Phenotypic Changes That TCR Vγ3+ Fetal Thymocytes Undergo During Their Maturation into Dendritic Epidermal T Cells

Phenotypic Changes That TCR V 1'3 + Fetal Thymocytes Undergo During Their Maturation into Dendritic Epidermal T Cells Elisabeth Payer, Raphaela Kutil, and Georg Stingl Division of Immunology, Allergy, and Infectious Diseases, Department of Dermatology, University of Vienna Medical School, Vienna International Research Cooperation Center, Vienna, Austria

Murine Thy-1 +. TCR Vy3NBt + dendritic epidennal

expression until or shortly after arrival in the epider­

T cells (DETC) express CD2 antigens. but differ from

mis. A similar loss of CDS expression by TCR Vy3+

most other T-cell subsets in their absence of CD4.

cells was observed in vitro under various culture con­

CDS. and CDS antigens. To determine whether neg­

ditions. To determine whether expression of CDS is

ativity for those antigens is an intrinsic feature of a

important for the maturation of DETC. we searched

given T-cell population or whether such triple-neg­

for these cells in the epidermis of CDS-deficient

ative T cells go through a maturational stage during

mice. There was no alteration in the number of

which they express these antigens. we determined the

Thy-1 +/TCR Vy3+ dendritic cells in the epidermis of

phenotype ofTCR Vy3+ fetal thymocytes. which are

CDS -/- mice. Even though the latter finding speaks

the precursor cells of DETC. We found that TCR

against a pivotal role of CDS during the maturation

Vy3 + fetal thymocytes at day 17 of gestation are

of DETC. the described cell system may serve as a

CD2+. CDS+. mosdy CDS+. and pardy CD4+. The

useful tool in further experiments aimed to clarify

expression of CDS is highest on early TCR Vy3+

the function of the CDS glycoprotein as well as the

thymocytes; these cells express intermediate levels of

mechanism(s) regulating its expression. Key words:

CDS when they leave the thymus and lose CDS

CD51CD21TCR "1/0.] Invest DennatoI105:54S-57S, 1995

CHARACTERIZATION AND ONTOGENY OF DENDRITIC

DETC was the finding that the thymus from fetuses at 14-1S d of

EPIDERMAL T CELLS (DETC)

gestation contains cells expressing exactly the same TCR V'Y3/V81 as DETC, which suggests that these thymocytes may be DETC

The murine epidermis contains two populations of bone-marrow­

precursor cells

derived dendritic cells, major histocompatibility complex (MHC)

resulted in the appearance of donor-type DETC in the recipients'

(DETC or Thy - 1 + DEC). The presence of dendritic Thy-l + cells in the epidermis was first reported in

19S3.

epidermis.

Besides Thy-1, these cells

contrast,

transplantation

or of fetal thymocytes depleted of TCR

CDS, CD4, CDS, and MHC class II antigens [1,2]. Because of this

to the generation of DETC

phenotype , it was not clear whether these cells are T cells or natural

that TCR

killer cells. The discovery of a TCR "118 on their surface 4 years

[3,4].

In

of

thymuses

from

newborn mice, or the injection of thymocytes from adult mice

express CD4S and asialo-GM1 antigens, but they are negative for

later finally established their T -cell lineage

Indeed, transplantation of fetal thymuses or

[5].

injection of fetal thymic suspensions into congenic adult mice

class 11+ Langerhans cells and Thy-1 + dendritic epidermal T cells

V'Y3+

[S,9].

V'Y3 +

cells, did not lead

These data clearly showed

fetal thymocytes are the actual DETC precursor

cells. Further research indicated that only stem cells from the fetal

However, they are

liver have the capacity to mature into TCR

very unusual T cells. T cells in peripheral organs such as lymph

V'Y3 +

cells and that the

fetal thymic milieu is crucial for the development of these cells

nodes and spleen express a wide variety of different TCR (mostly

[ 1 0 11 ] ,

TCR exl{3, with a few cells expressing TCR "1/8, both with a

.

The maturation of T cells involves a whole cascade of

different steps, as has been shown for TCR exl{3 cells (reviewed in

variety of different ex and (3 or "1 and 8 chains), thereby recognizing a wide range of different antigens. DETC, in contrast, almost

[12])

uniformly express TCR V'Y3/V81 with identical (canonical) se­

thymocytes acquire CDS and CD2 antigens early

.

It has been known since the late

19S0s

that most,

in

if not

all,

development

The epidermis is the only tissue in adult mice that

and go through a phase of CD4 and CDS coexpression before they

Their lack in the thymus of adult

mature into CD4 and CDS single-positive T cells [12-14). In

mice and the observation that adult bone-marrow-chimeric ani­

contrast, the developmental stages that TCR "118 cells undergo

quences

[5,6].

harbors TCR

V'Y3

cells

[5].

mals do not generate DETC

[7]

(especially in the fetal period) were not known, and became a

suggested that they have matura­

tional requirements different from those of other peripheral T

subject of research only recently

cells. A very important step in understanding the ontogeny of

CDS-, CD4-, and

CDS-,

[15,16].

Given that DETC are

we studied the expression of these

molecules on their fetal thymic precursor cells. In addition, because it was not known whether negativity for CDS is a stable trait of a

Reprint requests to: Dr. Elisabeth Payer, Laboratory of Immunology,

given cell, or if CDS- T cells (DETC, a CDS- subpopulation

NIAID, Bnilding 10, Room I1N321, 9000 Rockville Pike, Bethesda, MD

of intraepithelial lymphocytes

20892-1892.

eral blood T cells

Abbreviation: PE-SA, phycoerythrin-streptavidin.

0022-202X/95/S09.50

•

SSDI0022-202X(95)00150-]

•

Copyright

545

©

1995 by The

[1S])

[17] and a few

CDS - human periph­

go through a stage of CDS expression

Society for Investigative Dermatology, Inc.

VOL. 105, NO. 1, SUPPLEMENT, JULY 1995

CDS EXPRESSION ON DETC PRECURSOR CELLS

------ Iog FL 1

Figure

1. Immunofluorescence double labeling of epidermal ear

sheets prepared from adult B6PL mice by the NH.SCN separation technique. After acetone fixation, the sheets were incubated with bioti­ nylated anti-CD2 or anti-CDS MoAb, followed by Texas Red-streptavidin and FITC-Iabeled anti-TCR Vy3 MoAb. The TCR Vy3 + DETC

CD2+

(b)

but consistently CDS-

(a). Bar,

(A,B) are

SO /Lm.

=

TCR Vy3·FITC

Figure 2. Flow cytometric analysis ofTCR

55S

-------+

Vy3+ fetal thymocytes

at day 17 of gestation. B6PL fetal thymocytes were incubated with biotinylated anti-CDS

(A,B),

anti-CD2

(C),

anti-CD4

(D),

anti-CDS (E),

or control (F) MoAb, followed by PE-SA and FITC-Iabeled anti-TCR Vy3 MoAb: A total of 10,000 viable cells were recorded

(A).

Electronic gates

were then set to acquire at least SOOO TCR Vy3 + viable cells TCR Vy3+ cells expressed CDS

(A,B)

mately half expressed CD4 antigens

and CD2

(D), and

(C)

(B-F).

All

antigens, approxi­

90% expressed CDS antigens

(E).

during their development, we studied the regulation of CDS cell-surface expression. PHENOTYPIC DIFFERENCES BETWEEN TCR Vy3+ FETAL THYMOCYTES AND DETC

nous cell transfer, were CD5-; this indicates that the immediate DETC precursor is TCR Vy3+ ICDS-. These cells could either be TCR Vy3+ ICDS- during their entire development (unlikely in

Fig 2)

To compare the phenotype of DETC with that of their intrathymic

view of the results presented in

precursor cells, we first reevaluated the phenotype of DETC.

expression intrathymically. Because one might postulate that TCR

Immunofluorescence analysis of resident DETC in epidermal sheet

Vy3+ thymocytes down-regulate CDS expression at the end of

preparations as well as flow cytometric analysis of freshly prepared

gestation, we studied their anti-CDS reactivity from 15 d of

Figure 3

or could have lost CDS

DETC-enriched epidermal single-cell suspensions did not reveal

gestation until 2 d postpartum.

any detectable expression of CDS, CD4, or CD8 antigens on TCR

(Fig 1 and data not shown). In contrast, we found that (Fig 1) as well as freshly isolated DETC [19] expressed

sion was highest on TCR Vy3 + cells at 15 d of gestation. Although

Vy3+ cells

at day 16 and thereafter, a fraction of TCR Vy3+ cells had a

CD2, a glycoprotein expressed on all T cells and thymocytes

CDS - cells were never detected [19]. This suggests that TCR

studied to date [14]. Three ligands for CD2 have been identified:

Vy3+ cells leave the thymus at a ·stage of intermediate CDS

resident

reduced CDS cell-surface density

shows that CDS expres­

(Fig 3),

appreciable numbers of

CDS8 (LFA3), CDS9, and CD48 [20-22]. It remains to be seen

expression density. Analysis of CD5 expression on TCR Vy3 + cells

whether the interaction between CD2 and its ligands expressed on

in fetal blood, dermis, and epidermis (day 17 of gestation) showed

Langerhans cells and keratinocytes [23,24], in addition to homo­

that the level of CDS expression further decreased from the thymus

(Table I).

typic E-cadherin interactions (E-cadherins are expressed on Lan­

to the epidermis

gerhans cells, keratinocytes [25], and DETC; M. Udey, personal

on epidermal TCR Vy3 + cells at day 19 of gestation. These data,

CDS ultimately became undetectable

communication), helps to secure the intraepithelial position of

in conjunction with other findings-that release of DETC from

DETC.

their epidermal symbionts does not lead to CDS expression [26] and

Next we investigated the expression of the above antigens on the

that culture of freshly isolated DETC in phorbol myristate acetate

intrathymic DETC precursor cells at day 17 of gestation. TCR

(PMA) and ionomycin (PMA up-regulates CDS expression on T

V y3 + fetal thymocytes showed uniform expression of CD2 anti­

(Fig 2q and also reacted with anti-CDS monoclonal antibody (Fig 2A,B). Furthermore, most of these thymocytes were CD8+ (Fig 2E), and approximately 50% of them expressed low levels of CD4 antigens (Fig 2D). In summary, both our data and gens

(MoAb)

d.y 15

cloy IS

dly 17

day 18

day 19

the work by Leclercq et al [15] show that both TCR Vy3+ fetal thymocytes and DETC express CD2 antigens, but that these cells cillfer with respect to CDS, CD4, CD8, heat-stable antigen, and MEL-14 (L-selectin) expression. All of the latter antigens are present in the thymus but absent on DETC. INTENSITY OF CDS EXPRESSION DURING MATURATION OF TCR Vy3+ CELLS The transition from CD 5 + ITCR Vy3+ fetal thymocytes to CD 5- I TCR Vy3+ DETC was subsequently studied

in more detail.

Intravenous injection of fetal thymocyte suspensions containing TCR Vy3+ ICDS+ (but not TCR Vy3+ ICDS-) fetal thymocytes into athymic nude mice resulted in the appearance of numerous clusters of donor-type CD 5 - ITCR Vy3 + DETC in the recipients' epidermis. We observed that even the earliest donor-derived cells, which could be visualized in the epidermis 2 weeks after intrave-

------- TCRV,3-FrTC --------�

3. Flow cytometric analysis of CDS and CD2 expression on Vy3+ thymocytes obtained at 15-19 d of gestation. Fetal

Figure TCR

thymocyte suspensions were labeled with biotinylated anti-CDS or anti­ CD2 MoAb/PE-SA and FITC-Iabeled anti-TCR Vy3 MoAb, and only

FITC-Iabeled cells were acquired. CDS expressi�n was down-regulated on TCR V y3 + cells

(lane 1), whereas CD2 expression stayed constant (lane 2).

56S

PAYER ET AL

Table I.

THE JOURNAL OF

Intensity ofCD5 Expression on TCR

Vy3+

INVESTIGATIVE

DERMATOLOGY

Cells

in Various Tissues

Age of the Animal Day

Tissue

17 fetal

Thymus

Day 17 fetal

Blood

Day 17 fetal Day 17 fetal Day 18 fetal Day 19 fetal 8-12 Weeks a

Single-cell

Dennis Epidennis

Epidennis Epidermis Epidennis

MFI

MFI

CDS"

Control

15 11 6.4 4.9 3.9 2 3.3

1.6 1.8 1.8 2.1 2 2 3

suspensions were prepared from the indicated organs and

biotinylated anti-CDS

(FITC)-conjugated

or control MoAb followed by

anti-TCR

Vy3 MoAb

and

fluorescein

reacted with

isothiocyanate

phycoerythrin-streptavidin (PE-SA). cytometer on gated

CDS (or control) fluorescence intensity was measured with a flow

TCR Vy3 + cells using logaritlunic amplification. Data are given as the mean fluorescence intensity (MFI) of the respective staining.

cells [27]) also does not lead to CDS expression (data not shown)­ indicate that the a bsence of CDS molecules on DETC is not due to supp ressive signals pro vide d by other epidermal cells. CDS EXPRESSION ON

TCR V)'3+ THYMOCYTES

IN VITRO To understand the mechanism ( s )

FigUre

•. Imrnunolluorescence detection ofTCR Vy3+ cells in the

epidermis of a CDS-deficient C57BU6 mouse. Epidermal sheets were

incubated with biotinylated anti-TCR Vy3 MoAb. followed by Texas Red-streptavidin. Note that there is no difference in the shape or the density of the DETC in the epidennis of the CDS-deficient mouse compared with normal C57BL/6 (not shown) or B6PL mice (Fig 1). Bar, 50 /Lm.

regulating CDS cell-surface den­

sity on DETC precursor cells, we studied CDS expression on

thymocytes isolated froqJ. fetuses at day 17 of gestation and cultured under different conditions. Independent of whether these cells were maintained in plain m edium, in me dium supplemented with con­

canavalin A plus 10 U IL.-2, or in medium supplemented with 100 U IL-2 (a condition leading to the preferential proliferation ofTCR V),3+ cells [28]), or were studie d in int act thymic lobe explants, a complete or atleast substantial loss of CD 5 antigens on TCR V y3 + cells alw a ys occurred within 6-8 d [19]. Even the addition ofPMA and ionomy cin to the cultures c ould not pre vent the down­ regulation of CDS on these special thymocytes (Table D). This was surprising b e cause PMA enhances CDS expression on lymph node T cells [27] as well as on TCR-al {3-expressing fetal thymocytes (Table D). B ecause we were unable to up-regulate CDS expres­ sion on TCR Vy3+ fetal thymocytes and because sorted TCR ),Ia fe tal thymocytes lost CDS expression (ruling out down-regulatory signals delivered from other thymic cell populations), we conclude that TCR V)'3 + thymocytes may have an intrinsic program to down-regulate CDS and finally to lose expression of this antigen completely.

MATURATION OF DETC IN CDS-DEFICIENT MICE

The patterns of expression of CDS, CD8, and CD4 molecules within the thymus led to the speculation that these molecules play a role in the maturation andlor selection of these cells. We tested this hyp oth� sis with resp ect to the CDS molecule by studying the

Table ll. Intensity of CDS Expression After Itt Vitro Culture of Fetal Thyntocytes in Medium Supplemented With PMA apd Ionomycin Cell Population

Day

V y3 + FTa TCR a/{3+ FT" TCR a/{3+ LNb

382

TCR

d

0

Day

3

Day 7

295 :!: 123 1065 :!: 486 3462 :!: 327

:!: 116

226 :!: 25 965 :!: 262

72:!: 15 577 :!: 225 3029 :!: 764

Single-cell suspensions of retal thymocytes (FT)

were

cultured for

10 " g / ml

PMA

the indicated

and

500

anti-CDS/PE-SA and

period in

obtained at day 17 of gestation nonnal culture medium supplemented with

nglml ionomycin. Cells were then labeled

FTTC-Iabeled anti-TCR

Samples were analyzed on a flow cytotTleter. and

with biotinylated

Vy3 or anti-pan TCR al{3 MoAb. the mean fluorescence intensity of the

anti-CDS staining was recorded on gated FlTC-positive cells. Mean three independent experiments are shown. h Single-cell suspensions

6-8-week

B6PL mice.

were prepared

from lymph nodes

Cells were treated. stained.

and

values ± SD of

(LN) obtained from described in a.

analyzed as

DETC popula ti on in CDS-deficient mice [29].

Tarakhovsky et al the approach of homologous recombination to disrupt the CDS gene in a way that prevents the appearance of the CDS antigen on the cell surface. Mutant mice developed normally, and tp,e numbers and phenotypes ofT and B cells in the bone marrow, spleen , lymph nodes, and peritoneum of thes e mice m atched those of normal control mice [29]. We prepared epidermal sheets of these mice and studied their reactivity with anti-Thy-l and anti-TCR Vy3 MoAb. As can be seen in Fig 4, th er e was no alteration in the appearance and density ofTCR V1'3+ cells in the CD S -deficient mice compared with normal mice (see Fig 1), an d all Thy-l + cells coexpressed TCR Vy3. The possibility that DETC in CDS­ deficient mice have a functional defect cannot be excluded, but appears quite unlikely in view of studies by Tarakhovsky et al [29] showing no alterations in the immune function of their mice. Although these data argue against an important role for CDS in the maturation of DETC, other T cells, and B cells, it is conceivable that in th e setting of knockout animals, other molecules may have substituted for the function of the CDS molecules, and alterations may become visible only after the inactivation of a combination of [29] us ed

cell-surface markers.

Similar to the situation in CDS-deficient mice, no altera tions in the appearance of DETC or other TCR yl a cells have been observed in mice in which the interactions ofCD4 or CD8 antigens with MHC cla ss II or class I w e re interrupted as a result of inactiva tion of the latter genes [30,31]. This is not surprising be caus e the intriguing TCR homogeneity of these cell s is generated by an intrinsic program of the recombinatory machinery rather than from selection [32,33].

CONCLUDING REMARKS Our study of TCR V)'3 + fetal thymocytes as well as the studies Tatsumi et al [16] show that TCR ),Ia fetal thymocytes undergo several pheno typic changes during their maturation in the thymus. S ome of these changes are similar to the changes of TCR al{3 thymocy te s, e.g., the early expression of from Leclercq et al [15] and

heat-sta ble

antigen and loss of this molecule on mature cells With respect to CD4 and CD8 molecules, there are slight dilferences b et we en TCR al{3 and TCR yla thymocytes . Although the latter possess some anti-CD4 and anti-CD8 reactivity [34,35], they apparendy do not go through a stage of high CD4/CD8 doub le expression. Although this stage is important for the positive and negative selection of TCR al{3 cells, selection of TCR yla [15,16]'

VOL.

cells

CD5 EXPRESSION ON DETC PRECURSOR CELLS

105, NO. 1, SUPPLEMENT, JULY 1995

(if it

occurs at all) does not require this stage. However. the

regulation of CDS on TCR V")'3 + fetal thymocytes contrasts sharply with that of TCR

a/f3

cells. Whereas the latter population

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normal peripheral and epidermal T cells. We therefore assume that intrathymic expression of this glycoprotein on DETC precursor cells is not important for the maturation of these cells. but rather propose that the absence of this molecule on DETC is important for their fimction. CDS is associated with the TCR complex and can deliver stimulatory signals synergizing with TCR activation [3639]. In this context. it should be noted that the CDS - DETC as well as the TCR ")'/8, CDS_. CD2- subpopulation ofintraepithe­ lial lymphocytes exhibit a reduced proliferation in response to mitogens in the absence of exogenously added IL-2 [17.40.41]. One could therefore speculate that the absence of CDS on these

19. 20.

21.

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