In this study, we investigated the phenotypic attributes and antigen-presenting role of memory vs. naïve B cells from MS patients. While B cells from MS patients were phenotypically similar to those from healthy subjects and also showed similar patterns of LTα secretion (as a representative of pro-inflammatory cytokine release potential) in response to polyclonal stimuli, we found that memory B cells from a subset of MS patients, but not HD, elicited autologous CD4+ T cell proliferation in response to MBP and MOG. At the same time, both MS patients and HD elicited autologous CD4+ T cell proliferation in response to TT and GA. Thus, the differences were significantly centered on CNS-specific T cell responses, which are relevant to the pathogenesis of MS.
Our findings cannot be attributed to an enrichment of neuro-antigen reactive memory B cells in RRMS patients, since the neuro-antigen binding assay indicated that the frequencies of neuro-antigen binding memory B cells are similar in HD and RRMS patients. We cannot rule out, however, that other features of memory B cells from RRMS patients render them more capable of inducing neuro-antigen specific CD4+ T cell proliferation and cytokine secretion than their HD counterparts, including higher production of other inflammatory cytokines such as IL-1, IL-6, IL-8 and TNF, which are also readily produced by human B cells [36
], but were not measured in this series of experiments. It should also be noted that the ability of memory B cells from RRMS patients, but not HD to elicit CD4+ T cell proliferation in response to MBP was not attributable to general enrichment of MBP specific T cells in the RRMS patients, since the frequency of MBP specific T cells was similar in the HD and RRMS patient cohorts for this study. Others have also observed that the overall frequency of MBP specific T cells is similar between HD and RRMS patients [37
In contrast, the frequency of MOG specific T cells in the RRMS cohort was enriched in comparison to the HD cohort used for this study. Others have also observed that the frequency of MOG specific T cells is enriched in RRMS patients compared to HD [40
]. It is possible then, that the enhanced ability of memory and naïve B cells from RRMS patients, but not HD, to elicit CD4+ T cell proliferation and IFN-γ secretion in response to MOG is not due to enrichment of MOG-binding B cells in RRMS patients compared to HD, but may instead be due to enrichment of MOG specific T cells in the RRMS patients. Prior reports by others indicate that neuro-antigen-reactive memory T cells in particular are more prominent in RRMS patients than in HD [38
]. Thus, it is possible that B cells preferentially elicit proliferation and cytokine secretion by memory T cells, which may be most relevant to the underlying disease process. Determining the type of CD4+ T cell that is engaged by naïve and memory B cells in a MOG-specific manner is the focus of future studies by our laboratory.
The finding that MBP specific memory B cells are enriched in the peripheral blood of RRMS patients (compared to naïve) suggests that perhaps MBP specific B cells were primed in the periphery and have an effector function in the periphery as well. B cells could influence the pathogenesis of MS by priming T cells in secondary peripheral lymphoid organs, which then migrate to the CNS. B cells residing in the CNS may further promote CNS T cell activation, although this has not been formally tested. However, it should be noted that MBP specific memory B cells were also enriched in the peripheral blood of HD when compared to naïve B cells. In this scenario, HD peripheral memory B cells do not elicit CD4+ T cell proliferation in response to MBP. What impact removal of MBP-binding memory B cells has on the ability of the remaining B cell pool to elicit effector T cell function in response to MBP is under investigation in our laboratory.
GA-specific memory B cells, which are cross-reactive with MBP, may elicit the activation of MBP specific CD4+ T regulatory cells (Treg). In vitro treated CD4+
Treg show increased IL-10 production in response GA treatment [43
]. In addition, it was recently demonstrated that while untreated MS patients have reduced numbers of peripheral CD4+ Treg in comparison to HD, GA treatment restores both the number and functionality of these Treg [44
]. Perhaps this would suggest that memory B cells contribute most significantly to the expansion of CD4+ Treg in patients undergoing GA therapy.
Our data demonstrated that memory B cells from a subset of RRMS patients elicited IFN-γ secretion in response to MBP and MOG, but that memory B cells from HD did not elicit IFN-γ secretion in response to MBP and MOG. This data suggests that memory B cells from RRMS patients are more likely to promote activation of Th1 specific clones in response to MBP and MOG than memory B cells from HD. Of note, this response did not correlate with CD4+ T cell proliferation in individual patients (Supplementary Table 1
), and a comparison of MBP and MOG responders (defined as those patients whose B cells elicited IFN-γ secretion in response to MBP or MOG) in the RRMS and HD cohorts did not reach statistical significance. Other factors, such as the influence of CD8 T cells [45
] may explain the dichotomy in responses. Naïve B cells from 3 of 10 RRMS patients were also able to elicit IFN-γ secretion in response to MOG, and we cannot rule out that a small population of memory B cells that are CD27- [48
] may be present in our naïve B cell pools. Thus, it is possible that the IFN-γ secretion elicited by the naïve B cells from these 3 RRMS patients was mediated by CD27- memory B cells.
Interestingly, significant IFN-γ and IL-5 secretion was consistently observed in cultures where memory B cells from RRMS patients were presenting Tetanus Toxoid (TT) to T cells, suggesting that memory B cells are capable of stimulating both Th1 and Th2 TT reactive clones. Also of note, IFN-γ secretion in response to memory B cells from RRMS patients presenting TT was enhanced compared to memory B cells from HD presenting TT (). This is significant as it would suggest that TT specific T cell clones which do not typically secrete IFN-γ or show a predominant Th1 profile in HD [32
] have been skewed towards a Th1 phenotype in some RRMS patients. In fact, it was recently demonstrated that female RRMS patients show an exaggerated IFN-γ response to TT in PBMC as compared to control females [52
]. Our data may suggest that memory B cells from RRMS patients are likely candidates influencing this phenomenon.
In contrast to the increase in IFN-γ secretion in response to memory B cells presenting neuro-antigens or TT, IFN-γ secretion in response to memory B cells presenting GA was rarely observed ()(compare 6/9 TT responders to 2/11 GA responders, p=0.03). Yet CD4+ T cell proliferation in the presence of memory B cells and GA was robust in the majority of RRMS patients (). CD4+ T cell proliferation in the presence of memory B cells and GA was also robust in the majority of HD, yet IFN-γ was not observed in the majority of these cultures either. This data would suggest that memory B cells from RRMS patients and HD naïve to GA therapy maintain their capacity to elicit CD4+ T cell proliferation in response to GA, but that in most cases, are not able to drive T cell effector functions such as IFN-γ secretion. This was somewhat unexpected as the majority of GA-specific T cell lines derived from GA-naïve MS patients secrete IFN-γ readily [53
]. However, GA-specific T cell lines generated from MS patients that have been treated for several months with GA secrete significantly less IFN-γ [53
]. We would not expect that IFN-γ reactive T cell clones were absent in the MS patient cohort samples used in these studies since the patients were treatment naïve and in early stages of disease. It would be interesting to determine whether memory B cells from RRMS patients benefiting from GA therapy would additionally lose their capacity to elicit CD4+ T cell proliferation in response to GA, and the duration of GA therapy that is required to initiate this effect.
Of note, it has been documented that GA may not need to be processed since it binds directly to HLA-DR on the surface of APC [55
]. We were able to purify sufficient numbers of memory B cells from one patient to test whether GA processing by memory B cells was required to induce CD4+ T cell proliferation. Indeed, pretreatment of memory B cells with chloroquine, a lysosomotropic agent that prevents antigen processing, prior to co-culture with T cells and GA, reduced CD4+ T cell proliferation below the detection threshold in response to GA (data not shown).
Since sera from a subset of RRMS patients and HD contain MBP and MOG reactive antibodies [56
], we predicted that we would be able to detect memory B cells from RRMS patients and HD that would bind to MBP and MOG. Indeed, we detected memory B cells from RRMS patients and HD that bound MBP and MOG, yet only memory B cells from RRMS patients, but not from HD, elicited CD4+ T cell proliferation in response to MBP or MOG. As mentioned earlier in the discussion, this finding may be attributable to features of memory B cells from RRMS patients that are not present on memory B cells from HD or some differential enrichment of MBP- or MOG-specific memory T cells in the RRMS patients in comparison to HD. Thus, neuro-antigen binding memory B cells may have merit as a new target for immunotherapy.
However, naïve B cells from some RRMS patients, which bind to MOG at similar frequencies as memory B cells from RRMS patients, are also able to elicit CD4+ T cell proliferation in response to MOG. This finding is unique to MOG-reactive naïve B cells from RRMS patients since MBP-reactive naïve B cells from the same patients do not elicit appreciable CD4+ T cell proliferation in response to MBP. One possibility is that MOG-specific memory T cells enriched in RRMS patients can be reactivated by both memory and naïve B cells. Previous studies indicate that naïve B cells can elicit memory T cell activation [12
]. However, this hypothesis would apply only to MOG-specific responses, since naïve B cells do not elicit CD4+ T cell proliferation in response to MBP. It is also possible that if we had pre-activated naïve B cells from the RRMS patients, we may have also observed CD4+ T cell proliferation in response to MBP.
A second possibility is that MOG may be serving as a molecular mimic for an antigen recognized equally well by both the memory and naïve B cell repertoire, explaining the ability of both of these subtypes of B cells to bind and present MOG to T cells. For example, it has been demonstrated that MOG serum antibodies cross-react with epitopes present in the milk protein, butyrophilin [57
]. Additionally mice transgenic for IgH and IgL recognizing a conformationally specific epitope of MOG undergo receptor editing to endogenous light chains on both MOG sufficient and MOG deficient genetic backgrounds indicating some form of cross-reactivity of the MOG reactive antibodies to a distinct self protein [58
]. MOG could also potentially act as an auto-stimulatory TLR agonist in the context of RRMS as ribonucleoprotein particles do in the context of SLE [59
Speculation as to the development of neuro-antigen specific memory B cells in the context of RRMS is of great interest given the fact that autoreactive B cells are typically negatively selected during development [60
], or are prevented from accessing a germinal center reaction [61
]. Our results suggest that in this subset of RRMS patients whose memory B cells promoted CD4+ T cell expansion to MBP or MOG in vitro, either peripheral mechanisms of B cell tolerance failed or tolerance has been overcome through sensitization. Tolerance for example can be overcome by sensitization in a mouse model where B cells that express BCR specific for an endogenous neo-antigen are negatively selected during B cell development but none-the-less can develop a memory B cell repertoire that recognizes this neo-antigen after immunization [63
]. In addition, it was demonstrated that inherently autoreactive VH4-34 expressing B cells are not excluded from a germinal center reaction and can develop into memory B cells in the context of SLE [61
]. Whether this same principle explains the enrichment of neuro-antigen specific B cells in RRMS patients remains unexplored.
In conclusion, these studies identify MBP and MOG specific memory B cells as potent activators of neuro-antigen specific T cells from RRMS patients, but not HD. Thus, memory B cells may promote the exacerbation of RRMS by activating T cells in the periphery. Such studies provide a mechanistic explanation for why specific depletion of B cells from RRMS patients is beneficial for some MS patients and may indicate the need to investigate depletion of specific subsets (memory B cells) as a therapeutic strategy for patients whose memory B cells elicit CD4+ T cell proliferation and IFN-γ secretion in response to neuro-antigens.