IMPORTANCEThis study describes what is, to our knowledge, the previously unknown effect of glatiramer acetate therapy on B cells in patients with relapsing-remitting multiple sclerosis (MS).OBJECTIVE To determine whether glatiramer acetate therapy normalizes dysregulated B-cell proliferation and cytokine production in patients with MS. DESIGN, SETTING, AND PARTICIPANTS Twenty-two patients with MS who were receiving glatiramer acetate therapy and 22 treatment-naive patients with MS were recruited at The University of Texas Southwestern Medical Center MS clinic. Cell samples from healthy donors were obtained from HemaCare (Van Nuys, California) or Carter Blood Bank (Dallas, Texas). Treatment-naive patients with MS had not received any disease-modifying therapies for at least 3 months before the study.EXPOSURES Glatiramer acetate therapy for at least 3 months at the time of the study. MAIN OUTCOMES AND MEASURES B-cell phenotype and proliferation and immunoglobulin and cytokine secretion.RESULTS A restoration of interleukin 10 production by peripheral B cells was observed in patients undergoing glatiramer acetate therapy as well as a significant reduction of interleukin 6 production in a subset of patients who received therapy for less than 32 months. Furthermore, proliferation in response to high-dose CD40L was altered and immunoglobulin production was elevated in in vitro-activated B cells obtained from patients who received glatiramer acetate.CONCLUSIONS AND RELEVANCE Glatiramer acetate therapy remodels the composition of the B-cell compartment and influences cytokine secretion and immunoglobulin production. These data suggest that glatiramer acetate therapy affects several aspects of dysregulated B-cell function in MS that may contribute to the therapeutic mechanisms of glatiramer acetate.

Plasmablasts are a highly differentiated, antibody secreting B cell subset whose prevalence correlates with disease activity in Multiple Sclerosis (MS). For most patients experiencing partial transverse myelitis (PTM), plasmablasts are elevated in the blood at the first clinical presentation of disease (known as clinically isolated syndrome or CIS). In this study we found that many of these peripheral plasmablasts are autoreactive and recognize primarily gray matter targets in brain tissue. These plasmablasts express antibodies that over-utilize immunoglobulin heavy chain V-region subgroup 4 (VH4) genes, and the highly mutated VH4+ plasmablast antibodies recognize intracellular antigens of neurons and astrocytes. Most of the autoreactive, highly mutated VH4+ plasmablast antibodies recognize only a portion of cortical neurons, indicating that the response may be specific to neuronal subgroups or layers. Furthermore, CIS-PTM patients with this plasmablast response also exhibit modest reactivity toward neuroantigens in the plasma IgG antibody pool. Taken together, these data indicate that expanded VH4+ peripheral plasmablasts in early MS patients recognize brain gray matter antigens. Peripheral plasmablasts may be participating in the autoimmune response associated with MS, and provide an interesting avenue for investigating the expansion of autoreactive B cells at the time of the first documented clinical event.

*These authors contributed equally to the work in this manuscript.We have previously identified a distinct class of antibodies expressed by B cells in the cerebrospinal fluid (CSF) of early and established relapsing remitting multiple sclerosis (RRMS) patients that is not observed in healthy donors. These antibodies contain a unique pattern of mutations in six codons along VH4 antibody genes that we termed the antibody gene signature (AGS). In fact, patients who have such B cells in their CSF are identified as either having RRMS or developing RRMS in the future. As mutations in antibody genes increase antibody affinity for particular antigens, the goal for this study was to investigate whether AGS+ antibodies bind to brain tissue antigens. Single B cells were isolated from the CSF of 10 patients with early or established RRMS. We chose 32 of these B cells that expressed antibodies enriched for the AGS for further study. We generated monoclonal full-length recombinant human antibodies (rhAbs) and used both immunological assays and immunohistochemistry to investigate the capacity of these AGS+ rhAbs to bind brain tissue antigens. AGS+ rhAbs did not recognize myelin tracts in the corpus callosum. Instead, AGS+ rhAbs recognized neuronal nuclei and/or astrocytes, which are prevalent in the cortical gray matter. This pattern was unique to the AGS+ antibodies from early and established RRMS patients, as AGS+ antibodies from an early neuromyelitis optica patient did not display the same reactivity. Prevalence of CSF-derived B cells expressing AGS+ antibodies that bind to these cell types may be an indicator of gray matter-directed autoimmunity in early and established RRMS patients.

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The role of B cells in the pathoetiology of relapsing remitting multiple sclerosis is not well understood. B cells may contribute to MS by antibody secretion, cytokine production and antigen presentation. Antigen-specific B cells are highly efficient antigen presenting cells in the context of their cognate antigens. Since antibodies from multiple sclerosis patients bind to neuroantigens we hypothesized that neuroantigen specific memory B cells promote disease in multiple sclerosis patients by serving as antigen presenting cells for auto-reactive T cells. We observed that B cells from a subset of multiple sclerosis patients induce naïve and memory T cell proliferation in the presence of recombinant neuroantigens, myelin oligodendrocyte glycoprotein and myelin basic protein, but not MOG 35-55 peptide, in B-T cell co-cultures. Similarly, in the presence of neuroantigen proteins cells from multiple sclerosis patients secreted greater levels of IFN-γ, IL-6, -17A, -17F, -21, -22, -31, -33, TNF-α and soluble CD40L than healthy donors, while levels of IL-4 and IL-25 were similar. These data support a role for human B cells as antigen presenting cells in multiple sclerosis and highlight the role of B cells in the context of autoimmune disease beyond antibody production.