Background
The concept that multiple sclerosis (MS) is a T-cell-mediated disease has been changed and it is now widely accepted that B cells play a part in the pathogenesis of MS. [
1‐
3] Multiple roles of B cells have been elucidated emphasizing that B cells have dual contribution to autoimmunity [
1,
4‐
6]. Hence, the concept of regulatory B cells (Breg) has emerged, proving that B cell subset distribution is far more complex than the original concept. A novel Breg subset that was originally known as immature transitional B cells (CD19
+CD24
hiCD38
hi) has been described to have regulatory capacity through interleukin-10 production [
7]. Another study has found B cells highly expressing programmed death ligand-1 (CD19
+PD-L1
hi cells) that exert regulatory function through cell-to-cell contact via interaction of CD19
+PD-L1
hi cells with PD-1 on T cells, resulting in suppression of T follicular helper (Tfh) cell differentiation and expansion, which are the cell type known to be involved in the relapse of MS patients [
8,
9].
Alemtuzumab is a highly effective treatment in relapsing MS. It provides a long-lasting suppression of disease activity by altering the proportion of lymphocyte subsets with preferential increase of regulatory T cells (Treg) [
10,
11]. In contrast to alemtuzumab’s documented efficacy, alemtuzumab’s mechanism of action is not fully understood and information about the composition of the repopulating B cell pool, especially Breg, is scarce.
Here, we pinpoint deficiency of CD19+CD24hiCD38hi and CD19+PD-L1hi cells during relapse and subsequent expansion following alemtuzumab infusion. We also highlight the possible clinical implication of CD19+CD24hiCD38hi cells.
Methods
Study population
For cross-sectional study, 20 MS patients during relapse (MS-relapse) and 17 MS patients in remission (MS-remission) and 11 healthy controls (HC) were included. For longitudinal analysis, 11 patients who were treated with alemtuzumab were included. All MS patients fulfilled the 2010 McDonald’s criteria [
12]. Current study was approved by the Institutional Review Board, and informed consent was obtained from each subject. Demographic and clinical characteristics of participants are summarized in Table
1.
Table 1
Demographic and clinical characteristics of MS patients in relapse and remission
Age (years, mean ± SD) | 34.5 ± 9.4 | 36.9 ± 5.2 |
Women:men (n:n) | 16:4 | 10:7 |
Onset age (years, mean ± SD) | 30.4 ± 22.0 | 29.8 ± 7.3 |
Disease duration (years, mean ± SD) | 12 ± 24.9 | 6 ± 5.9 |
EDSS (mean ± SD) | 2.9 ± 1.9 | 1.8 ± 1.9 |
Flow cytometry
For cross-sectional study, fresh peripheral blood mononuclear cells (PBMCs) were surface stained with monoclonal antibodies against CD19-APC-cy7, CD27-FITC, CD24-BV421, CD38-BV510, and PD-L1(CD274)-PE-cy7 (BD Biosciences). For longitudinal study, frozen PBMCs, collected from 11 RRMS patients undergoing alemtuzumab at baseline and 6, 9, and 12 months, were surface stained with monoclonal antibodies as stated above.
Statistical analysis
We performed analysis of significance in Prism (GraphPad, La Jolla, USA). For cross-sectional data, one-way ANOVA analysis with Tukey’s multiple comparison post hoc analysis was performed to compare the frequency of Bregs between HC, MS-relapse, and MS-remission. Unpaired t test was used to compare the absolute number of Bregs between MS-relapse and MS-remission. Repeated measures ANOVA with Tukey’s multiple comparison post hoc analysis was performed for longitudinal analysis. A p value of < 0.05 was considered statistically significant. All values show mean ± SEM.
Discussion
Little is known about the repopulating B cell pool following alemtuzumab. Cases of severely exacerbated central nervous system inflammation in alemtuzumab-treated MS patients have reported B-cell driven pathology, further emphasizing the importance of B cell study in alemtuzumab-treated patients [
13‐
15]. We show that deficiency of CD19
+CD24
hiCD38
hi cells and CD19
+PD-L1
hi cells in the peripheral blood of relapsing MS patients are restored following alemtuzumab and that B cell distribution shifts towards naïve phenotype. In fact, the frequency of Bregs with disparate regulatory mechanisms exceeded baseline level, which may underlie the long-lasting suppression of disease activity.
Interestingly, both frequency and absolute number of CD19
+CD24
hiCD38
hi cells are reduced during and prior to relapse in an alemtuzumab-treated patient. CD19
+CD24
hiCD38
hi cells are known to maintain Tregs and limit the differentiation of T helper 1 (Th1) and T helper 17 (Th17) cells [
16]. The deficiency of CD19
+CD24
hiCD38
hi cells could have a significant impact on the regulation of pathology. Indeed, recent studies have found that transitional B cells are impaired in various immune-related disorders [
7,
16‐
18], although conflicting results were observed in MS. [
19,
20] In the early phase of alemtuzumab therapy, CD19
+ B cells repopulate earlier than CD4+ T cells and immature B cells dominate the repopulated CD19+ B cells [
21]; the extensive repopulation of CD19
+CD24
hiCD38
hi cells following alemtuzumab may contribute to the expansion of Tregs while suppressing differentiation of naïve CD4
+ T cells into Th1 and Th17 cells and hence, contributing to the efficacy of alemtuzumab.
A recent study described that CD19
+PD-L1
hi cells are capable of suppressing Tfh cell differentiation and expansion through interaction with PD-1 on activated T cells [
8]. Interaction causes an increase in signal transducer and activator of transcription 5 expression, a known suppressor of Tfh-cell development and expansion. Since Tfh cells aid germinal center formation and hence, involved in the formation of memory B cells and plasma cells, Tfh cells were thought to have pathogenic role in the B-cell-mediated autoimmune diseases. There has been several reports on Tfh cell involvement in MS. [
22‐
25] Most of all, there has been report that CCR7
+ ICOS
+ circulating memory Tfh cells are increased in MS patients during relapse, but decreased in patients during remission [
9]. This finding is in line with our results, where CD19
+PD-L1
hi cells are decreased in MS patients during relapse, but restored during remission. Hence, suppression of Tfh cell differentiation and proliferation by CD19
+PD-L1
hi cells may be impaired due to CD19 + PD-L1hi cell deficiency, contributing to enhancement of disease activity.
This study is limited by its small sample size. In addition, we report that the marked reduction of CD19
+CD24
hiCD38
hi cells during relapse in an alemtuzumab-treated patient was observed in only 2 relapses of a single case. Therefore, in order to decipher the critical role of CD19
+CD24
hiCD38
hi cells in the long-term disease suppression of MS and in the mechanism of action of alemtuzumab, further longitudinal study with larger number of patients on how CD19
+CD24
hiCD38
hi cells and other lymphocytes (including Tregs) change during relapse, is required. The extensive expansion of CD19
+CD24
hiCD38
hi cells was maintained until 9 months post-alemtuzumab, whereas CD19
+PD-L1
hi cells were maintained until the end of the cycle. Further work needs to be established to explain the difference in the results. In addition, a recent study has reported that hyperrepopulation of immature B cells post-alemtuzumab in the absence of adequate regulation by T cells increases the risk of secondary autoimmunity [
21]. Due to vast composition of immature B cells and lack of understanding of their function, it remains to be further elucidated which specific immature B cell subset would be responsible for the secondary autoimmunity. Lastly, further functional study on the CD19
+CD24
hiCD38
hi cells would clarify the mechanism of action of alemtuzumab.