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CD40 interacts with CD40L and plays an essential role in immune regulation and homeostasis. Recent research findings, however, support a pathogenic role of CD40 in a number of autoimmune diseases. We previously showed that memory B cells from relapsing-remitting multiple sclerosis (RRMS) patients exhibited enhanced proliferation with CD40 stimulation compared with healthy donors. In this study, we used a multiparameter phosflow approach to analyze the phosphorylation status of NF-κB and three major MAPKs (P38, ERK, and JNK), the essential components of signaling pathways downstream of CD40 engagement in B cells from MS patients. We found that memory and naive B cells from RRMS and secondary progressive MS patients exhibited a significantly elevated level of phosphorylated NF-κB (p-P65) following CD40 stimulation compared with healthy donor controls. Combination therapy with IFN-β-1a (Avonex) and mycophenolate mofetil (Cellcept) modulated the hyperphosphorylation of P65 in B cells of RRMS patients at levels similar to healthy donor controls. Lower disease activity after the combination therapy correlated with the reduced phosphorylation of P65 following CD40 stimulation in treated patients. Additionally, glatiramer acetate treatment also significantly reduced CD40-mediated P65 phosphorylation in RRMS patients, suggesting that reducing CD40-mediated p-P65 induction may be a general mechanism by which some current therapies modulate MS disease.

My lab’s recent work has led to the discovery that antibodies expressed by memory B cells in the cerebrospinal fluid (CSF) of patients with Multiple Sclerosis recognize neurons and astrocytes in the brain. Genetic analysis further revealed that only certain heavy chain genes accommodate binding to these brain targets, and that memory B cells from control cohorts rarely express these antibody genes. In fact, in a recent clinical trial, we demonstrated that the prevalence of these antibody genes expressed by memory B cells in the CSF support identification of patients who either have MS or will develop MS in the future with 89–94% accuracy. This Antibody Gene Signature (AGS) is a novel biomarker that heralds the advent of antibody genetics as a means to support categorization of patients early in their disease course. Our goal for this project was to determine whether antibodies expressed by activated memory B cells that have initiated antibody production (called “plasmablasts”) in the peripheral blood of MS patients also recognize neurons and astrocytes. We cloned and expressed more than 50 antibodies expressed by peripheral plasmablasts of early MS patients and control cohorts. We found that antibodies expressed by peripheral plasmablasts from patients who develop MS bind to neurons and astrocytes in the brain. Those plasmablasts that did not express antibody heavy chains associated with the AGS did not bind to brain targets. Interestingly, only those patients in our cohort who have an elevated frequency of peripheral plasmablasts continue to accumulate brain MRI activity. It is possible that brain-reactive peripheral plasmablasts contribute to the brain damage associated with MS.

Plasmablasts are a transient B cell subtype that secretes large amounts of antibody and arises in response to antigen exposure and T cell help. Once plasmablasts form they die off in a few days or differentiate into a long lived plasma cells, and therefore are only present during an active immune response. In our laboratory a subset of Clinically Isolated Syndrome (CIS) patients presenting with Transverse Myelitis (TM) symptoms at high risk to develop Multiple Sclerosis (MS) also have an elevated level of plasmablasts in the peripheral blood and/or the cerebrospinal fluid. Interestingly, only those patients in our cohort who have elevated plasmablast levels continue to accumulate brain MRI activity. The antibody genetics of plasmablasts from CIS TM patients are altered in comparison to those of plasmablasts responding to influenza from otherwise healthy individuals. There is increased evidence of receptor editing in CIS patients, as well as enrichment of the Antibody Gene Signature (AGS), which predicts whether CIS patients will convert to MS later in life. Antibodies from CIS plasmablasts that are enriched in overall mutations and AGS mutations recognize mouse brain by ELISA and histology, binding to multiple different cell types. By flow cytometry we have shown that many of these antibodies recognize mouse astrocyte and neuron cell lines. If these antibodies are able to bind brain tissue in vivo, they could exacerbate brain damage and contribute to disease in CIS patients.

Patients with relapsing-remitting multiple sclerosis (RRMS), or who will convert to RRMS, have cerebrospinal fluid (CSF) B cells that accumulate specific replacement mutations in their rearranged VH4 gene segments as identified by single-cell Sanger sequencing. To harness this observation in a clinical setting, we have developed a diagnostic test to extract and score this genetic information from bulk CSF lymphocytes by next-generation DNA sequencing (NGS). Our current aim is to determine the accuracy of this test on a cohort of patients with varying times of RRMS onset and disease modifying therapies, as well as on a diverse cohort of patients with other neurological diseases (OND) that generally mimic the presentation of MS. Our initial testing on 39 patients has shown an accuracy of 84% in distinguishing RRMS from OND patients. As we continue to expand the size of the cohorts being tested, we will also focus on identifying differences between sub-cohorts of patients at different stages of RRMS or with particular OND diagnoses. With a growing need for a faster and more accurate diagnosis of RRMS, MSPrecise may be a useful addition to the existing diagnostic toolkit that clinicians use to achieve this goal.

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.

We have previously identified a distinct mutation pattern in the B cell receptors of cerebrospinal fluid B cells that can predict conversion to multiple sclerosis. This antibody gene signature, developed using Sanger sequencing on singly-sorted B cells, is limited in its clinical application by the time and effort required to obtain such single-cell repertoires. In order to increase the throughput of antibody gene signature evaluation for patients, we used 454 sequencing on bulk-sorted cerebrospinal fluid B cell pellets. We focused our analysis on identifying the mutation and distribution profiles of the repertoires obtained via both sequencing methods. Here we outline the additional filtering and data processing steps used to correct for 454 platform bias and compare the 454 and Sanger sequencing-derived repertoires which include multiple sclerosis samples sequenced by both methods in parallel. Although we observe variable amounts of background mutation and uneven sequence amplification noise by 454 sequencing for our matched samples, most of the antibody gene signature scores remain above the multiple sclerosis conversion threshold established by Sanger sequencing. We expect that additional improvements directed at the sample preparation, data filtering and sequence clustering methods presented here will help further improve the reliability of next generation sequencing-based multiple sclerosis conversion diagnosis.

My lab’s recent work on the antibody genetics of multiple sclerosis (MS) patients has led to the astonishing discovery that patterns of mutation accumulation into the antibody genes of B cells in the central nervous system (CNS) of MS patients are unique and can be used to predict clinically isolated syndrome (CIS) conversion to MS with 94% accuracy. This Antibody Gene Signature (AGS) is a novel, potentially diagnostic biomarker that is currently in clinical trials and heralds the advent of antibody genetics as a means to categorize patients early in their disease course in order to provide prompt treatment and prevent extensive CNS damage. Our goal for this project was to determine what antibodies expressed by these B cells from MS patients reacted against. We cloned and expressed more than 30 antibodies that contain the AGS-MS from cerebrospinal fluid derived single B cells of MS and CIS patients, and tested their capacity to bind to brain tissue. We found that these AGS-MS enriched antibodies bind to neurons and astrocytes that reside in the brain gray matter tissue with high specificity. Some of these antibodies recognize both neurons and astrocytes in the gray matter, whereas others are specific for a single cell type. We hypothesize that AGS-enriched rhAbs and B cells expressing these antibodies could be participating in gray matter disease pathology in both early CIS and later MS stages.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS) that afflicts more than 400,000 people in the US. Although the etiology of the disease is unknown, pathogenic T cells are thought to underlie MS immune pathology. In contrast to the current paradigm, we recently showed that MS patients harbor CNS-specific CD8+ T regulatory cells (CD8 Tregs) that are deficient during disease relapse. In the current study, we demonstrate that the neuroantigen-specific CD8 Tregs were cytolytic and eliminated pathogenic CD4+ T cells. Sorting of CD8+ T cells using an array of surface cellular markers revealed that the CD8 Tregs were terminally differentiated (CD27-, CD45RO-). The CD8 Treg-mediated suppression was perforin, granzyme B, and interferon-γ-dependent. Interestingly, we found that MS patients with acute disease exacerbation displayed a significant loss (averaging 25%) in the terminally differentiated CD8+ T cells, with a concurrent loss in perforin and granzyme B expression. In order to restore the regulatory potential of impaired CD8 Tregs during exacerbation, we pre-treated exacerbation-derived bulk CD8+ T cells with the cytokine IL-12 and significantly increased the suppressive capability of the cells by ~48% through upregulation of granzyme B and perforin. Our studies uncover the immune suppressive mechanism of neuroantigen-specific CD8 Tregs, and may contribute to the design of clinically relevant immune therapies for MS patients.

Daily subcutaneous administration of FDA-approved glatiramer acetate (GA) has beneficial effects on clinical course of relapsing remitting multiple sclerosis (RRMS). Although mechanism of GA-action has been widely investigated and partially understood, immediate immune dynamics following GA-therapy and their significance are unknown. In the present study we characterized immediate effects of GA on phenotype, quantity and function of immune cell subsets in MS patients. PBMC from RRMS patients, either untreated or initiating GA, and healthy donors were obtained at time of admission and subsequently at 4h, 12h, 24h and 72h after first GA-injection. Prominent changes in immune cells were detected within 4-12h post first GA-injection. T-cell modulation included significantly decreased CD4/CD8 ratio, perturbation in homeostasis of predominantly CD8+ T-cells, significant enhancement in CD8+ T-cell mediated suppression and inhibitory potential of induced CD4-suppressors. Changes in APCs were restricted to monocytes and included reduced stimulatory capacity in MLR and significantly increased IL-10 and TNF-α. Our study provides first evidence that GA induced rapid immunologic changes even after first dose. Interestingly, these responses are not restricted to APCs but also include complex modulation of T-cell functionality. Long-term studies will evaluate sustenance of these effects over time and their significance in clinical efficacy of therapy.

Multiple Sclerosis (MS) is the leading disease of the central nervous system and a significant, costly cause of disability in young adults. Patients at risk for MS often present with a single demyelinating event, typically called a “clinically isolated syndrome” (CIS). A CIS can occur in the brain, spinal cord (Acute Partial Transverse Myelitis, APTM), or optic nerve (Optic Neuritis, ON). Our laboratory discovered a unique pattern of antibody gene mutation accumulation (i.e. antibody gene signature, or “AGS”) that is exclusive to B cells from the cerebrospinal fluid (CSF) and brain lesions of MS patients and CIS patients that develop MS in the future. Our goal for this study was to determine whether CIS and MS patient B cells that carry this pattern of somatic hypermutation accumulation in their antibody genes react to brain tissue. Thirty-four antibody genes of B cells isolated from the CSF of MS and CIS patients carrying this AGS pattern were cloned into antibody expression vectors to generate recombinant full-length human antibodies (rhAbs). Preliminary DAB staining using a subgroup of these rhAbs on cryo-sectioned mouse brain demonstrates binding to CNS tissue. Cortical staining of varied intensities was found among the rhAbs tested. Patterns of staining and details of antigen reactivity will be presented. This result provides promise in continuing to identify the auto-antigens that are recognized by the AGS B cells shared among patients at risk to convert to MS.

Multiple Sclerosis (MS) is an immune-mediated, demyelinating disorder of the CNS. Glatiramer acetate (GA/Copaxone) is an FDA-approved treatment for relapsing-remitting MS that induces functional changes in antigen-presenting cells (APC) and CD4 and CD8 T-cells. We have previously shown that treatment-naïve MS patients have deficient CD8 T-cell responses to GA, which are restored after GA therapy. This is associated with enhanced cytotoxic/suppressive function in these cells. In this study, we assessed the immediate immunologic changes induced by GA therapy in treatment-naïve MS patients by multiparametric monitoring of T-cell and APC subsets prior to and at 4, 12, 24, 72 hrs of treatment. In untreated healthy control subjects quantitative and functional readouts showed stable dynamics over time. MS patients showed greater proportions of memory/effector CD4 and CD8 populations at baseline, which did not vary. Similarly, cytokine production by APC subsets was also stable. Interestingly, as early as 4 to 12 hrs after the initial GA injection, we observed significantly enhanced suppressive ability in CD8 T-cells. This rapid functional change consistently occurred in GA, as well as an IFN-b-treated control cohort. These studies show that the induction of T-cell suppressive function is an important early event during therapy. Future studies will delineate whether these changes are predictive of sustained longterm immunologic and clinical responsiveness.

Multiple Sclerosis (MS) is the leading disease of the central nervous system and a significant, costly cause of disability in young adults. Patients at risk for MS often present with a single demyelinating event, typically called a “clinically isolated syndrome” (CIS). A CIS can occur in the brain, spinal cord (Acute Partial Transverse Myelitis, APTM), or optic nerve (Optic Neuritis, ON) at similar rates and present a challenging diagnostic and therapeutic dilemma. Stratifying CIS patients most likely to develop MS is a complicated process, but desirable since treatment with the appropriate immunomodulatory agents early in the disease course can delay long-term disability. Thus, a primary focus of our laboratory has been to develop a completely novel type of biomarker that can identify CIS patients that will develop MS. This unique biomarker is based on a pattern of antibody gene mutations (i.e. antibody gene signature or “AGS”) that is exclusive to B cells from the cerebrospinal fluid and brain lesions of MS patients that initially presented with either TM or ON. In fact, the AGS is consistently elevated in patients with one attack of ON that go on to develop definite MS, and can predict conversion to MS with 91% accuracy. More recently, we are testing the hypothesis that the AGS serves as a predictor for conversion to MS in CIS patients that present with TM. Preliminary data demonstrate that the AGS is also prevalent in CIS patients presenting with TM.

Recent evidence suggests that B and T cell interactions may be paramount in relapsing remitting multiple sclerosis (RRMS) disease pathogenesis. We hypothesized that memory B cell pools from RRMS patients may specifically harbor a subset of potent neuro-antigen presenting cells that support neuro-antigen reactive T cell expansion and activation. To assess the potential influence of neuro-antigen specific memory B cells from RRMS patients on autologous CD4+ T cell activation, we compared CD80 and HLA-DR expression, IL-10 and LTα secretion, neuro-antigen binding capacity, and functional neuro-antigen presentation by memory B cells from RRMS patients and compared these parameters to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HDs). We identified memory B cells from a subset of RRMS patients that were functionally capable of presenting neuro-antigen to autologous T cells. Notwithstanding the fact that the phenotypic parameters that promote efficient antigen presentation were observed to be similar between RRMS and HDs memory B cells, a corresponding capability to present neuro-antigen was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B cell pool in RRMS harbors neuro-antigen specific clones that can activate T cells.