Here, we demonstrated that B cells from patients with RRMS present cytotoxic behavior. Moreover, NTZ-treatment seems to restrain these cells in the peripheral blood effectively. These results may present a novel pathophysiological mechanism for the disease as well as for its treatment.
Until now, a vast collection of studies has consistently demonstrated the enhancement of cytotoxic activity during MS. Despite a possible role of GzmB in regulatory pathways , CD8 + T cells are a classical cytotoxic subset that can induce direct axonal and/or neuron injury through the MHC class I-mediated manner and GzmB releasing . Relapses during RRMS are associated with increased CD8+ T lymphocytes in CSF . Noteworthy, cytotoxic behavior seems not to be restricted to these classical CD8+ T lymphocytes. Recently, Larochelle and colleagues described the migratory capacity of CD8+GzmB+ and CD4+GzmB+ T lymphocytes into the CNS-parenchyma from fatal MS relapse after NTZ discontinuation . Moreover, our group also has previously identified the cytotoxic activity of B cells in neuroinflammatory diseases .
Our results demonstrated a slight but significant increase percentage of GzmB-producing B cells in untreated patients with RRMS in comparison with healthy individuals. Those cytotoxic B cells are capable of producing GzmB as well as to release the protease after IL-21 and CpG stimulation. Of note, untreated RRMS patients are significantly younger and with shorter time of disease diagnosis than treated RRMS patients. Many authors have proposed that B cells would take a later role in MS pathophysiology, especially in the formation of ectopically leukocytes aggregates . Therefore, it is plausible that during the early stage of the disease, the cytotoxic activity of B cells is less prominent. Nevertheless, it is difficult to test this possibility since the patients are treated at a very early stage upon diagnosis.
Regarding treatment, our data showed that FTY-treated RRMS patients present a significantly higher percentage of CD19+GzmB+ in comparison with healthy, untreated or GA-treated RRMS patients. The effect of FTY over the B cell population seems to be a lesser extent than T cells. On the one hand, the treatment with FTY inhibits the pro-inflammatory B cell function favoring the regulatory phenotype [22, 23]. On the other hand, FTY-treatment does affect the T-independent response . In this context, human cytotoxic B cells differentiate upon incomplete T-cell help [11, 25]. Thus, it is possible that FTY-treatment does not act over those cells, which would explain the increased proportion of granzyme B-expressing cells in the peripheral blood.
Besides, NTZ-treated RRMS patients present a consistent and significant retrains of CD8+ T lymphocytes and cytotoxic B cells in the peripheral blood. The restrain of activated T cells is the rationale of the NTZ treatment . In fact, during fatal rebound following NTZ withdrawal CNS-infiltrating lymphocytes, mainly CD8+ T cells, express high amounts of GzmB. Curiously, monocytes/macrophages, and B cells were enriched in the CNS parenchyma compared to the CSF . Here, we demonstrated that GzmB-expressing B cells are preferentially CD49d positive, which would explain the consistent restrain of cytotoxic B cells by NTZ treatment.
In contrast, NTZ promotes activation and pro-inflammatory enhancement of B cells in vitro . Moreover, those B cells with an inflammatory profile increase in the PB of NTZ-treated RRMS patients progressively . Nevertheless, the restrain of those pro-inflammatory B cells in the PB seem to be crucial for the effectiveness of the treatment.
Runx3 is a master regulator of the cytotoxic program in CD8+ T lymphocytes and natural killer cells [27, 28]. The ectopic expression of Runx3 has been extensively associated with non-classical cytotoxic cells [13, 16, 29]. Our data demonstrated a correlation between the expression of GzmB and Runx3 by B cell, which was independent of the treatment. Mechanistically, the ectopic expression of Runx3 by RRMS patient's B cells might explain the enhancement of cytotoxic activity. Of note, the expression of Runx3 is required for the proliferation of immortalized B cell during the Epstein-Barr virus (EBV) infection, which has been extensively associated with MS . Noteworthy, the ectopic expression of Runx3 by dendritic cells regulates the activity of the CD49d gene promoter .
Collectively, our findings support that beyond antigen presentation to T cells, releasing of lytic factors, secretion of anti and pro-inflammatory cytokines, cytotoxic behavior might emerge in the context of antibody-independent functions developed by B cells during MS. In this context, anti-CD20 monoclonal antibodies have been reported as a viable option for escalation, aiming to prevent relapses after NTZ therapy in MS patients . Anti-CD20 monoclonal antibodies main deplete naïve and memory B cells, preserving antibody-secreting B cells, which is confirmed by the sustained levels of immunoglobulins in the CSF from MS treated patients . The future investigation might reveal whether memory B cells responsible for antigen presentation, secretion of pro-inflammatory cytokines, and even, GzmB-expressing subsets are targeted by anti-CD20 depleting therapies during MS.
We are aware that the size of the cohort and the transversal nature of our study did not allow us to understand the clinical relevance of our finds better. Moreover, although we were able to establish a strong correlation between Runx3 and GzmB expression by B cells, there are not enough tools to imply causality to the phenomenon. In vitro generation of cytotoxic B cells will be necessary to clarify the role of Runx3 expression in the ectopic cytotoxic activity of these cells.