *** + +
** *
* **
100 100
D
CLA
CD8 IFNg+CLA+
100
50
N
AD N
Infants
E
AD N
AD
N
AD
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
0
CD4 IL13+CLA+
40
* ** *** ***
40
30 30
Children Adolescents
Adults
*
**
Infants
F
IL-13 CLA
CD4 IL13+CLA
20 20
+ + *** ***
AD
Children Adolescents
N
AD
0
**
15
AD N
AD N
Infants
AD
N
CD8 IL13+CLA+
Children Adolescents
*** ***
60
5
10
0
6 *** *** *** ***
N
AD N
AD N
AD
N
AD
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
0
Infants
I
IFN-
Children Adolescents
*
50 40
40 30
30
+*
*** *
AD N
Infants
J
IFN-
*
*** **
N
AD
0
*** *
** *** ***
100
100
*** ***
** *
*** *
** **
+*
** **
AD N
Infants
AD N
AD
K IFN-
Children Adolescents
N
AD
4
0
AD
Adults
IL-13 CLA * * *
+ *
+
N
AD N
AD N
Infants
L
IFN-
Children Adolescents
*** *
*
N
AD
Adults
CLA /IL-13 CLA
CD8 IFNg+CLA /IL13+CLA
*
**
75
AD
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
Adults
CLA /IL-13 CLA
CD8 IFNg+CLA+/IL13+CLA+
75
1000
1000
**
50
**
N
0
N
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
Adults
CLA /IL-13 CLA
CD4 IFNg+CLA /IL13+CLA
150
*** *
AD
Children Adolescents
150
*
20
50
** +
500
500
20
50
25
50
+*
10
0
Adults
CLA /IL-13 CLA
CD4 IFNg+CLA+/IL13+CLA+
AD N
AD
2
0
N
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
AD N
Children Adolescents
*** *** *** * *** * *** *** *** *** + * + + *
2
+
20
0
0
**
AD N
CD8 IL13+CLA
4
20
** **
** *
5
10
N
6
** **
10
20
** ***
* **
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
Infants
H
40
** **
0
Adults
40
15
AD
IL-13 CLA
60
+
*** ***
0
N
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
G
*
**
** ***
50
Adults
IL-13 CLA
10
20
AD N
*** ***
50
0
N AD N
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
*** ***
CLA ** **
+
100
50
0
0
IFN-
150
+ +
+
CD8 IFNg+CLA
150
25
0
Frequency (%)
CD8 T cells
0
0
25
*** ***
+*
**
AD N Infants.AD
Infants
AD N
AD N
Children.Normal Children.AD Adolescents.NormalAdolescents.AD Adults.Normal
AD Adults.AD
Children Adolescents Adults
0 0
0
N
Infants.Normal
N
Infants.Normal
AD N Infants.AD
Infants
AD N
AD N
Children.Normal Children.AD Adolescents.NormalAdolescents.AD Adults.Normal
Children Adolescents
AD
Adults.AD
Adults
0
0
N
Infants.Normal
AD N Infants.AD
Infants
AD N
AD
N
Children.Normal Children.AD Adolescents.NormalAdolescents.AD Adults.Normal
Children Adolescents
AD
Adults.AD
Adults
N
Infants.Normal
AD N Infants.AD
Infants
AD N
AD N
Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal
AD
Adults.AD
Children Adolescents Adults
Figure E5
A
CD4 T cells
CD4 IL9+CLA+
80
Frequency (%)
80
** **
60 60
*** ** ** *
** *
40
B
IL-9 CLA
*** ***
CD4 IL9+CLA
*** ***
7.5
N
AD N
AD N
Frequency (%)
AD
CD4 IL17+CLA+
Children Adolescents
N
100
0
AD
**
N
AD N
Infants
CD4 IL17+CLA
7.5
20
**
5
5.0
+
10
AD N
AD
N
Children Adolescents
AD
**
*** **
*** *** +
AD N
Infants
AD N
AD
CD4 IL22+CLA+
Children Adolescents
N
AD
*** ***
+ + +
40 40
+* 20
AD N
AD
N
AD
CD4 IL22+CLA
*** *** *** ** *** ***
15
** **
10
10
*** ***
*
** ***
*** ***
AD N
AD N
AD
N
AD
Infants
Children Adolescents
Adults
N
0
AD N Infants.AD
Infants
CD8 IL22+CLA+
AD N
0
AD
*** *
+
AD
N
AD
Infants
Children Adolescents
Adults
0
N
AD
**
N
Infants.Normal
Adults.AD
Adults
*** **
**
**
AD N
Infants.AD
CD8 IL22+CLA
*** *** *** *** ***
6 6
** ***
4 4
+
AD Adults.AD
Adults
IL-22 CLA
8
**
AD N
Children Adolescents
8
+*
**
AD N
Children.Normal Children.AD Adolescents.NormalAdolescents.AD Adults.Normal
Infants
L
*** *
*** ***
* **
2 2
0
AD N
*** *
0.5
*** ***
30
AD N
AD
Adults
0.5
IL-22 CLA
30
N
*
1.0
Children Adolescents
**
60
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
*** *
1
*
60
+
*** ***
1.5
Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal
90
*** ***
N
IL-17 CLA
CD8 IL17+CLA
1.5
+
90
0
N
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
**
AD
0.0
120
*
AD N
Children Adolescents
2.0
+
**
AD N
Infants
2
+
Infants.Normal
120
5
0
0
K
IL-22 CLA
N
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
**
+
+ +
Adults
5
0
AD
Adults
H
5.0
Children Adolescents
0
IL-17 CLA
5
AD N
Infants
20
*** **
20
N
20
*** ***
AD N
0.0
15
+ +
CD8 IL17+CLA+
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
J
+*
AD N
2.5
0
+
3
2.5
Adults
IL-22 CLA
60
6
3
Children Adolescents
7.5
0.0
N
AD N
7.5
2.5
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
N
10
2.5
0
6
+
10.0
** **
**
9
0
Infants
G
*
9
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
Adults
IL-17 CLA ** *
7.5
20
10
*** ***
+ +
+
IL-9 CLA
CD8 IL9+CLA
+
0
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
F
+*
+
50 0
** *
** *
50
Adults
IL-17 CLA
30
Frequency (%)
100
0.0
30
60
*** *
2.5
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
*** ***
150
** *
D
IL-9 CLA
CD8 IL9+CLA+
150
2.5
Infants
I
**
+*
0
0
** + + +*
5
20
E
+
5.0
20 0
C
IL-9 CLA +
7.5
*** **
40
CD8 T cells
0 0
N
AD N
AD N
AD
N
AD
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
Infants
Children Adolescents
Adults
N
AD N
AD N
AD
N
AD
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
Infants
Children Adolescents
Adults
Figure E6
●●
●
● ● ●● ●
● ●
● ●
●
● ● ●● ●
●
0
●● ● ●●●
●
●
● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●●● ● ● ● ● ● ●●●●● ● ● ●● ●● ●● ● ● ● ● ● ● ●● ●● ●● ● ● ● ● ● ●● ●● ● ● ● ● ●● ● ● ● ● ● ●●● ● ● ●● ● ●● ● ● ● ●●● ● ● ●● ● ●● ● ●●●● ●● ● ● ●● ● ● ●
20
●
● ●● ● ●
●
●
● ●●
● ● ●
●
● ●
● ●
● ● ● ●
●
●
● ● ●
●
●
●● ●
●● ● ● ●
●
● ● ●
●
● ● ● ●● ● ●●
●
● ● ●
0
20
40
60
80
r=−0.038 (CI: 0.045−0.43) ● p=0.71
p=0.71
p=0.0093 ●
●
● ●
●●
60
●
●
●
● ● ●
● ●
40
●
● ● ● ● ●
● ● ●
●
● ●
● ●
0
●● ● ● ● ● ●● ●● ● ● ●● ●● ● ● ●● ● ●● ●● ● ● ●● ● ● ●● ●● ●● ●● ● ● ● ● ● ● ● ● ● ●● ● ●●●● ● ● ● ● ● ● ●●● ● ●● ●● ● ●●● ● ● ●● ● ● ● ●● ● ●● ● ●● ● ● ●● ●● ● ● ●● ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ●
0
80
● ●
● ● ● ● ●
●
20
●
●
●
● ●
●
●●
●
● ●
● ●
●
●
●
●
●
● ● ●●●
●●
●
●
●
● ● ●
● ● ●
●
●
● ●
●
●
●
●
●
●
●
●
●
20
40
Age Age
60
100
● ●
15
50
●
● ●
25
0
● ●
●
● ●
●
● ● ●●● ●● ●● ● ● ● ●● ● ● ● ● ● ●
●
●
● ● ●● ●● ● ● ●●● ●●
● ●
● ●● ● ●● ● ● ● ●●
40
●
● ●
60
40
● ● ●
●
●
●
●●
20
●
●
● ●● ● ● ●● ●
● ● ● ●● ● ● ● ● ● ●●● ● ● ●● ● ●●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●
● ● ●
0
60
● ● ●
●
● ●
●
●
●
●
●
●
● ●
● ●
●
●
●
●
0
●
●
●
●
●
●
● ●●● ●● ● ● ● ● ● ● ● ●
●● ● ● ● ● ●
●
●
40
6 ●
15
●
20
●
●
● ●
● ●●
3
● ● ●
10
r=0.28 (CI: 0.056−0.47) r=0.28 (CI: 0.056–0.47) p=0.015 p=0.015
● ●
●
● ● ● ●● ● ●
0
● ● ●● ● ● ● ● ● ● ● ● ● ●●
0
●●
● ● ● ● ●●
●
● ● ●● ●
●
●
5
●● ●
●
● ● ●
●
●
● ●
●
●
●
●
0
Duration.of.disease
● ● ●
●
●
10
●
●
●
●
●
●
●
●
15
Duration of Disease
●● ● ● ● ● ● ● ●● ● ●● ● ● ●● ● ● ●● ● ●●● ●●
0
●● ● ● ● ● ●● ● ● ●
●
● ●
●
●
5
●
● ●
●
● ● ●●
● ●
● ●
● ●
● ● ●
10
●
●
● ● ●●
●
●
● ● ●
● ●
●
0
15
5
10
15
Duration.of.disease
L
Duration.of.disease vs. CD4 IFNg+CLA−
●
40
r=0.58 (CI: 0.41−0.71) r=0.58 (CI: 0.4–0.7) p=2.7e−08 ● p=0.000002
●
●
● ●
●
● ● ● ●
●
0 5
K Duration.of.disease vs. CD4 IL22+CLA+ ●
●
●
●
● ●
●
Duration of Disease
●
2
●
●
Duration.of.disease
●
●
●
● ●
●●
●
●
0
●
●
●
● ●
●
● ● ●
10
Duration.of.disease
10
● ●
● ●
Duration of Disease
● ●
● ●●
●●
0 15
●
● ● ● ●●
0
●
●
●
● ● ●● ● ● ● ● ● ●●
● ●
●
●
●
● ●
●
20
●
●
● ●
● ● ●
●
●
● ● ● ●
●
●
● ● ●
●
● ● ●
●
● ●
●● ●●●
● ●●
●
●
●
●
●
● ●
●
● ●
●
CD4⁺IFNγ⁺CLA ⁻ CD4 IFNg+CLA−
●
80
●
● ●
●
20
10
CD4⁺IL22⁺CLA⁺ CD4 IL22+CLA+
CD4⁺IL9⁺ CD4 IL9+
CD4⁺IL13⁺ CD4 IL13+
●
60
●
●
4 ●
●
Duration.of.disease vs. Pruritus.ADQ
● ● ●●● ● ●
●
p=0.0018 ●
●
Age Age
●
9
●
● ●
● ●● ● ● ● ●
●
●
● ●
● ●
● ●
●
●
20
30
●
●
●
● ●
20
● ● ● ●● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ●● ●●● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ●● ●●●●● ●●● ● ●● ●●●● ●● ●
●
●
●
r=0.35 (CI: 0.14−0.53) r=0.35 (CI: 0.14–0.53) p=0.0018
p=0.058
●
●
● ● ●
●
J Duration.of.disease vs. CD4 IL9+
r=0.22 (CI: −0.0077−0.42) r=0.22 (CI:● -0.0077–0.42) p=0.058
● ●●
●
●●
Duration of Disease
Duration.of.disease vs. CD4 IL13+
●
●●
●
●
●
●
Duration.of.disease
Duration of Disease
●
●
● ●●
●
● ●
●
●
●
●
● ●
r=0.22 (CI: −0.0064−0.43) r=0.22 (CI:●-0.0064–0.43) p=0.057 ● p=0.057 ● ●
●
● ●● ● ● ●
40
●
●
5
Duration.of.disease
● ● ●
H ●
●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●
●
0
●
●
●
0
15
40
●
●
●
●
●
80
●
●
80
●
10
●
●
● ● ● ● ●
●
●
●● ● ●
5
● ●
●
● ● ● ●● ● ●● ●
●●
●
● ●●● ● ●●● ●● ●● ●
0
●● ●
20
●
● ●
● ●
● ●● ●● ● ● ●● ● ● ● ● ●● ● ● ●●● ●● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ●● ●●●● ● ●● ●●● ● ●● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ●●● ●●● ●● ● ●● ●●● ●● ● ●●● ●● ●● ●
●
●
5
●
●
r=0.15 (CI: −0.072−0.36) r=0.15 (CI: -0.072-0.36) p=0.18 p=0.18 ●
60
EASI EASI
●
●
●
● ● ● ● ● ● ●
G Duration.of.disease vs. SCORAD
SCORAD SCORAD
● ●● ● ●
●
●
● ●
●
●
10
●
●
●
p=0.00065
● ●●
●
Age Age
●
0
●
12
●
Age vs. CD8 EM HLADR+/CLA+ r=0.41 (CI: 0.23−0.55) r=0.026 (CI: 0.22−0.56) r=0.41 (CI: 0.23–0.55) r=0.026 (CI: 0.22-0.56) ● p=1.6e−05 p=0.8 ● ● ● ● ● p=1.6e-05 p=0.8
Age Age
r=0.38 (CI: 0.17−0.55) r=0.38 p=0.00065 (CI: 0.17–0.55)
p=1.1e-56
I
75
80
F Duration.of.disease vs. EASI
r=0.98 (CI: 0.97−0.99) r=0.98 (CI: 0.97–0.99) p=1.1e−56
●
r=−0.059 (CI: 0.24−0.58) p=0.57
Age Age
EDuration.of.disease vs. Age
0
r=0.42 (CI: 0.25–0.57) r=-0.059 (CI: 0.24-0.58) p=7.3e-06 p=0.57 r=0.42 (CI: 0.25−0.57) ● p=7.3e−06
HLA-DR⁺CLA⁺CD8⁺ Tem cells CD8 EM HLADR+/CLA+
●
●●
40
p=0.16
● p=0.0056
D
C Age vs. CD8 CM HLDR+/CLA+
r=0.24 (CI: 0.062–0.41) r=-0.038 (CI: 0.045–0.43) r=0.24 (CI: 0.062−0.41) p=0.0093
Pruritus ADQ Pruritus.ADQ
60
r=−0.14 (CI: 0.064−0.44) ● p=0.16
CD8 CM HLDR+/CLA+ HLA-DR⁺CLA⁺CD8⁺ Tcm cells
B Age vs. CD4 EM ICOS+/CLA+
r=0.26 (CI: 0.078–0.43) r=-0.14 (CI: 0.064–0.44) r=0.26 (CI: 0.078−0.43) p=0.0056
ICOS⁺CLA⁺CD4⁺ Tem cells CD4 EM ICOS+/CLA+
ICOS⁺CLA⁺CD4⁺ Tcm cells CD4 CM ICOS+/CLA+
A Age vs. CD4 CM ICOS+/CLA+
●
30 ● ● ●● ●
●
●
●
●
0
5
10
15
Duration.of.disease Duration of Disease
●
●
●
● ●
●
● ●
●
●
●
●
● ●
● ●
●
●● ● ● ● ●
● ●
●
●
●
● ●
●
● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ●● ●
0
●
●
●
●
10
●
● ●
●
20
●
● ● ●
●
5
● ●
10
15
Duration.of.disease Duration of Disease
CD4⁺ T cells
A Age vs. CD4 IFNg+CLA+ r=0.44 (CI: 0.28–0.57) r=0.44 (CI: 0.28−0.57) ● p=5.5e−07 ●
p=0.013
10
0
●
●
● ●
●
p=4.7e-09
● p=0.1
●
●
●
●
● ●
●
● ● ● ● ● ● ● ● ● ● ●●●● ● ● ● ● ● ● ●● ● ●● ●● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●●● ●● ● ●● ● ● ● ● ● ● ●● ●● ● ●● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ●●● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ●●● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ●● ● ● ● ● ● ●● ● ●●● ● ● ● ● ● ● ● ●●● ● ● ●● ● ● ●● ● ●
0
20
40
●
●
●
40
●
● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●●● ●● ● ● ● ●●● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●●●●● ●●● ● ● ● ●● ● ●● ●● ● ●●● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ●● ●●● ● ● ● ● ● ●● ● ● ●●● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ●● ● ● ● ●● ●● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ●● ● ●
20
80
0
20
40
Age Age
40
20
●
r=−0.14 (CI: −0.32−0.036) ● p=0.12
p=0.12
p=0.41
75 ●
● ●
●
● ●
●● ● ●
● ● ● ●● ●● ● ● ● ● ● ● ●●● ● ● ● ● ● ●● ● ● ● ●● ● ●● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ●●
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●
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●
●
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●
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● ●● ●● ● ●● ●● ●● ● ●●●●● ● ● ●●● ● ●● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ●● ● ●● ● ● ● ● ● ● ●●●● ●● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ●
● ● ● ●
● ● ● ● ●
●
●
● ●
●
50
25
● ● ● ●
0
●
40
60
80
● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ●● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ●● ●●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ●● ●● ●● ● ● ● ● ● ●● ●● ●● ● ● ● ● ● ●● ● ●
0
20
40
●
●
60
80
Age Age
H
r=-0.4 (CI: -0.54–-0.24) r=-0.21 (CI: -0.56–-0.23)
Age vs. CD8 IL9+CLA−
(CI: ● -0.071–0.29) p=0.23 8 r=-0.11 r=0.11 (CI: −0.071−0.29)
r=−0.21 (CI: −0.56−−0.23) p=0.036
p=5e-06 ●
●
●
●
●
●
●
●
●●
● ● ●●
●
●
●
●●
20
● ● ●
● ●
r=−0.4 (CI: −0.54−−0.24) ● p=5e−06
4
p=8.2e-05
●
●
G Age vs. CD8 IL9+CLA+
p=0.35
r=0.38 (CI: 0.36−0.65) r=0.38 (CI: ● 0.36-0.65) ● p=8.2e−05
●
●
Age Age
r=−0.094 (CI: −0.33−0.052) p=0.35
●
●
●
● ● ● ●
●●
0
80
r=-0.14 (CI: -0.32–0.036) r=0.-0.094 (CI: -0.33–0.052)
r=−0.082 (CI: −0.28−0.11) p=0.41
p=0.34
60
●
● ● ● ● ●
0
F Age vs. CD4 IL9+CLA−
r=-0.088 (CI: -0.26–0.093) r=-0.082 (CI: -0.28–0.11) 40
● ● ●● ●
●
p=1.3e-09
●
● ●
Age Age
EAge vs. CD4 IL9+CLA+ r=−0.088 (CI: −0.26−0.093) ● p=0.34
●
●
0
60
●● ● ●
● ● ●
Age vs. CD8 IFNg+CLA− r=0.52 (CI: 0.37−0.64) r=0.52 (CI: 0.37–0.64) ● p=1.3e−09
r=0.18 (CI: −0.047−0.34) p=0.076 ●
p=0.076
● ●
●
CD8 IFNg+CLA+ IFN-γ⁺CLA⁺
30
p=1.4e-15
●
IFN-γ⁺CLA⁻ CD4 IFNg+CLA−
IFN-γ⁺CLA⁺ CD4 IFNg+CLA+
●
r=0.15 (CI: -0.031–0.32) r=0.18 (CI: -0.047-0.34)
60
● ●
●
r=0.54 (CI: 0.52-0.75)
r=0.15 (CI: −0.031−0.32) ● p=0.1
r=0.54 (CI: 0.52−0.75) ● p=4.7e−09
60
●
D
CAge vs. CD8 IFNg+CLA+
r=0.65 (CI: 0.53–0.74) r=0.65 (CI: 0.53−0.74) p=1.4e−15
r=0.25 (CI: 0.26–0.58)
40
20
B Age vs. CD4 IFNg+CLA−
r=0.25 (CI: 0.26−0.58) p=0.013 ●
p=5.5e-07
CD8⁺ T cells
IFN-γ⁺CLA⁻ CD8 IFNg+CLA−
Figure E7
p=0.23
p=0.036
r=0.21 (CI: −0.088−0.3) r=0.21 (CI:p=0.033 -0.088–0.3)
p=0.033
60
10
0
0
20
40
2
● ●
● ● ●●
●
0
60
●
● ●
1
●
●
80
●
●
● ● ●
● ● ● ● ● ● ● ●● ●●●● ● ● ●●● ● ● ●● ● ●● ● ● ● ● ● ● ●●● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ●● ●●● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●●●●●●● ● ●●● ● ● ● ●●● ● ● ●● ● ●●● ●● ● ● ●●● ● ●● ● ●● ● ●● ●●●●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●●●●● ● ●● ●● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ●●● ● ● ● ● ●● ● ● ●
●
0
20
40
Age
Age vs. CD4 IL17+CLA+
r=0.2 (CI: 0.019−0.36) 15 r=0.2 ● (CI: 0.019–0.36) p=0.031
p=0.031
p=0.37
p=7.6e-08
●●
5
0
IL-17⁺CLA⁻ CD4 IL17+CLA−
IL-17⁺CLA⁺ CD4 IL17+CLA+
●
●
● ● ●
●
● ●
● ●
●
● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ●● ● ● ● ●● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ●● ● ●● ● ●●● ● ● ●● ● ●● ● ● ● ● ● ● ●●●● ● ●● ●● ●●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ●●● ● ● ●● ● ●● ● ● ●● ● ● ●● ● ● ●● ● ● ●●● ● ● ● ● ● ●● ● ●● ● ●●● ● ●● ● ●● ● ● ● ● ● ● ●● ● ●●●● ● ●●●● ● ● ●● ● ●● ● ● ●● ● ● ● ●● ● ● ● ●● ●● ●
0
20
40
Age
Age
60
0
80
20
40
60
r=0.27 (CI: 0.3−0.61) r=0.27 (CI: 0.3-0.61) p=0.0063
p=0.0063
●
2
● ● ● ● ● ● ●
● ● ●
1
●
0
●
●
●
●
●
● ● ●
● ● ●
20
● ●
●● ●● ●
●
●
●
●
● ●
●
● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ●●● ●● ● ● ● ●● ● ● ●● ● ● ●● ● ● ● ● ●● ● ●● ●● ●●● ●● ● ●● ●● ● ● ● ● ● ● ● ●●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●
0
●
●
●●
●
60
0
80
20
40
●
● ● ● ●
●
60
80
Age
Age
L
Age vs. CD8 IL17+CLA−
p=0.27
60
r=0.46 (CI: 0.31−0.59) r=0.42 (CI: 0.29−0.6) r=0.46 (CI: 0.31–0.59) r=0.42 (CI: 0.29-0.6) p=1.1e−07 ● p=9.6e−06 p=1.1e-07 p=9.6e-06
●
3 ●
● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ●● ●●● ●● ●● ● ● ● ● ● ●●●● ● ● ●●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●●●● ● ● ●●● ● ●● ● ● ● ●● ● ● ● ●● ● ●● ● ●● ● ● ●●●●● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ●● ●
30
●
● ● ● ● ●
● ● ● ●
●
r=0.11 (CI: −0.057−0.33) p=0.27
p=0.12
●
●
● ●
2
●
● ●
●
r=0.14 (CI: -0.038–0.31) r=0.11 (CI: -0.057-0.33) r=0.14 (CI: −0.038−0.31) ● p=0.12
●
●
80
●
●
Age
K Age vs. CD8 IL17+CLA+
r=0.47 (CI: 0.31−0.6) r=0.47 (CI: 0.31–0.6) p=7.6e−08 ●
●
4
0
J Age vs. CD4 IL17+CLA−
r=-0.091 (CI: 0.00018–0.38)
6
●
Age
r=−0.091 (CI: 0.00018−0.38) p=0.37
●
●
20
● ● ● ● ● ● ●●● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ●● ● ●● ● ● ●● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ●● ●●● ●● ●● ● ●● ● ● ● ● ●● ● ● ●●● ●● ●●● ● ●● ● ● ● ● ● ●●● ● ● ●●●● ●● ● ● ●● ●●●●● ● ● ● ●●● ● ● ● ●● ●● ● ● ●●● ●●● ● ● ● ● ● ●●● ● ● ●●● ● ● ●● ● ● ●●● ● ● ●● ● ● ● ● ● ● ●● ●●●●●●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●
Age
4
10
●
Age
Age
I
●
● ● ●
0 ●
●
● ●
40
●
●
●
IL-9⁺CLA⁻
● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ●●● ● ●● ●● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ●● ● ● ●●● ● ● ● ● ● ● ●● ● ●● ● ●● ● ● ● ● ●● ●● ● ● ●● ●● ● ● ●●●● ● ● ● ● ●●● ● ● ●●● ● ●●●● ●● ● ●● ● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●●●● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ●●
● ●● ●
●
● ●
CD8 IL9+CLA−
●●
●
IL-17⁺CLA⁺ CD8 IL17+CLA+
20
●
●
3
● ● ●●
20
●
10
● ● ● ●● ●●● ● ●
●
●●
●
40
Age
Age
●
●
● ●
● ● ● ●● ●
●● ● ● ● ●
● ●
● ●
●
60
●●
●
●
80
IL-17⁺CLA⁻ CD8 IL17+CLA−
●
● ●
IL-9⁺CLA⁺
●
●
CD8 IL9+CLA+
IL-9⁺CLA⁻
●
CD4 IL9+CLA−
IL-9⁺CLA⁺
CD4 IL9+CLA+
●
30
●
●
●
0
●
●
● ●
20
20
●
● ● ● ● ● ● ● ● ●● ● ●● ●● ● ● ●● ●● ● ●● ● ● ● ●●●● ● ● ●● ● ●●● ● ●●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ●● ●●● ●● ● ●● ●● ●● ●● ● ● ● ● ● ● ● ●●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ●●● ● ● ● ●●●● ● ● ●●●● ● ●● ● ● ●●● ● ●●
0
40
40
Age
Age
60
●
●
0 80
● ● ● ●● ● ● ● ● ●● ●●●●● ●● ● ● ● ● ● ● ●●● ● ● ● ●●● ● ●● ● ●●● ●●●● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ●● ●●●●● ●●●●● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ●●● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ●
0
20
40
Age
Age
60
●
●
80
Figure E8 A SCORAD vs. CD4 IL13+CLA+
B SCORAD vs. CD8 IL9+CLA−
●
4
●
●
p=0.0034
●
●
●
●●
● ●
● ●
●
●
●
●
●
●
● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ● ● ● ●● ●● ●● ● ●● ● ●● ● ● ●● ● ● ● ● ●
● ● ● ● ● ● ● ● ●
●
●
●
●
●
●●
●
●
●
●
●
40
60
● ●
●
●
2 ●
●
● ● ● ●
● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ●● ● ● ●● ● ● ● ● ●● ●●● ● ● ● ●● ● ● ●● ● ●●● ● ● ● ● ● ●●● ● ●● ● ● ● ● ●● ● ● ●
● ● ● ●●
●
●
●
●
0
r=0.24 (CI: 0.021−0.45) r=0.24 (CI:● 0.021–0.45) p=0.033
●
p=0.033
●
4
80
20
100
40
60
Pruritus ADQ Pruritus.ADQ
● ● ●
5.0 ● ●
● ●
●
●
●
●
● ●
● ●
20
●
● ● ●● ● ● ● ● ● ●● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ●●● ● ● ● ●● ● ●
10
●
● ●●
10.0
● ● ●
●
●
● ●●
● ● ●
●
●
● ●●
●
●
●
●
●
●
●● ●
●
● ● ● ● ●● ●● ● ● ●
●
25
●
●
●● ●
●
●
●
20
40
60
H Pruritus.ADQ vs. Eosinophils r=0.39 (CI: 0.15−0.58) r=0.39 p=2.7e−07 (CI: 0.15–0.58) p=2.7e-07
● ● ●
5.0 ● ●
●
2.5
●
0.0 40
60
●
●
0
●
● ●
●
●
●
●
● ● ● ● ● ● ● ● ● ● ●
10
● ●
●
15
● ● ● ● ● ●
●
20
●
●
●
5
● ●
●
●
●
●
●
●
●
● ● ● ●
● ●
●
●
●
●
● ●
0 10
30
20
30
Pruritus ADQ Pruritus.ADQ
L
K TEWL.NL vs. CD4 IL22+ 10.0
●
●
Pruritus ADQ ●
● ●
● ●
●
● ●
● ●
Pruritus.ADQ
r=0.15 (CI: −0.11−0.38) r=0.15 (CI: -0.11–0.38) ● p=0.06 p=0.6
●
●
10
●
● ●
●
● ● ●
● ●
● ●
● ●
●
●
● ● ●
●
● ● ● ● ● ● ●
●
●
●
● ● ●
●
r=0.16 (CI: −0.098−0.39) r=0.16 (CI: -0.098–0.39) ● p=0.045 p=0.045
10.0
● ●●
● ●
50
TEWL.LS TEWL LS
75
●
●
● ●
● ●
●
● ● ●
●
● ●
●
●
●
● ● ● ● ● ● ●● ●
● ●
0.0 100
TEWL.LS vs. CD4 IL22+ r=0.28 (CI: 0.035−0.49) r=0.28 (CI: 0.035–0.49) p=0.00025 p=0.00025
●
●
●
20
●
30
40
0.0
50
SCORAD SCORAD
●
●
● ●
● ●
● ●
●
●
●
● ●
● ● ● ●● ●● ●
●
● ● ● ●● ● ● ● ● ● ●● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ●
●●
● ● ●
● ● ● ●
●
● ● ● ● ●
●
●
●
●
●
●
●
● ●
●
●
● ●
● ●●
40
40
TEWL NL TEWL.NL
60
● ● ●
0
● ●
20
●
40
TEWL NL TEWL.NL
60
80
● ●
● ●
●
50
50
75
100
●
●
●
●
●
● ● ●●
● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●
60
●● ●
●● ● ●
40
● ●
● ● ●
●
●
● ●
●
●
●
●
●
●
●●
20 100
● ●
●
●
●
TEWL.LS TEWL LS
●
● ●
●
0 0
●
TEWL.LSLS TEWL
●
●
●
●
●
●
● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ●● ● ● ● ●● ●●● ● ● ● ●
20
●
●
● ●
● ●
●
r=−0.063 (CI: −0.26−0.13) r=-0.063 (CI: -0.26–0.13) ● p=0.42 ● ● p=0.42 ● ●
● ●
●
●
P TEWL.LS vs. SCORAD
●
●
80
25
●
●
●
●
● ● ● ● ● ●●●● ● ● ● ● ●● ● ●● ● ● ● ● ●●
50
●
●
●
●●
20
40
●
●
●● ●
●
●
●
30
TEWL.NLNL TEWL
80
● ●
20
● ●
● ●
●
0.0
●
●
● ●
●
●
●
●● ●
●
●
● ● ● ● ●
●
0 20
● ●● ●
40
●
●
● ●
● ●
r=0.076 (CI: −0.12−0.27) r=0.076 (CI: -0.12–0.27) ● p=0.45 p=0.45 ●
60
●
●
●
●
● ● ● ● ●
● ●
●
●
60
● ●
●
O TEWL.LS vs. EASI
● ●
●
●
5.0
● ● ● ● ● ●●
●●
10
● ●
2.5 ●●
● ● ●
7.5
●
●
●
● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●
●●
40
●
● ● ● ●●
●
● ●
TEWL.NL TEWL NL
●
● ●● ●
●
● ●
● ●
●
●
●
r=0.18 (CI: −0.015−0.37) r=0.18 (CI: -0.015–0.37) ● p=0.019 ● p=0.019 ● ●
80
●
●●
● ● ● ●
10
●
●
2.5
●
●
●
●
NTEWL.NL vs. SCORAD
r=0.29 (CI: 0.11–0.46)● p=0.0027
●
● ●
●● ●
●
●
5.0
●
●
● ●
●
●● ●
●
●
5.0
●
●
●
●
●
2.5 ●
7.5
● ●
r=0.29 (CI: 0.11−0.46) p=0.0027
0
● ●
●
EASI EASI
7.5
●
7.5
MTEWL.NL vs. EASI
●
●
● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ●● ● ● ●● ● ● ●● ● ●● ● ● ●●●●● ● ● ● ● ●
0
CD4 IL22+ CD4⁺IL-22⁺
● ●
20
● ● ● ● ● ●
100
SCORAD SCORAD
CD4 CLA+ CD4⁺CLA⁺
●
●
●
1
●
80
●
20
● ●
●
●
60
●
J TEWL.NL vs. CD4 CLA+ ●
●
0.0
●
●
●
●
●
EASI EASI
●
2.5
●
●
0
60
r=−0.28 (CI: −0.49−−0.035) r=-0.28 (CI:● -0.49–-0.35) p=0.00026 p=0.00026 ●
●
60
●
● ●
r=0.17 (CI: −0.067−0.38) r=0.17 (CI: -0.067–0.38) ● p=0.031 p=0.031
●
TEWL.LS vs. CD4 CLA+
5.0
40
●●
●
●
●
●
2
20
●
EASI
7.5
20
●
●
EASI
10.0
●
10.0
●
●
● ●
40
●
●
SCORAD
●
● ● ●
0
20
I
●
●
●●
●
●●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ●● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●
CD4 IL22+ CD4⁺IL-22⁺
0.0
●
●
10
G Pruritus.ADQ vs. CD4 IL22+
●
● ● ●● ● ● ● ● ● ● ● ● ●
●
●
●
●●
●
●
● ● ● ●●●
● ● ●● ● ● ● ●● ● ●● ● ● ●● ● ●● ● ● ● ● ● ●● ● ●● ● ● ● ●● ●● ● ●
●
●
●
●
100
●
30
7.5
●
● ●
● ●
3
SCORAD
r=0.46 (CI: 0.3−0.59) r=0.46 (CI: 0.3–0.59) ● ● ● ●● p=4.4e−07 p=4.4e-07 ●
●
●
80
●
●
●
●
F EASI vs. Pruritus.ADQ ●
● ●
●
SCORAD
p=0.0086
2.5
●
20
SCORAD
r=0.3 (CI: 0.079−0.49) r=0.3 (CI: 0.079–0.49) ● p=0.0086
● ● ● ● ● ●
● ●
●
●
0
EEASI vs. CD4 IL22+
● ●
●
●
●
SCORAD SCORAD
10.0
●
●
1
EASI EASI
10
● ●
●
●
●
CD4 IL22+ CD4⁺IL-22⁺
20
3
EASI vs. CD4 IL9+CLA−
●
CD4⁺IL-9⁺CLA⁻ CD4 IL9+CLA−
●
●
20
CD4 IL22+ CD4⁺IL-22⁺
r=0.23 (CI: 0.047−0.39) r=0.23 (CI: 0.047–0.39) p=0.0034
Eosinophils Eosinophils
●
●
0
CD4 CLA+ CD4⁺CLA⁺
30
CD4⁺IL-22⁺CLA⁺ CD4 IL22+CLA+
● ●
●
●
EASI EASI
D
CSCORAD vs. CD4 IL22+CLA+
r=0.28 (CI: 0.099−0.44) r=0.28 ● p=0.00031 (CI: 0.099–0.44) p=0.00031
● ●
CD8⁺IL-9⁺CLA⁻ CD8 IL9+CLA−
CD4⁺IL-13⁺CLA⁺ CD4 IL13+CLA+
30
r=0.2 (CI: 0.016−0.37) r=0.2 p=0.011 (CI: 0.016–0.37) p=0.011
150
0
50
100
TEWL.LSLS TEWL
150
Figure E9 60 60
Frequency (%)
B
CD25 CD127 Tregs
T Regs (CD25+CD127 )
*** *** *** **
40 40
** **
*** ***
* **
** **
**
** **
*** ***
20 20
*** ***
** ** 80 80
N
AD
N
AD
N
AD
Infants
Children
N
Adolescents
CAge vs. T Regs (CD25+CD127
AD
** **
*** ***
+
** * +
N
r=0.2 (CI: 0.015 0.38) r=0.2 (CI: 0.015–0.38) p=0.034
p=0.034
)
r= 0.092 (CI: 0.0017 0.39) p=0.38
r=-0.092 (CI: -0.0017–0.39)
10
N
Infants
AD
Children
N
AD
Adolescents
N
AD
Adults
D Age vs. Tregs CCR4+CLA+ 60
p=0.38
20
AD
Infants.NormalInfants.AD Children.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.NormalAdults.AD
Adults
CCR4 CLA Tregs Tregs CCR4+CLA+
CD25 CD127 Tregs T Regs (CD25+CD127 )
** *
40 40
Infants.NormalInfants.ADChildren.Normal Children.AD Adolescents.Normal Adolescents.AD Adults.Normal Adults.AD
0
** * + *
00
00
30
CCR4 CLA Tregs
Tregs CCR4+CLA+
120 120
* **
Frequency (%)
A
r=0.23 (CI: 0.043 0.4) r=0.23 p=0.016 (CI: 0.043–0.4)
r=0.4 (CI: 0.029 0.41) r=0.4 (CI:p=5e 0.029-0.41) 05
p=0.016
p=5e-05
40
20
0 0
20
40
Age
Age
60
80
0
20
40
Age
Age
60
80
1 1
Supplementary Materials and Methods
2
Isolation of Peripheral blood mononuclear cells and multiparameter flow
3
cytometry blood analyses
4
Peripheral blood mononuclear cells (PBMCs) were obtained by gradient
5
centrifugation with Ficoll-Paque Plus (Amersham Biosciences, Pittsburgh, PA).
6
The blood was laid under the Ficoll gradient; after spinning, the PBMCs were
7
collected at the interface between the plasma and the Ficoll gradient and washed
8
with PBS before staining for flow cytometry analysis. For T-cell surface staining,
9
PBMCs were washed and incubated for 30 minutes in ice with fluorochrome-
10
conjugated monoclonal antibodies (mAbs) to cell surface molecules (CD3-Pe AF
11
610, CD8-V-500, CD4-Qdot800, CD45RO-PeCy7, CLA-FITC, ICOS-
12
PerCpCy5.5, CCR7-Alexa Fluor 700, HLA-DR-APC-H7, CCR4-BV 421, CD25-
13
PE, CD127-BV 650, Live/dead-Blue Dye). Then, the cells were fixed in ice with
14
4% paraformaldehyde (BD Biosciences, San Jose, CA) for 20 minutes. For
15
intracellular cytokine staining, whole blood was activated with 25 ng/mL of
16
phorbol 12-myristate 13-acetate (PMA) and 2 µg/mL of ionomycin in the
17
presence of 10 µg/mL brefeldin A (protein transporter inhibitor) for 4 hours at
18
37°C. All reagents were purchased from Sigma Aldric h (St. Louis, MO). Non-
19
activated controls were only treated with brefeldin A. After this incubation, EDTA
20
(2 mM; Invitrogen, Grand Island, NY) was added for 15 minutes at room
21
temperature (RT) to stop activation. The blood was then incubated with FACS
22
lysing solution for 10 minutes at RT and washed with PBS twice. Then, the cells
23
were incubated for 20 minutes with fluorochrome-conjugated mAbs to cell
2 24
surface molecules (CD3-Pe AF 610, CD4-Qdot800, CLA-FITC). Subsequently,
25
the cells were permeabilized (Perm/Wash; BD Biosciences, San Jose, CA) and
26
incubated for 20 min with fluorochrome conjugated mAbs (IL-13-PerCpCy5.5, IL-
27
22-PeCy7, IL-9-Pe, IFN-γ- Alexa Fluor 700 and IL-17-APC efluor 780) in 5%
28
mouse serum (eBioscience, San Diego, CA). All incubations for cell surface and
29
intracellular staining were done at RT. Appropriate FMO-isotype controls were
30
used. Following incubations, cells were washed and subsequently acquired with
31
a BD LSR II flow cytometer (BD Biosciences) and analyzed by flow-Jo software
32
(Treestar, Ashland, OR).
33
3 34
Supplementary Figure legends
35
Figure E1. After gating on CD3+ viable T-cells using flow cytometry, CD3+CD4+
36
and CD3+CD8+ were defined and analyzed separately. CCR7 and CD45RO were
37
used to differentiate memory subsets within the CD4 and CD8 populations.
38
CCR7+CD45RO- defined naïve cells, CCR7+CD45RO+ central memory T-cells
39
(Tcm), CCR7-CD45RO+ effector memory T-cells (Tem) and CCR7-CD45RO-
40
Effector/Temra/terminally differentiated T-cells (A-C). We then further defined
41
activated ICOS/HLA-DR activated Tcm/Tem memory subset, using CLA to
42
segregate skin-homing (CLA+) versus systemic (CLA-) subsets, for each memory
43
population. Gating strategy for (A) CD3 vs. FSC-A, (B) CD4 vs. CD8, and (C)
44
CCR7 vs. CD45RO cells. (D-G) CD4+/CD8+ Tem (CD45RO+CCR7-) and Tcm
45
(CD45RO+CCR7+) subset frequencies and (H-K) HLA-DR+CLA+/-
46
CD45RO+CCR7+/- in AD and their respective controls across ages. Bar plots
47
represent means (black)/medians (red) ± SEMs. AD, Atopic dermatitis; CLA,
48
Cutaneous lymphocyte antigen; CCR, C-C Motif Chemokine Receptor 7; FSC-A,
49
Forward Scatter-Area; SEM, Standard error of the mean/median; Tcm, central
50
memory T-cells; Tem, effector memory T-cells. P-values are designated
51
as ***<0.001, **<0.01, *<0.05, and +<0.1.
52
Figure E2. (A-H) Pie charts showing the distribution of CD4+/CD8+ naïve, Tcm,
53
Tem, and effector T-cells in infants, children, adolescents and adults. (I-P)
54
Spearman correlation scatter plots (linear regression [red=AD/blue=control line]
55
with their CI [in gray]) for frequencies (%) of naïve/effector/TCM/TEM T-cells in the
56
CD4+ (I-J, M-N) and CD8+ (K-L, O-P) compartments versus age in AD patients
4 57
(red dots/line) and controls (blue dots/line). AD, Atopic dermatitis; CI, Confidence
58
interval; r, Spearman correlation coefficient with CI and associated P-value; Tcm,
59
central memory T-cells; Tem, effector memory T-cells.
60
Figure E3. Gating strategy of IFN-γ, IL-13, and IL-22 versus CLA positivity in
61
CD4+ and CD8+ non-activated and PMA/ionomycin activated T-cells in a
62
representative patient with AD. CLA, Cutaneous lymphocyte antigen.
63
Figure E4. A-D) IFN-γ+, (E-H) IL-13+, and (I-L) IFN-γ+/IL-13+ cytokine
64
frequencies in CLA+, CLA-, CD4+ and CD8+ T-cells in AD and their respective
65
controls across ages. The ratio between the % of IFN-γ+CLA+CD4+ (or CD8+) T
66
cells and the % of their IL-13+ counterpart was calculated for each sample but
67
was not multiplied by 100 and is therefore unitless. Bar plots represent means
68
(black)/medians (red) ± SEMs. AD, Atopic dermatitis; CLA, Cutaneous
69
lymphocyte antigen; SEM, Standard error of the mean/median. P-values are
70
designated as ***<0.001, **<0.01, *<0.05, and +<0.1.
71
Figure E5. A-D) IL-9+, (E-H) IL-17+ and (I-L) IL-22+ frequencies in CLA+, CLA-,
72
CD4+ and CD8+ T-cells in AD and their respective controls across ages. Bar plots
73
represent means (black)/medians (red) ± SEMs. AD, Atopic dermatitis; CLA,
74
Cutaneous lymphocyte antigen; SEM, Standard error of the mean/median. P-
75
values are designated as ***<0.001, **<0.01, *<0.05, and +<0.1.
76
Figure E6. Spearman correlation scatter plots (linear regression
77
[red=AD/blue=control line] with their CI [in gray]) for (A-D) frequencies (%) of
78
ICOS+ and HLA-DR+ activated CLA+/CD4+/CD8+ Tem (CD45RO+CCR7-) and
79
Tcm (CD45RO+CCR7+) T-cells versus age and (E-L) disease duration versus
5 80
clinical scores and various cytokine-producing T-cell subsets in AD patients (red
81
dots/line) and control subjects (blue dots/line). CI, Confidence interval; CLA,
82
Cutaneous lymphocyte antigen; EASI, Eczema area and severity index; r,
83
Spearman correlation coefficient with CI and associated P-value; SCORAD,
84
SCORing of Atopic Dermatitis.
85
Figure E7. Spearman correlation scatter plots (linear regression
86
[red=AD/blue=control line] with their CI [in gray]) for frequencies (%) of
87
CD4+/CD8+, CLA+/CLA- (A-D) IFN-γ +, (E-H) IL-9+, and (I-L) IL-17+ producing T-
88
cells versus age in AD patients (red dots/line) and controls (blue dots/line). CI,
89
Confidence interval; CLA, Cutaneous lymphocyte antigen; r, Spearman
90
correlation coefficient with CI and associated P-value.
91
Figure E8. Spearman correlation scatter plots (linear regression
92
[red=AD/blue=control line] with their CI [in gray]) for (A-C) SCORAD, (D-F) EASI,
93
(G-H) Pruritus, and (I-P) TEWL versus clinical measures and frequencies (%) of
94
various T-cell subsets. Dots colors represent different AD patient ages from
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infancy to adulthood as shown in Figure 1. CI, Confidence interval; CLA,
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Cutaneous lymphocyte antigen; EASI, Eczema area and severity index; LS,
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Lesional; NL, Non-lesional; SCORAD, SCORing of Atopic Dermatitis; TEWL,
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Transepidermal water loss.
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Figure E9. A) Total and B) skin-homing Treg frequencies in AD and their
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respective controls across ages and (C-D) Spearman correlation scatter plots
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(linear regression [blue=control/red=AD lines] with their CI [in gray]) for Treg
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frequency (%) versus age. Bar plots represent means (black)/medians (red) ±
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SEMs. r, Spearman correlation coefficient with CI and associated P-value. AD,
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Atopic dermatitis; CI, Confidence interval; CLA, Cutaneous lymphocyte antigen;
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SEM, Standard error of the mean/median; Tregs, T-regulatory cells. P-values are
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designated as ***<0.001, **<0.01, *<0.05, and +<0.1.