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Early hematopoiesis in multiple sclerosis patients
Journal of Neuroimmunology 299 (2016) 158–163
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Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Early hematopoiesis in multiple sclerosis patients Daniel Jons a,⁎, Maria Kneider a, Linda Fogelstrand b,c, Anders Jeppsson d, Stefan Jacobsson b, Oluf Andersen a a
Section of Clinical Neuroscience, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Sweden Department of Clinical Chemistry, Sahlgrenska University Hospital, Sweden c Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Sweden d Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Sweden b
a r t i c l e
i n f o
Article history: Received 26 May 2016 Received in revised form 17 August 2016 Accepted 7 September 2016 Keywords: Multiple sclerosis B cell Precursor NKT cell Bone marrow FACS
a b s t r a c t Contemporary evidence supports that MS immunopathology starts in the peripheral lymphatic system. However, the site and character of crucial initiating events are unknown. We examined subsets of the first stages of blood cells in the bone marrow of 9 MS patients and 11 neurologically healthy controls using FACS analysis. The proportion of natural killer T cells was lower (P = 0.045) in the bone marrow of MS patients, but proportions of hematogenous stem cells, myeloblasts, and B cell precursor subsets in the bone marrow did not differ between MS patients and controls. In this pilot study with a limited number of samples we found no deviation of the early B cell lineage in bone marrow from MS patients. © 2016 Elsevier B.V. All rights reserved.
1. Introduction MS is a chronic inflammatory disorder characterized by a long preclinical stage and a lifelong developing autoimmune process in the central nervous system, mainly targeting myelin. A complex of genetic and epidemiological risk factors and interactions are established. However, pivotal events turning the balance towards immunopathology are unknown, and new hypotheses on their site and character continue to be presented. They include influence from metabolites induced by gut microbiota (Rothhammer et al., 2016) and priming of lymphocytes by modified proteins in the lungs (Hedstrom et al., 2013, Odoardi et al., 2012). A few unconfirmed studies suggested a role for the bone marrow (BM) as a reservoir for latent viruses in MS (Fredrikson et al., 1989, Goswami et al., 1984, Kam-Hansen et al., 1988, Mitchell et al., 1978) (see Fig. 1). Supported by analogy with experimental autoimmune encephalomyelitis and extensive immunohistological data, multiple sclerosis (MS) is considered a T cell–driven disease (Carbajal et al., 2015). Deviations in the proportion of T cell and natural killer (NKT) cell subsets in MS patients have been reported (Sellebjerg et al., 2012, Svenningsson et al., 1995), but B cells also have important roles in MS pathogenesis
⁎ Corresponding author at: Section of Clinical Neuroscience, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Sweden. E-mail address: [email protected] (D. Jons).
http://dx.doi.org/10.1016/j.jneuroim.2016.09.004 0165-5728/© 2016 Elsevier B.V. All rights reserved.
(Disanto et al., 2012) and are the only cells specifically targeted by effective MS pharmaceuticals (Sorensen and Blinkenberg 2016). The specificity of the majority of the oligoclonal IgGs in cerebrospinal fluid (CSF) and brain plaques (Mehta et al., 1981) of MS patients is unknown; however, there is a characteristic MS antiviral specificity in a minor IgG fraction in each patient (Hottenrott et al., 2015, Reiber et al., 1998). Distinctive features in MS neuropathology are structures in the brain similar to the immunoglobulin-secreting region of lymph nodes (Prineas 1979) and ectopic B cell follicles in the meninges (Howell et al., 2011, Serafini et al., 2004). According to contemporary hypotheses, these persistent B cell clones may arise through i) assistance from T follicular helper cells affected by primary immunopathology (Tangye et al., 2013) or pathological T cell–B cell interaction (Romme Christensen et al., 2013); ii) immortalization from infection with Epstein–Barr virus (EBV), which establishes a chronic infection in B cells and is an accepted risk factor for MS that is most pronounced when it manifests as infectious mononucleosis (Bagert 2009, Jons et al., 2015); and iii) genetic germline predisposition influencing the B cell lineage. The present pilot study focuses on the early B cell lineage in MS patients. NKT cells are implicated in the control of autoimmunity (O'Keeffe et al., 2015), and they may be involved in the defense against viruses, including EBV (Chung et al., 2013). They originate from the bone marrow (BM) but mature in peripheral lymphoid tissues in adults, and their affinity to the BM in pathological conditions is unknown. Several tissues including white blood cells, macrophages and possibly microglia in the brain are constantly rebuilt from the bone marrow.
D. Jons et al. / Journal of Neuroimmunology 299 (2016) 158–163
We here hypothesize that disturbances in the early hematopoiesis, either genetic or caused by viral infections, are at the core of MS pathogenesis. Previous studies on BM in MS patients were small and their methods diverged. B-cell activation and immunoglobulin production was addressed (Fredrikson et al. 1991). Only one study has focused on the immunophenotypes of BM cells, showing a reduced cellular content that was thought to be induced by ongoing immunosuppressive treatment (Carrai et al., 2013). We conducted an exploratory flow cytometry study of the BM of MS patients and neurologically healthy controls, examining basic mononuclear cell types and early lineage of BM cells in these two populations, particularly focusing on the proportions of stem cells, B cell precursors and NKT cells in MS. 2. Materials and methods 2.1. Patients and controls We included nine MS patients (6 women, 3 men) with a median age of 51 years (range 41–56 years). Six had relapsing–remitting MS (RRMS), and the other three were in the secondary progressive phase (SPMS) (Table 1). The RRMS patients were clinically stationary during at least a year. One of the SPMS patients were in an active phase with a superimposed attack one year earlier, and MRI activity. Exclusion criteria were disease-modifying or experimental therapy during the last year and immunosuppressive therapy potentially associated with BM suppression. Glatiramer acetate (GA) therapy was ongoing in the RRMS cases, but the SPMS cases were untreated. Eleven neurologically healthy control patients (4 women, 7 men), scheduled for sternumsplitting thoracotomy for non-inflammatory cardiac disorders, were consecutively included. Median age for controls was 40 years (range 22–59 years). One cubic centimeter of BM was aspirated by crista puncture in patients and by sternal puncture immediately before sternotomy in controls (see Table 2b). The study was approved by the Research Ethics Committee of Gothenburg 2010 (188-10). 2.2. Flow cytometry Peripheral blood was collected in EDTA tubes and BM aspirate samples in saline-containing EDTA tubes at the Departments of Hematology (patients) and Cardiothoracic Surgery (controls) and transported to the Clinical Chemical Laboratory of the Sahlgrenska University Hospital. Within 24 h, erythrocytes were lysed using NH4Cl, and cells were stained with the following monoclonal antibodies: CD105 and CD117 (Beckman Coulter, Marseille, France), CCR7 (R&D Systems, Oxon, UK), simultest (Cytognos, Salamanca, Spain), and the following from BD Biosciences (San José, CA): V450-conjugated anti-CD4 (clone RPA-T4), V450-conjugated anti-CD20 (clone L27), V500-conjugated anti-CD45 (clone HI30), simultest CD8 + lambda-FITC/CD56 + kappa-PE (ref CYT-SLPC-50), PerCP-Cy5,5–conjugated anti-CD5 (clone L17F12), PE-Cy7–conjugated anti-CD19 (clone SJ25C1), PE-Cy7–conjugated anti-TCR γ (clone 11F2), APC-conjugated anti-CD3 (clone SK7), APCH7–conjugated CD38 (clone Hb7), V450-conjugated anti-HLA-DR (clone L243), FITC-conjugated anti-CD36 (clone CLB-IVC7), PE-conjugated anti-CD105 (clone 1G2), PerCP-Cy5,5–conjugated anti-CD34 (clone 8G12), PC7-conjugated anti-CD117 (clone 104D2D1), APC-conjugated anti-CD33 (clone P67,6), APC-H7–conjugated CD71 (clone MA712), V450-conjugated anti-CD4 (clone RPA-T4), FITC-conjugated anti-CD27 (clone L128), PE-conjugated anti-CCR7 (clone 150,503), PerCP-Cy5,5–conjugated anti-smCD3 (clone SK7), PE-Cy7–conjugated anti-CD45R0 (clone UCHL1), APC-conjugated anti-CD45RA (clone HI100), APC-H7-conjugated CD8 (clone SK1), FITC-conjugated antiCD5 (clone L17F12), and PE-Cy7–conjugated anti-HLA-DR (clone L243) (see Table 3b).
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All cells were analyzed on a FACS Canto II (BD), with at least 30,000 acquired events per tube. Data files were analyzed using FacsDIVA software (BD). Myeloblasts were defined by low SSC, dim (D) expression of CD45, and expression of CD34 and CD117. More mature myeloid progenitors were defined by expression of SSC, CD45 + D, CD34-, and CD117 +. Hematogones were defined by low SSC, CD19 +, CD38 +, and CD45 + D (Chantepie et al., 2013). Early hematogones (stage 1) were defined by very low SSC, CD45 + D, CD34+, and CD117-. The proportion of late hematogones (stages 2–3) was calculated by subtraction of the proportion of early hematogones from total hematogones. Mature B cells were defined by low SSC, CD45 + B, CD19 +, and CD20 + and confirmed expression of light chains (kappa/lambda). T cells were defined as low SSC, CD45 + B, CD3+, and CD19- and further subdivided based on expression of CD4, CD8, CD27, CD45RA, CD45R0, and CD56, with NKT cells defined by expression of CD3 + and CD56 +. Granulocytes and monocytes were defined based on CD45 and SSC properties and erythroblasts based on FSC, SSC, CD45−/+D expression, and concomitant expression of CD71 and CD36.
2.3. Statistical analysis All statistical analyses were performed using SPSS (IBM Corp., Armonk, NY). Data are presented as medians and ranges. Differences in the proportions of different cell types between MS patients and control individuals were analyzed by the Mann–Whitney U test. P b 0.05 was regarded as statistically significant.
3. Results 3.1. Similarity of proportion of B cell precursors and mature B cells between patients and controls in BM and peripheral blood The proportion of mature B cells did not differ between patients and controls, and the groups did not differ regarding the proportions of either total hematogones or stage 1 (early) or stages 2–3 (late) hematogones in the BM. In the B cell subsets, MS patients and controls did not differ in the proportion of CD5+ cells (Table 2a, b). MS patients and controls also did not differ regarding the kappa/lambda ratio in either blood or BM.
3.2. No deviation in MS patients regarding major leukocyte subsets in the BM or blood When the frequencies of monocytes, granulocytes, B and T lymphocytes, and NK cells were assessed in BM and peripheral blood, patients and controls did not differ. Neither were there any differences between patients and controls in the proportion of myeloblasts (Tables 3a and 3b). The proportion of erythroblasts in peripheral blood was 1.70% in MS patients and 0.20% in the controls (P = 0.038), but no difference in this proportion was found in the BM.
3.3. T cell subsets in BM and peripheral blood In agreement with previously published findings, we found no differences in proportions of CD4 + and CD8 + T cells or in subsets of these cells for CD27+, CD45R0, or CD45RA (Table 4a and 4b). In BM, the proportion of NKT cells was lower in the MS group than in controls (3.5% and 7.1%, respectively, P = 0.046) (Table 2a). A similar tendency was identified in blood but with no significant difference (2.4% and 7.5%, respectively, P = 0.18). When comparing only the RRMS patients with controls, the proportions of NKT cells were 2.1% and 7.1% in the BM, respectively (P = 0.005), and 1.6% and 7.5% in blood, respectively (P = 0.027).
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D. Jons et al. / Journal of Neuroimmunology 299 (2016) 158–163
Fig. 1. Dot plots of bone marrow and blood sample from one individual with MS. Plot a), c) and e) show bone marrow and b), d) and f) blood. Hematogones are depicted in blue and mature B cells in red. In plot a) and b) all cells are included, showing B cells and hematogones among the CD45bright and CD45dimcells with low SSC. In plots c) to f) CD19+ cells are shown, showing gating of hematogones and mature B cells based on expression of CD20 and CD38. Plot e) shows that the majority of hematogones do not express light chain (kappa/lambda), in contrast to the mature B cells, which display a normal kappa to lambda ratio (e) and f).
4. Discussion There is overwhelming evidence that the immunological aberrations of MS pathogenesis start in the peripheral lymphatic system, at an unknown site (Ramagopalan et al., 2010). Evidence for influence from peripheral sites such as gut microbiota and T cell priming in the lungs was proposed (Hedstrom et al., 2013, Odoardi et al., 2012, Rothhammer et al., 2016). The BM is a source of several cell lineages of which at least one – macrophages - populates the brain in adult life (Cogle et al.,
2004). The present explorative BM study was incited as a follow-up to unconfirmed BM studies suggesting the presence of latent viruses in MS patients (Goswami et al., 1984). We found it reasonable to perform a pilot study with a small cohort to examine the basic cell proportions prior to viral studies. This investigation using a powerful FACS technique showed no deviation of the early hematopoiesis in a small group of MS patients. The study was timely considering the evolving interest in the role of B cells and EBV infection in MS (Disanto et al., 2012, Michel et al.,
D. Jons et al. / Journal of Neuroimmunology 299 (2016) 158–163 Table 1 Patients.
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Table 2b Flow cytometric analysis of peripheral blood, B cell populations.
Patient
Sex
Age
MS type
Disease duration (years)
EDSS
Therapy
Patients
MS1 MS2 MS3 MS4 MS5 MS6 MS7 MS8 MS9
F M M F M F F F F
41 45 52 53 48 50 56 45 52
RRMS SPMS RRMS RRMS RRMS RRMS SPMS RRMS SPMS
14 13 16 8 9 8 20 10 20
2.5 6.5 3,5 2 3 1.0 5.5 1.5 6
GA none GA GA GA GA none GA none
Median
25%
1.33 14.10
1.27 1.53 1.43 8.70 14.70 13.40
1–9: patient serial number. F, female; M, male; RRMS, relapsing-remitting MS; SPMS, secondary progressive MS; EDSS, Expanded Disability Status Scale; GA, glatiramer acetate. See text for further information on MS course at time for bone marrow aspiration.
2015). MS is characterized by B cell proliferation, hypermutation, oligoclonal restriction, and development of germ centers with dividing B cells locally in the meninges and brain parenchyma (Márquez and Horwitz 2015). EBV infection results in a lifelong chronic persistence in a small proportion (1–50/1,000,000) of an individual's B cells (Santpere et al., 2014), and EBV-induced transformation events in B cells occur more frequently in MS patients (Torring et al., 2014). Some CNS lymphomas are preceded by MS-like multifocal demyelination (Kvarta et al., 2016). Our aim was to examine the first stages of myeloid and lymphoid differentiation using established markers. To investigate the early B cell lineage, we examined different stages of hematogones. Morphologically, the most immature of these BM cells are similar to lymphoblasts while the most mature resemble B cells. Their immunophenotypic features, as used in the present study, have been characterized (Chantepie et al., 2013). An increase in hematogones has been reported in lymphomas, and in case reports of autoimmune diseases and viral infections (Moreno-Madrid et al., 2008). In addition, the CD5 + B cells, which may have a fine-tuning or suppressive effect through the receptors of T and B cells, have been implicated in autoimmunity (Sigal 2012). However, in the present study, the proportion of these cells did not differ between MS patients and controls. Thus, we found a normal cellular state in the early B cell lineage in the BM. This is important information for procedures like immunoablation followed by hematopoietic stem cell transplantation, increasingly used as a treatment for MS, as previously discussed (de Oliveira et al., 2015, Papadaki et al., 2005). In the present material, the group of NKT cells defined by CD3 + CD56+ was significantly lower in BM from MS patients than in healthy controls, particularly for patients with RRMS. In peripheral blood the NKT cells was significantly lower for the RRMS group only. This result is consistent with previous reports from peripheral blood and CSF in RRMS patients where significant decrease was reported in the level of NKT cells, as defined by CD3 + CD16/CD56 + (Svenningsson et al., 1995). One type of NKT cell, known as iNKT, has a semi-invariant T cell receptor that recognizes CD1d–glycolipid complexes (Borg et al., 2007). MS patients may carry several autoantibodies directed against
Kappa/lambda B1 cells (CD5+) (% of B cells)
Controls 75%
Median
P value 25%
75%
1.33 1.46 0.87 7.45 17.90 0.81
All values are medians with 25th and 75th percentiles. Comparison of values between patients and healthy controls was performed with the Mann–Whitney U test. P b 0.05 was considered statistically significant.
glycolipids (Haghighi et al., 2013), in particular the glycosphingolipids sulfatide and GalCer, which are major constituents of myelin. When antigen-presenting cells present sulfatide via CD1d to the iNKT cell, the iNKT cell is reported to exert an inhibitory function (Duarte et al., 2004). Individuals who lack iNKT cells have an immunodeficiency syndrome characterized by fulminant EBV infection. Transformation of B cells by EBV results in attenuation of CD1d expression with an inability to activate iNKT cells. Furthermore, herpes viruses may have evolved mechanisms to suppress surface CD1d expression as an adaptation for evading immunosurveillance by iNKT cells (Chung et al., 2013). We have been unable to find reports regarding the presence of NKT cells in the BM, and further studies of NKT cell subtypes are warranted. It is possible that the decreased proportion of NKT cells in MS patients is related primarily or secondarily to the course of EBV infection in this group. A major limitation of the present pilot study is the small number of patients and neurologically healthy controls. The results need validation with a larger material. Another limitation is that the populations are not matched for age or gender. The definitions of subgroups relied on FACS analysis with a considerable, still limited number of determinants. A caveat in BM studies is possible contamination with peripheral blood. Although we took care to avoid an admixture of blood with the BM specimens by aspirating only 1 cm3 of BM, contamination cannot be completely excluded. Furthermore, the risk that therapy with GA influenced the results must be considered; however, even though GA therapy has been reported to influence the expression of ICAM 3, CD25, and other surface molecules on immune cells, the major blood cell proportions have been essentially similar in previous studies (Kala et al., 2011, Sellner et al., 2013). 5. Conclusions This exploratory pilot study identified no deviations in the early B cell lineage to anticipate later B cell–associated events in MS patients, including formation of germinal centers and development of immunoglobulin producing plasma cells in the meninges and cerebral parenchyma. We found that the proportion of NKT cells in MS patients was reduced also in the BM. A possible association between reduced NKT cell counts and EBV infection, an established
Table 2a Flow cytometric analysis of bone marrow, B cell populations. Patients
Kappa/lambda Hematogones (% of B cells) Hematogones (% of WBCs) Stage 1 hematogones (% of WBCs) Stages 2–3 hematogones (% of WBCs) B1 cells (CD5+) (% of B cells)
Controls
P value
Median
25%
75%
Median
25%
75%
1.40 33.90 1.31 0.08 1.23 11.60
1.35 32.00 0.52 0.07 0.51 9.40
1.42 40.60 1.67 0.19 1.34 14.70
1.36 25.10 0.89 0.07 0.79 11.60
1.25 17.70 0.42 0.03 0.39 9.90
1.57 48.50 1.77 0.11 1.46 13.60
All values are medians with 25th and 75th percentiles. WBC, white blood cells. Comparison of values between patients and healthy controls was performed with the Mann–Whitney U test. P b 0.05 was considered statistically significant.
0.66 0.46 0.71 0.66 0.82 0.94
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Table 3a Flow cytometric analysis of bone marrow major leukocyte subsets. Patients
White blood cells (% of all events) Lymphocytes (% of white blood cells) B cells (% of lymphocytes) T cells (% of lymphocytes) NK cells (% of parent) NKT cells (% of parent) Monocytes (% of white blood cells) Granulocytes (% of parent) Erythroblasts (% of parent) Myeloblasts (% of parent)
Controls
P value
Median
25%
75%
Median
25%
75%
75%
89.60 19.80 15.70 63.60 16.40 3.50 4.30 79.10 76.40 13.40
82.70 16.10 14.90 56.10 13.50 1.80 3.10 71.80 70.90 8.80
90.80 23.50 20.80 70.80 21.00 7.00 4.40 82.60 82.50 18.70
85.50 14.30 21.60 58.80 14.00 7.10 4.30 82.10 79.10 18.50
78.70 9.70 11.30 52.00 9.10 3.60 3.80 73.00 74.00 13.90
87.10 21.40 26.20 68.50 25.80 13.00 5.30 83.90 84.30 27.30
0.37 0.15 0.37 0.71 0.71 0.046* 0.29 0.60 0.23 0.13
All values are medians with 25th and 75th percentiles. Comparison of values between patients and healthy controls was performed with the Mann–Whitney U test. P b 0.05 was considered statistically significant.
Table 3b Flow cytometric analysis of peripheral blood, major leukocyte subsets. Patients
White blood cells (% of all events) Lymphocytes (% of white blood cells) B cells (% of lymphocytes) T cells (% of lymphocytes) NK cells (% of parent) NKT cells (% of parent) Monocytes (% of white blood cells) Granulocytes (% of parent) Erythroblasts (% of parent)
Controls
P value
Median
25%
75%
Median
25%
75%
94.80 19.70 11.80 71.90 13.90 2.40 3.60 76.50 1.70
93.20 16.90 7.50 69.00 13.30 1.30 3.30 72.50 0.30
95.30 20.60 13.70 75.60 14.00 7.20 4.00 77.50 2.40
92.40 15.90 11.60 73.30 11.50 7.50 3.50 80.60 0.20
91.50 14.60 9.05 70.50 7.30 2.45 2.75 74.00 0.10
96.45 24.35 12.30 80.60 14.60 9.15 4.20 83.60 0.35
0.96 0.60 0.84 0.60 0.60 0.18 0.97 0.34 0.038*
All values are medians with 25th and 75th percentiles. Comparison of values between patients and healthy controls was performed with the Mann–Whitney U test. P b 0.05 was considered statistically significant.
risk factor for MS, needs further investigation. In this study, the BM was not confirmed as a candidate site for early crucial events in MS. However, our conclusions must be interpreted with caution, considering the small number of patients and neurologically healthy controls.
Conflicts of interest The authors declare that there are no conflicts of interest.
Acknowledgments
This is a timely report considering the recent interest in B cell depleting therapies in Multiple Sclerosis. It is original work not published or considered for publication elsewhere.
The study was supported by grants from the Research Foundation of Neuro Sweden, Stockholm, The Research Foundation of the Gothenburg MS Society, Björnsson's Research Foundation, Göteborg, Sweden, and from the Västra Götaland region, Sweden (VGFOUREG-570441). We thank the staff at the section for flow cytometry at Department of Clinical Chemistry at Sahlgrenska University hospital for excellent technical assistance.
Table 4a Flow cytometric analysis of bone marrow, T cell populations.
Table 4b Flow cytometric analysis of peripheral blood, T cell populations.
Author declaration
Patients
CD4+ CD8+ CD4+ Cell fractions: CD27+ CD45R0 CD45RA CD8+ Cell fractions: CD27+ CD45R0 CD45RA
Controls
P value
Median
25%
75%
Median
25%
75%
53.00 41.20
45.50 36.30
61.10 49.80
53.10 44.90
36.90 37.10
58.20 51.30
0.55 0.71
87.50 46.00 33.50
86.60 30.90 26.30
93.00 48.90 45.00
86.00 38.80 35.00
80.50 33.80 27.20
93.20 55.00 41.00
0.26 1.00 1.00
68.00 18.00 44.60
60.10 12.20 33.50
76.70 34.10 62.30
65.40 22.30 47.30
58.90 13.10 32.10
84.20 23.50 62.30
0.88 0.94 0.94
All values are medians with 25th and 75th percentiles. Comparison of values between patients and healthy controls was performed with the Mann–Whitney U test. P b 0.05 was considered statistically significant.
Patients
CD4+ CD8+ CD4+ cell fractions: CD27+ CD45R0 CD45RA CD8+ cell fractions: CD27+ CD45R0 CD45RA
Controls
P value
Median
25%
75%
Median
25%
75%
57.1 36.3
49 31.6
66 42.5
57.9 36.6
54.7 23.05
71.95 39.55
0.239 0.305
88.6 46.8 26.8
85.6 38.4 19.9
92 55.4 42
91.6 37.8 39
87.75 33.4 29.65
93.8 47.2 41.2
0.494 0.470 0.648
60.9 17 47.8
51.4 9.6 31.7
71.7 30 66.8
67 15.8 51.2
60.15 10.5 40.45
80.95 18.2 57.5
0.447 0.676 0.909
All values are medians with 25th and 75th percentiles. Comparison of values between patients and healthy controls was performed with the Mann–Whitney U test. P b 0.05 was considered statistically significant.
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Márquez, A.C., Horwitz, M.S., 2015. The role of latently infected B cells in CNS autoimmunity. Front. Immunol. 6, 1–8. Mehta, P.D., Frisch, S., Thormar, H., Tourtellotte, W.W., Wisniewski, H.M., 1981. Bound antibody in multiple sclerosis brains. J. Neurol. Sci. 49, 91–98. Michel, L., Touil, H., Pikor, N.B., Gommerman, J.L., Prat, A., Bar-Or, A., 2015. B cells in the multiple sclerosis central nervous system: trafficking and contribution to CNS-compartmentalized inflammation. Front. Immunol. 6, 636. Mitchell, D.N., Porterfield, J.S., Micheletti, R., Lange, L.S., Goswami, K.K., Taylor, P., et al., 1978. Isolation of an infectious agent from bone-marrows of patients with multiple sclerosis. Lancet 2, 387–391. Moreno-Madrid, F., Uberos, J., Diaz-Molina, M., Ramirez-Arredondo, A., Jimenez-Gamiz, P., Molina-Carballo, A., 2008. The presence of precursors of benign pre-B lymphoblasts (hematogones) in the bone marrow of a paediatric patient with cytomegalovirus infection. Clin Med Oncol. 2, 437–439. Odoardi, F., Sie, C., Streyl, K., Ulaganathan, V.K., Schlager, C., Lodygin, D., et al., 2012. T cells become licensed in the lung to enter the central nervous system. Nature 488, 675–679. O'Keeffe, J., Podbielska, M., Hogan, E.L., 2015. Invariant natural killer T cells and their ligands: focus on multiple sclerosis. Immunology 145, 468–475. Papadaki, H.A., Tsagournisakis, M., Mastorodemos, V., Pontikoglou, C., Damianaki, A., Pyrovolaki, K., et al., 2005. Normal bone marrow hematopoietic stem cell reserves and normal stromal cell function support the use of autologous stem cell transplantation in patients with multiple sclerosis. Bone Marrow Transplant. 36, 1053–1063. Prineas, J.W., 1979. Multiple sclerosis: presence of lymphatic capillaries and lymphoid tissue in the brain and spinal cord. Science 203, 1123–1125. Ramagopalan, S.V., Dobson, R., Meier, U.C., Giovannoni, G., 2010. Multiple sclerosis: risk factors, prodromes, and potential causal pathways. Lancet Neurol. 9, 727–739. Reiber, H., Ungefehr, S., Jacobi, C., 1998. The intrathecal, polyspecific and oligoclonal immune response in multiple sclerosis. Mult. Scler. 4, 111–117. Romme Christensen, J., Bornsen, L., Ratzer, R., Piehl, F., Khademi, M., Olsson, T., et al., 2013. Systemic inflammation in progressive multiple sclerosis involves follicular T-helper, Th17- and activated B-cells and correlates with progression. PLoS One 8, e57820. Rothhammer, V., Mascanfroni, I.D., Bunse, L., Takenaka, M.C., Kenison, J.E., Mayo, L., et al., 2016. Type I interferons and microbial metabolites of tryptophan modulate astrocyte activity and central nervous system inflammation via the aryl hydrocarbon receptor. Nat. Med. Santpere, G., Darre, F., Blanco, S., Alcami, A., Villoslada, P., Mar Alba, M., et al., 2014. Genome-wide analysis of wild-type Epstein-Barr virus genomes derived from healthy individuals of the 1,000 genomes project. Genome Biol Evol. 6, 846–860. Sellebjerg, F., Krakauer, M., Khademi, M., Olsson, T., Sorensen, P.S., 2012. FOXP3, CBLB and ITCH gene expression and cytotoxic T lymphocyte antigen 4 expression on CD4(+) CD25(high) T cells in multiple sclerosis. Clin. Exp. Immunol. 170, 149–155. Sellner, J., Koczi, W., Harrer, A., Oppermann, K., Obregon-Castrillo, E., Pilz, G., et al., 2013. Glatiramer acetate attenuates the pro-migratory profile of adhesion molecules on various immune cell subsets in multiple sclerosis. Clin. Exp. Immunol. 173, 381–389. Serafini, B., Rosicarelli, B., Magliozzi, R., Stigliano, E., Aloisi, F., 2004. Detection of ectopic Bcell follicles with germinal centers in the meninges of patients with secondary progressive multiple sclerosis. Brain Pathol. 14, 164–174. Sigal, L.H., 2012. Basic science for the clinician 54: CD5. J. Clin. Rheumatol. 18, 83–88. Sorensen, P.S., Blinkenberg, M., 2016. The potential role for ocrelizumab in the treatment of multiple sclerosis: current evidence and future prospects. Ther. Adv. Neurol. Disord. 9, 44–52. Svenningsson, A., Andersen, O., Hansson, G.K., Stemme, S., 1995. Reduced frequency of memory CD8+ T lymphocytes in cerebrospinal fluid and blood of patients with multiple sclerosis. Autoimmunity 21, 231–239. Tangye, S.G., Ma, C.S., Brink, R., Deenick, E.K., 2013. The good, the bad and the ugly - TFH cells in human health and disease. Nat. Rev. Immunol. 13, 412–426. Torring, C., Andreasen, C., Gehr, N., Bjerg, L., Petersen, T., Hollsberg, P., 2014. Higher incidence of Epstein-Barr virus-induced lymphocyte transformation in multiple sclerosis. Acta Neurol. Scand. 130, 90–96.
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Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Early hematopoiesis in multiple sclerosis patients Daniel Jons a,⁎, Maria Kneider a, Linda Fogelstrand b,c, Anders Jeppsson d, Stefan Jacobsson b, Oluf Andersen a a
Section of Clinical Neuroscience, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Sweden Department of Clinical Chemistry, Sahlgrenska University Hospital, Sweden c Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Sweden d Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Sweden b
a r t i c l e
i n f o
Article history: Received 26 May 2016 Received in revised form 17 August 2016 Accepted 7 September 2016 Keywords: Multiple sclerosis B cell Precursor NKT cell Bone marrow FACS
a b s t r a c t Contemporary evidence supports that MS immunopathology starts in the peripheral lymphatic system. However, the site and character of crucial initiating events are unknown. We examined subsets of the first stages of blood cells in the bone marrow of 9 MS patients and 11 neurologically healthy controls using FACS analysis. The proportion of natural killer T cells was lower (P = 0.045) in the bone marrow of MS patients, but proportions of hematogenous stem cells, myeloblasts, and B cell precursor subsets in the bone marrow did not differ between MS patients and controls. In this pilot study with a limited number of samples we found no deviation of the early B cell lineage in bone marrow from MS patients. © 2016 Elsevier B.V. All rights reserved.
1. Introduction MS is a chronic inflammatory disorder characterized by a long preclinical stage and a lifelong developing autoimmune process in the central nervous system, mainly targeting myelin. A complex of genetic and epidemiological risk factors and interactions are established. However, pivotal events turning the balance towards immunopathology are unknown, and new hypotheses on their site and character continue to be presented. They include influence from metabolites induced by gut microbiota (Rothhammer et al., 2016) and priming of lymphocytes by modified proteins in the lungs (Hedstrom et al., 2013, Odoardi et al., 2012). A few unconfirmed studies suggested a role for the bone marrow (BM) as a reservoir for latent viruses in MS (Fredrikson et al., 1989, Goswami et al., 1984, Kam-Hansen et al., 1988, Mitchell et al., 1978) (see Fig. 1). Supported by analogy with experimental autoimmune encephalomyelitis and extensive immunohistological data, multiple sclerosis (MS) is considered a T cell–driven disease (Carbajal et al., 2015). Deviations in the proportion of T cell and natural killer (NKT) cell subsets in MS patients have been reported (Sellebjerg et al., 2012, Svenningsson et al., 1995), but B cells also have important roles in MS pathogenesis
⁎ Corresponding author at: Section of Clinical Neuroscience, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Sweden. E-mail address: [email protected] (D. Jons).
http://dx.doi.org/10.1016/j.jneuroim.2016.09.004 0165-5728/© 2016 Elsevier B.V. All rights reserved.
(Disanto et al., 2012) and are the only cells specifically targeted by effective MS pharmaceuticals (Sorensen and Blinkenberg 2016). The specificity of the majority of the oligoclonal IgGs in cerebrospinal fluid (CSF) and brain plaques (Mehta et al., 1981) of MS patients is unknown; however, there is a characteristic MS antiviral specificity in a minor IgG fraction in each patient (Hottenrott et al., 2015, Reiber et al., 1998). Distinctive features in MS neuropathology are structures in the brain similar to the immunoglobulin-secreting region of lymph nodes (Prineas 1979) and ectopic B cell follicles in the meninges (Howell et al., 2011, Serafini et al., 2004). According to contemporary hypotheses, these persistent B cell clones may arise through i) assistance from T follicular helper cells affected by primary immunopathology (Tangye et al., 2013) or pathological T cell–B cell interaction (Romme Christensen et al., 2013); ii) immortalization from infection with Epstein–Barr virus (EBV), which establishes a chronic infection in B cells and is an accepted risk factor for MS that is most pronounced when it manifests as infectious mononucleosis (Bagert 2009, Jons et al., 2015); and iii) genetic germline predisposition influencing the B cell lineage. The present pilot study focuses on the early B cell lineage in MS patients. NKT cells are implicated in the control of autoimmunity (O'Keeffe et al., 2015), and they may be involved in the defense against viruses, including EBV (Chung et al., 2013). They originate from the bone marrow (BM) but mature in peripheral lymphoid tissues in adults, and their affinity to the BM in pathological conditions is unknown. Several tissues including white blood cells, macrophages and possibly microglia in the brain are constantly rebuilt from the bone marrow.
D. Jons et al. / Journal of Neuroimmunology 299 (2016) 158–163
We here hypothesize that disturbances in the early hematopoiesis, either genetic or caused by viral infections, are at the core of MS pathogenesis. Previous studies on BM in MS patients were small and their methods diverged. B-cell activation and immunoglobulin production was addressed (Fredrikson et al. 1991). Only one study has focused on the immunophenotypes of BM cells, showing a reduced cellular content that was thought to be induced by ongoing immunosuppressive treatment (Carrai et al., 2013). We conducted an exploratory flow cytometry study of the BM of MS patients and neurologically healthy controls, examining basic mononuclear cell types and early lineage of BM cells in these two populations, particularly focusing on the proportions of stem cells, B cell precursors and NKT cells in MS. 2. Materials and methods 2.1. Patients and controls We included nine MS patients (6 women, 3 men) with a median age of 51 years (range 41–56 years). Six had relapsing–remitting MS (RRMS), and the other three were in the secondary progressive phase (SPMS) (Table 1). The RRMS patients were clinically stationary during at least a year. One of the SPMS patients were in an active phase with a superimposed attack one year earlier, and MRI activity. Exclusion criteria were disease-modifying or experimental therapy during the last year and immunosuppressive therapy potentially associated with BM suppression. Glatiramer acetate (GA) therapy was ongoing in the RRMS cases, but the SPMS cases were untreated. Eleven neurologically healthy control patients (4 women, 7 men), scheduled for sternumsplitting thoracotomy for non-inflammatory cardiac disorders, were consecutively included. Median age for controls was 40 years (range 22–59 years). One cubic centimeter of BM was aspirated by crista puncture in patients and by sternal puncture immediately before sternotomy in controls (see Table 2b). The study was approved by the Research Ethics Committee of Gothenburg 2010 (188-10). 2.2. Flow cytometry Peripheral blood was collected in EDTA tubes and BM aspirate samples in saline-containing EDTA tubes at the Departments of Hematology (patients) and Cardiothoracic Surgery (controls) and transported to the Clinical Chemical Laboratory of the Sahlgrenska University Hospital. Within 24 h, erythrocytes were lysed using NH4Cl, and cells were stained with the following monoclonal antibodies: CD105 and CD117 (Beckman Coulter, Marseille, France), CCR7 (R&D Systems, Oxon, UK), simultest (Cytognos, Salamanca, Spain), and the following from BD Biosciences (San José, CA): V450-conjugated anti-CD4 (clone RPA-T4), V450-conjugated anti-CD20 (clone L27), V500-conjugated anti-CD45 (clone HI30), simultest CD8 + lambda-FITC/CD56 + kappa-PE (ref CYT-SLPC-50), PerCP-Cy5,5–conjugated anti-CD5 (clone L17F12), PE-Cy7–conjugated anti-CD19 (clone SJ25C1), PE-Cy7–conjugated anti-TCR γ (clone 11F2), APC-conjugated anti-CD3 (clone SK7), APCH7–conjugated CD38 (clone Hb7), V450-conjugated anti-HLA-DR (clone L243), FITC-conjugated anti-CD36 (clone CLB-IVC7), PE-conjugated anti-CD105 (clone 1G2), PerCP-Cy5,5–conjugated anti-CD34 (clone 8G12), PC7-conjugated anti-CD117 (clone 104D2D1), APC-conjugated anti-CD33 (clone P67,6), APC-H7–conjugated CD71 (clone MA712), V450-conjugated anti-CD4 (clone RPA-T4), FITC-conjugated anti-CD27 (clone L128), PE-conjugated anti-CCR7 (clone 150,503), PerCP-Cy5,5–conjugated anti-smCD3 (clone SK7), PE-Cy7–conjugated anti-CD45R0 (clone UCHL1), APC-conjugated anti-CD45RA (clone HI100), APC-H7-conjugated CD8 (clone SK1), FITC-conjugated antiCD5 (clone L17F12), and PE-Cy7–conjugated anti-HLA-DR (clone L243) (see Table 3b).
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All cells were analyzed on a FACS Canto II (BD), with at least 30,000 acquired events per tube. Data files were analyzed using FacsDIVA software (BD). Myeloblasts were defined by low SSC, dim (D) expression of CD45, and expression of CD34 and CD117. More mature myeloid progenitors were defined by expression of SSC, CD45 + D, CD34-, and CD117 +. Hematogones were defined by low SSC, CD19 +, CD38 +, and CD45 + D (Chantepie et al., 2013). Early hematogones (stage 1) were defined by very low SSC, CD45 + D, CD34+, and CD117-. The proportion of late hematogones (stages 2–3) was calculated by subtraction of the proportion of early hematogones from total hematogones. Mature B cells were defined by low SSC, CD45 + B, CD19 +, and CD20 + and confirmed expression of light chains (kappa/lambda). T cells were defined as low SSC, CD45 + B, CD3+, and CD19- and further subdivided based on expression of CD4, CD8, CD27, CD45RA, CD45R0, and CD56, with NKT cells defined by expression of CD3 + and CD56 +. Granulocytes and monocytes were defined based on CD45 and SSC properties and erythroblasts based on FSC, SSC, CD45−/+D expression, and concomitant expression of CD71 and CD36.
2.3. Statistical analysis All statistical analyses were performed using SPSS (IBM Corp., Armonk, NY). Data are presented as medians and ranges. Differences in the proportions of different cell types between MS patients and control individuals were analyzed by the Mann–Whitney U test. P b 0.05 was regarded as statistically significant.
3. Results 3.1. Similarity of proportion of B cell precursors and mature B cells between patients and controls in BM and peripheral blood The proportion of mature B cells did not differ between patients and controls, and the groups did not differ regarding the proportions of either total hematogones or stage 1 (early) or stages 2–3 (late) hematogones in the BM. In the B cell subsets, MS patients and controls did not differ in the proportion of CD5+ cells (Table 2a, b). MS patients and controls also did not differ regarding the kappa/lambda ratio in either blood or BM.
3.2. No deviation in MS patients regarding major leukocyte subsets in the BM or blood When the frequencies of monocytes, granulocytes, B and T lymphocytes, and NK cells were assessed in BM and peripheral blood, patients and controls did not differ. Neither were there any differences between patients and controls in the proportion of myeloblasts (Tables 3a and 3b). The proportion of erythroblasts in peripheral blood was 1.70% in MS patients and 0.20% in the controls (P = 0.038), but no difference in this proportion was found in the BM.
3.3. T cell subsets in BM and peripheral blood In agreement with previously published findings, we found no differences in proportions of CD4 + and CD8 + T cells or in subsets of these cells for CD27+, CD45R0, or CD45RA (Table 4a and 4b). In BM, the proportion of NKT cells was lower in the MS group than in controls (3.5% and 7.1%, respectively, P = 0.046) (Table 2a). A similar tendency was identified in blood but with no significant difference (2.4% and 7.5%, respectively, P = 0.18). When comparing only the RRMS patients with controls, the proportions of NKT cells were 2.1% and 7.1% in the BM, respectively (P = 0.005), and 1.6% and 7.5% in blood, respectively (P = 0.027).
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D. Jons et al. / Journal of Neuroimmunology 299 (2016) 158–163
Fig. 1. Dot plots of bone marrow and blood sample from one individual with MS. Plot a), c) and e) show bone marrow and b), d) and f) blood. Hematogones are depicted in blue and mature B cells in red. In plot a) and b) all cells are included, showing B cells and hematogones among the CD45bright and CD45dimcells with low SSC. In plots c) to f) CD19+ cells are shown, showing gating of hematogones and mature B cells based on expression of CD20 and CD38. Plot e) shows that the majority of hematogones do not express light chain (kappa/lambda), in contrast to the mature B cells, which display a normal kappa to lambda ratio (e) and f).
4. Discussion There is overwhelming evidence that the immunological aberrations of MS pathogenesis start in the peripheral lymphatic system, at an unknown site (Ramagopalan et al., 2010). Evidence for influence from peripheral sites such as gut microbiota and T cell priming in the lungs was proposed (Hedstrom et al., 2013, Odoardi et al., 2012, Rothhammer et al., 2016). The BM is a source of several cell lineages of which at least one – macrophages - populates the brain in adult life (Cogle et al.,
2004). The present explorative BM study was incited as a follow-up to unconfirmed BM studies suggesting the presence of latent viruses in MS patients (Goswami et al., 1984). We found it reasonable to perform a pilot study with a small cohort to examine the basic cell proportions prior to viral studies. This investigation using a powerful FACS technique showed no deviation of the early hematopoiesis in a small group of MS patients. The study was timely considering the evolving interest in the role of B cells and EBV infection in MS (Disanto et al., 2012, Michel et al.,
D. Jons et al. / Journal of Neuroimmunology 299 (2016) 158–163 Table 1 Patients.
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Table 2b Flow cytometric analysis of peripheral blood, B cell populations.
Patient
Sex
Age
MS type
Disease duration (years)
EDSS
Therapy
Patients
MS1 MS2 MS3 MS4 MS5 MS6 MS7 MS8 MS9
F M M F M F F F F
41 45 52 53 48 50 56 45 52
RRMS SPMS RRMS RRMS RRMS RRMS SPMS RRMS SPMS
14 13 16 8 9 8 20 10 20
2.5 6.5 3,5 2 3 1.0 5.5 1.5 6
GA none GA GA GA GA none GA none
Median
25%
1.33 14.10
1.27 1.53 1.43 8.70 14.70 13.40
1–9: patient serial number. F, female; M, male; RRMS, relapsing-remitting MS; SPMS, secondary progressive MS; EDSS, Expanded Disability Status Scale; GA, glatiramer acetate. See text for further information on MS course at time for bone marrow aspiration.
2015). MS is characterized by B cell proliferation, hypermutation, oligoclonal restriction, and development of germ centers with dividing B cells locally in the meninges and brain parenchyma (Márquez and Horwitz 2015). EBV infection results in a lifelong chronic persistence in a small proportion (1–50/1,000,000) of an individual's B cells (Santpere et al., 2014), and EBV-induced transformation events in B cells occur more frequently in MS patients (Torring et al., 2014). Some CNS lymphomas are preceded by MS-like multifocal demyelination (Kvarta et al., 2016). Our aim was to examine the first stages of myeloid and lymphoid differentiation using established markers. To investigate the early B cell lineage, we examined different stages of hematogones. Morphologically, the most immature of these BM cells are similar to lymphoblasts while the most mature resemble B cells. Their immunophenotypic features, as used in the present study, have been characterized (Chantepie et al., 2013). An increase in hematogones has been reported in lymphomas, and in case reports of autoimmune diseases and viral infections (Moreno-Madrid et al., 2008). In addition, the CD5 + B cells, which may have a fine-tuning or suppressive effect through the receptors of T and B cells, have been implicated in autoimmunity (Sigal 2012). However, in the present study, the proportion of these cells did not differ between MS patients and controls. Thus, we found a normal cellular state in the early B cell lineage in the BM. This is important information for procedures like immunoablation followed by hematopoietic stem cell transplantation, increasingly used as a treatment for MS, as previously discussed (de Oliveira et al., 2015, Papadaki et al., 2005). In the present material, the group of NKT cells defined by CD3 + CD56+ was significantly lower in BM from MS patients than in healthy controls, particularly for patients with RRMS. In peripheral blood the NKT cells was significantly lower for the RRMS group only. This result is consistent with previous reports from peripheral blood and CSF in RRMS patients where significant decrease was reported in the level of NKT cells, as defined by CD3 + CD16/CD56 + (Svenningsson et al., 1995). One type of NKT cell, known as iNKT, has a semi-invariant T cell receptor that recognizes CD1d–glycolipid complexes (Borg et al., 2007). MS patients may carry several autoantibodies directed against
Kappa/lambda B1 cells (CD5+) (% of B cells)
Controls 75%
Median
P value 25%
75%
1.33 1.46 0.87 7.45 17.90 0.81
All values are medians with 25th and 75th percentiles. Comparison of values between patients and healthy controls was performed with the Mann–Whitney U test. P b 0.05 was considered statistically significant.
glycolipids (Haghighi et al., 2013), in particular the glycosphingolipids sulfatide and GalCer, which are major constituents of myelin. When antigen-presenting cells present sulfatide via CD1d to the iNKT cell, the iNKT cell is reported to exert an inhibitory function (Duarte et al., 2004). Individuals who lack iNKT cells have an immunodeficiency syndrome characterized by fulminant EBV infection. Transformation of B cells by EBV results in attenuation of CD1d expression with an inability to activate iNKT cells. Furthermore, herpes viruses may have evolved mechanisms to suppress surface CD1d expression as an adaptation for evading immunosurveillance by iNKT cells (Chung et al., 2013). We have been unable to find reports regarding the presence of NKT cells in the BM, and further studies of NKT cell subtypes are warranted. It is possible that the decreased proportion of NKT cells in MS patients is related primarily or secondarily to the course of EBV infection in this group. A major limitation of the present pilot study is the small number of patients and neurologically healthy controls. The results need validation with a larger material. Another limitation is that the populations are not matched for age or gender. The definitions of subgroups relied on FACS analysis with a considerable, still limited number of determinants. A caveat in BM studies is possible contamination with peripheral blood. Although we took care to avoid an admixture of blood with the BM specimens by aspirating only 1 cm3 of BM, contamination cannot be completely excluded. Furthermore, the risk that therapy with GA influenced the results must be considered; however, even though GA therapy has been reported to influence the expression of ICAM 3, CD25, and other surface molecules on immune cells, the major blood cell proportions have been essentially similar in previous studies (Kala et al., 2011, Sellner et al., 2013). 5. Conclusions This exploratory pilot study identified no deviations in the early B cell lineage to anticipate later B cell–associated events in MS patients, including formation of germinal centers and development of immunoglobulin producing plasma cells in the meninges and cerebral parenchyma. We found that the proportion of NKT cells in MS patients was reduced also in the BM. A possible association between reduced NKT cell counts and EBV infection, an established
Table 2a Flow cytometric analysis of bone marrow, B cell populations. Patients
Kappa/lambda Hematogones (% of B cells) Hematogones (% of WBCs) Stage 1 hematogones (% of WBCs) Stages 2–3 hematogones (% of WBCs) B1 cells (CD5+) (% of B cells)
Controls
P value
Median
25%
75%
Median
25%
75%
1.40 33.90 1.31 0.08 1.23 11.60
1.35 32.00 0.52 0.07 0.51 9.40
1.42 40.60 1.67 0.19 1.34 14.70
1.36 25.10 0.89 0.07 0.79 11.60
1.25 17.70 0.42 0.03 0.39 9.90
1.57 48.50 1.77 0.11 1.46 13.60
All values are medians with 25th and 75th percentiles. WBC, white blood cells. Comparison of values between patients and healthy controls was performed with the Mann–Whitney U test. P b 0.05 was considered statistically significant.
0.66 0.46 0.71 0.66 0.82 0.94
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Table 3a Flow cytometric analysis of bone marrow major leukocyte subsets. Patients
White blood cells (% of all events) Lymphocytes (% of white blood cells) B cells (% of lymphocytes) T cells (% of lymphocytes) NK cells (% of parent) NKT cells (% of parent) Monocytes (% of white blood cells) Granulocytes (% of parent) Erythroblasts (% of parent) Myeloblasts (% of parent)
Controls
P value
Median
25%
75%
Median
25%
75%
75%
89.60 19.80 15.70 63.60 16.40 3.50 4.30 79.10 76.40 13.40
82.70 16.10 14.90 56.10 13.50 1.80 3.10 71.80 70.90 8.80
90.80 23.50 20.80 70.80 21.00 7.00 4.40 82.60 82.50 18.70
85.50 14.30 21.60 58.80 14.00 7.10 4.30 82.10 79.10 18.50
78.70 9.70 11.30 52.00 9.10 3.60 3.80 73.00 74.00 13.90
87.10 21.40 26.20 68.50 25.80 13.00 5.30 83.90 84.30 27.30
0.37 0.15 0.37 0.71 0.71 0.046* 0.29 0.60 0.23 0.13
All values are medians with 25th and 75th percentiles. Comparison of values between patients and healthy controls was performed with the Mann–Whitney U test. P b 0.05 was considered statistically significant.
Table 3b Flow cytometric analysis of peripheral blood, major leukocyte subsets. Patients
White blood cells (% of all events) Lymphocytes (% of white blood cells) B cells (% of lymphocytes) T cells (% of lymphocytes) NK cells (% of parent) NKT cells (% of parent) Monocytes (% of white blood cells) Granulocytes (% of parent) Erythroblasts (% of parent)
Controls
P value
Median
25%
75%
Median
25%
75%
94.80 19.70 11.80 71.90 13.90 2.40 3.60 76.50 1.70
93.20 16.90 7.50 69.00 13.30 1.30 3.30 72.50 0.30
95.30 20.60 13.70 75.60 14.00 7.20 4.00 77.50 2.40
92.40 15.90 11.60 73.30 11.50 7.50 3.50 80.60 0.20
91.50 14.60 9.05 70.50 7.30 2.45 2.75 74.00 0.10
96.45 24.35 12.30 80.60 14.60 9.15 4.20 83.60 0.35
0.96 0.60 0.84 0.60 0.60 0.18 0.97 0.34 0.038*
All values are medians with 25th and 75th percentiles. Comparison of values between patients and healthy controls was performed with the Mann–Whitney U test. P b 0.05 was considered statistically significant.
risk factor for MS, needs further investigation. In this study, the BM was not confirmed as a candidate site for early crucial events in MS. However, our conclusions must be interpreted with caution, considering the small number of patients and neurologically healthy controls.
Conflicts of interest The authors declare that there are no conflicts of interest.
Acknowledgments
This is a timely report considering the recent interest in B cell depleting therapies in Multiple Sclerosis. It is original work not published or considered for publication elsewhere.
The study was supported by grants from the Research Foundation of Neuro Sweden, Stockholm, The Research Foundation of the Gothenburg MS Society, Björnsson's Research Foundation, Göteborg, Sweden, and from the Västra Götaland region, Sweden (VGFOUREG-570441). We thank the staff at the section for flow cytometry at Department of Clinical Chemistry at Sahlgrenska University hospital for excellent technical assistance.
Table 4a Flow cytometric analysis of bone marrow, T cell populations.
Table 4b Flow cytometric analysis of peripheral blood, T cell populations.
Author declaration
Patients
CD4+ CD8+ CD4+ Cell fractions: CD27+ CD45R0 CD45RA CD8+ Cell fractions: CD27+ CD45R0 CD45RA
Controls
P value
Median
25%
75%
Median
25%
75%
53.00 41.20
45.50 36.30
61.10 49.80
53.10 44.90
36.90 37.10
58.20 51.30
0.55 0.71
87.50 46.00 33.50
86.60 30.90 26.30
93.00 48.90 45.00
86.00 38.80 35.00
80.50 33.80 27.20
93.20 55.00 41.00
0.26 1.00 1.00
68.00 18.00 44.60
60.10 12.20 33.50
76.70 34.10 62.30
65.40 22.30 47.30
58.90 13.10 32.10
84.20 23.50 62.30
0.88 0.94 0.94
All values are medians with 25th and 75th percentiles. Comparison of values between patients and healthy controls was performed with the Mann–Whitney U test. P b 0.05 was considered statistically significant.
Patients
CD4+ CD8+ CD4+ cell fractions: CD27+ CD45R0 CD45RA CD8+ cell fractions: CD27+ CD45R0 CD45RA
Controls
P value
Median
25%
75%
Median
25%
75%
57.1 36.3
49 31.6
66 42.5
57.9 36.6
54.7 23.05
71.95 39.55
0.239 0.305
88.6 46.8 26.8
85.6 38.4 19.9
92 55.4 42
91.6 37.8 39
87.75 33.4 29.65
93.8 47.2 41.2
0.494 0.470 0.648
60.9 17 47.8
51.4 9.6 31.7
71.7 30 66.8
67 15.8 51.2
60.15 10.5 40.45
80.95 18.2 57.5
0.447 0.676 0.909
All values are medians with 25th and 75th percentiles. Comparison of values between patients and healthy controls was performed with the Mann–Whitney U test. P b 0.05 was considered statistically significant.
D. Jons et al. / Journal of Neuroimmunology 299 (2016) 158–163
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