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ObjectiveWe investigated the predictive value of the enzyme-linked immunospot technique (ELISPOT) in identifying patients with relapsing-remitting multiple sclerosis (RRMS) who will respond to treatment with glatiramer acetate (GA) or interferon-β (IFN-β), based on the brain-reactive B-cell activity of peripheral blood cells.MethodsIn this retrospective, cross-sectional, real-world multicenter study, we identified patients with RRMS in the NeuroTransData MS registry and stratified them based on their documented treatment response (relapse-free in the first 12 months of treatment) to GA or IFN-β. The GA group comprised 73 patients who responded to GA and 35 nonresponders. The IFN-β group comprised 62 responders to IFN-β and 37 nonresponders. Patients with previous or current therapy affecting B-cell activity were excluded. We polyclonally stimulated mononuclear cells from peripheral blood samples (collected after participant selection) and investigated brain-reactive B-cell activity after incubation on brain tissue lysate-coated ELISPOT plates. Validity metrics of the ELISPOT testing results were calculated (Python 3.6.8) in relation to the clinical responsiveness in the 2 treatment groups.ResultsThe ELISPOT B-cell activity assay showed a sensitivity of 0.74, a specificity of 0.76, a positive predictive value of 0.78, a negative predictive value of 0.28, and a diagnostic OR of 8.99 in predicting clinical response to GA vs IFN-β therapy in patients with RRMS.ConclusionMeasurement of brain-reactive B-cell activity by ELISPOT provides clinically meaningful predictive probabilities of individual patients' treatment response to GA or IFN-β. The assay has the potential to improve the selection of optimal first-line treatment for individual patients with RRMS.Classification of EvidenceThis study provides Class II evidence that in patients with RRMS, the brain reactivity of their peripheral-blood B cells predicts clinical response to GA and IFN-β.

Background MP4-induced experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS), which enables targeted research on B cells, currently much discussed protagonists in MS pathogenesis. Here, we used this model to study the impact of the S1P1 receptor modulator FTY720 (fingolimod) on the autoreactive B cell and antibody response both in the periphery and the central nervous system (CNS). Methods MP4-immunized mice were treated orally with FTY720 for 30 days at the peak of disease or 50 days after EAE onset. The subsequent disease course was monitored and the MP4-specific B cell/antibody response was measured by ELISPOT and ELISA. RNA sequencing was performed to determine any effects on B cell-relevant gene expression. S1P1 receptor expression by peripheral T and B cells, B cell subset distribution in the spleen and B cell infiltration into the CNS were studied by flow cytometry. The formation of B cell aggregates and of tertiary lymphoid organs (TLOs) was evaluated by histology and immunohistochemistry. Potential direct effects of FTY720 on B cell aggregation were studied in vitro. Results FTY720 significantly attenuated clinical EAE when treatment was initiated at the peak of EAE. While there was a significant reduction in the number of T cells in the blood after FTY720 treatment, B cells were only slightly diminished. Yet, there was evidence for the modulation of B cell receptor-mediated signaling upon FTY720 treatment. In addition, we detected a significant increase in the percentage of B220+ B cells in the spleen both in acute and chronic EAE. Whereas acute treatment completely abrogated B cell aggregate formation in the CNS, the numbers of infiltrating B cells and plasma cells were comparable between vehicle- and FTY720-treated mice. In addition, there was no effect on already developed aggregates in chronic EAE. In vitro B cell aggregation assays suggested the absence of a direct effect of FTY720 on B cell aggregation. However, FTY720 impacted the evolution of B cell aggregates into TLOs. Conclusions The data suggest differential effects of FTY720 on the B cell compartment in MP4-induced EAE. Electronic supplementary material The online version of this article (doi:10.1186/s12974-017-0924-4) contains supplementary material, which is available to authorized users.

B cell aggregates in the central nervous system (CNS) have been associated with rapid disease progression in patients with multiple sclerosis (MS). Here we demonstrate a key role of carcinoembryogenic antigen-related cell adhesion molecule1 (CEACAM1) in B cell aggregate formation in MS patients and a B cell-dependent mouse model of MS. CEACAM1 expression was increased on peripheral blood B cells and CEACAM1(+) B cells were present in brain infiltrates of MS patients. Administration of the anti-CEACAM1 antibody T84.1 was efficient in blocking aggregation of B cells derived from MS patients. Along these lines, application of the monoclonal anti-CEACAM1 antibody mCC1 was able to inhibit CNS B cell aggregate formation and significantly attenuated established MS-like disease in mice in the absence of any adverse effects. CEACAM1 was co-expressed with the regulator molecule T cell immunoglobulin and mucin domain -3 (TIM-3) on B cells, a novel molecule that has recently been described to induce anergy in T cells. Interestingly, elevated coexpression on B cells coincided with an autoreactive T helper cell phenotype in MS patients. Overall, these data identify CEACAM1 as a clinically highly interesting target in MS pathogenesis and open new therapeutic avenues for the treatment of the disease. CA extern

B cells have only recently begun to attract attention in the immunopathology of multiple sclerosis (MS). Suitable markers for the prediction of treatment success with immunomodulatory drugs are still missing. Here we evaluated the B cell response to brain antigens in n = 34 relapsing-remitting MS (RRMS) patients treated with glatiramer acetate (GA) using the enzyme-linked immunospot technique (ELISPOT). Our data demonstrate that patients can be subdivided into responders that show brain-specific B cell reactivity in the blood and patients without this reactivity. Only in patients that classified as B cell responders, there was a significant positive correlation between treatment duration and the time since last relapse in our study. This correlation was GA-specific because it was absent in a control group that consisted of interferon-ß (IFN-β)-treated RRMS patients (n = 23). These data suggest that GA has an effect on brain-reactive B cells in a subset of patients and that only this subset benefits from treatment. The detection of brain-reactive B cells is likely to be a suitable tool to identify drug responders.

Multiple sclerosis (MS) is an autoimmune disease caused by the infiltration of autoreactive lymphocytes into the central nervous system (CNS). The influence of B cells has been underestimated for a long time. Recently, it has become more apparent that B cells can fundamentally contribute to the pathogenesis of MS in terms of antigen presentation, co-stimulation, cytokine production, ectopic lymphoneogenesis, and antibody secretion (1–3). Along these lines, B cell depletion in MS patients with a relapsing-remitting disease course was able to reduce inflammatory brain lesions and clinical relapses (4) and B cell activation influences T cell polarization in the animal model of MS (5). Great effort has been made to utilize B cells for dividing patients into subgroups and to predict treatment responses in individual patients to specific drugs (6). Especially the detection of autoantibodies against CNS antigens is a crucial research subject due to its capability to be used as disease-specific biomarkers for the diagnosis and prognosis of MS. To this end, in a clinical study published in 2012 in the New England Journal of Medicine, Srivastava and colleagues screened serum samples aiming to identify CNS-specific antibodies in MS (7). The authors identified the glial potassium channel KIR4.1 as one of the serum antibody targets in MS patients. Subsequently, an enzyme-linked immunosorbent assay (ELISA) large-scale screening with 397 MS patients, 329 persons with other neurological diseases, and 59 healthy donors was performed. Antibodies against KIR4.1 were observed in 46.9% of patients with MS, but were essentially absent in people with other neurological diseases and healthy donors. Based on these data, Srivastava et al. concluded that KIR4.1 is a CNS-specific target of the autoantibody response in a subgroup of patients with MS. We would like to carefully raise the possibility that the serum IgG reactivity against KIR4.1 might present an epiphenomenon and there could be an additional target for autoreactive B cells beyond the CNS. It has to be considered that KIR4.1 is not only expressed in the CNS, but also on parietal cells of the gastric mucosal epithelium (8). The KIR4.1 channel expression is essential for the parietal cell control of acid secretion (9, 10). It is well known that there is a connection between the gastrointestinal immune response and CNS autoimmunity (11, 12). There are several possible mechanisms including molecular mimicry, determinant spreading, or bystander activation explaining the link between gastrointestinal immune response and MS (13). As recently published by Banati et al. (11), antibodies against parietal cells are also present in the sera of patients with MS. The presence of anti-parietal cell antibodies was most frequently associated with gastrointestinal complaints of the MS patients (11). Thus, it is possible that an impaired gastric mucosa, which can appear in cases of a frequent corticosteroid therapy, could extensively expose the parietal cell KIR4.1 to B cells. Corticosteroids augment gastroduodenal permeability and high doses are associated with macroscopic mucosal lesions (14). Therefore, we consider it as necessary to include the history of gastrointestinal complaints in the anamnesis of the study subjects before defining KIR4.1 as a CNS target of serum antibodies. In the setting of patients with an initial demyelinating event who have not received corticosteroids, one could readily exclude an impact of the MS medication on the gut. Finding anti-KIR4.1 antibodies in such patients in combination with gut impairments could raise the possibility that the gut itself is also a target of the autoimmune process. While further investigations are awaited to clearly analyze the impact of immune responses against antigens of gastric parietal cells on the finding of KIR4.1-reactive antibodies in patients with MS, the diagnostic potential of serum KIR4.1 antibodies may be highly clinically relevant. Therefore, the sensitivity and specificity of the initial findings of serum KIR4.1 antibodies in a subgroup of MS patients should be confirmed in studies from independent researchers. Furthermore, the pathogenicity of KIR4.1 antibodies needs to be clarified. Only 2 of 19 serum IgG positive MS patients also had detectable levels of KIR4.1 antibodies in the cerebrospinal fluid (CSF) (7). By analogy, aquaporin-4 antibodies were not readily detected in the CSF in neuromyelitis optica patients (15). Immunhistochemical investigations by Lennon et al. (16) showed that serum aquaporin-4 antibodies in patients with neuromyelitis optica bind to CNS tissue as well as to parietal cells of the gastric mucosa (16). However, it is beyond doubt that aquaporin-4 antibodies are highly pathogenic in neuromyelitis optica since they were studied extensively. Kinoshita and colleagues have demonstrated the pathogenic role of aquaporin-4 antibodies in vivo (17). They obtained serum IgG from patients with neuromyelitis optica and detected astrocytic damage in rats after passive transfer of aquaporin-4 containing IgG. In vivo experiments of this kind could delineate whether KIR4.1 antibodies are only a disease marker or if they play a central pathogenic role. To this end, KIR4.1 antibody containing serum could be injected into mice and subsequent histological and immunohistochemical investigations could show potential resulting pathological effects in CNS and gut. Furthermore, astrocytes should be incubated with KIR4.1 antibody containing serum and cell viability assays may demonstrate whether KIR4.1-reactive IgG induces astrocytic cytotoxicity.

B cells are increasingly coming into play in the pathogenesis of multiple sclerosis (MS). Here, we screened peripheral blood mononuclear cells (PBMC) from patients with clinically isolated syndrome (CIS), MS, other non-inflammatory neurological, inflammatory neurological or autoimmune diseases, and healthy donors for their B cell reactivity to CNS antigen using the enzyme-linked immunospot technique (ELISPOT) after 96h of polyclonal stimulation. Our data show that nine of 15 patients with CIS (60.0%) and 53 of 67 patients with definite MS (79.1%) displayed CNS-reactive B cells, compared to none of the control donors. The presence of CNS-reactive B cells in the blood of the majority of patients with MS or at risk to develop MS along with their absence in control subjects suggests that they might be indicative of a B cell-dependent subpopulation of the disease.

In this study we aimed to investigate whether treatment with an immune modulatory drug had an effect on the distribution of B cell subpopulations in patients with remitting-relapsing multiple sclerosis (RRMS). We investigated the first-line drugs glatiramer acetate, interferon-β and natalizumab. Our data show that the frequency of the CD27(+)CD43(+) B1 cell subset was significantly diminished in RRMS patients compared to healthy subjects and that this subset was unaffected by treatment. Regardless of their true nature, we believe that these cells are part of the autoimmune disease pattern.