In multiple sclerosis (MS4) B cell depleting therapy using monoclonal anti-CD20 antibodies, including rituximab (RTX) and ocrelizumab (OCR), effectively reduces disease activity. Based on indirect evidence, it is generally believed that elimination of the antigen presenting capabilities and antigen non-specific immune functions of B cells underlie the therapeutic efficacy. However, a small subset of T lymphocytes (T cells) was shown to also express CD20, but controversy prevails surrounding the true existence of this T cell subpopulation. Using single-cell imaging flow cytometry and expression profiling of sorted lymphocyte subsets, we unequivocally demonstrate the existence of CD3+CD20dim T cells. We show that in MS patients increased levels of CD3+CD20dim T cells are effectively depleted by RTX. The pathological relevance of this T cell subset in MS remains to be determined. However, given their potential pro-inflammatory functionality, depletion of CD20-expressing T cells may also contribute to the therapeutic effect of RTX and other monoclonal antibodies targeting CD20.

Clemastine fumarate as a remyelinating therapy for multiple sclerosis (ReBUILD): a randomised, controlled, double-blind, crossover trial.

In multiple sclerosis (MS) pathogenic B cells likely act on both sides of the blood-brain barrier (BBB). However, it is unclear whether antigen-experienced B cells are shared between the CNS and the peripheral blood (PB) compartments. We applied deep repertoire sequencing of IgG heavy chain variable region genes (IgG-VH) in paired cerebrospinal fluid and PB samples from patients with MS and other neurological diseases to identify related B cells that are common to both compartments. For the first time to our knowledge, we found that a restricted pool of clonally related B cells participated in robust bidirectional exchange across the BBB. Some clusters of related IgG-VH appeared to have undergone active diversification primarily in the CNS, while others have undergone active diversification in the periphery or in both compartments in parallel. B cells are strong candidates for autoimmune effector cells in MS, and these findings suggest that CNS-directed autoimmunity may be triggered and supported on both sides of the BBB. These data also provide a powerful approach to identify and monitor B cells in the PB that correspond to clonally amplified populations in the CNS in MS and other inflammatory states.

A diverse antibody repertoire is essential for an effective adaptive immune response to novel molecular surfaces. Although past studies have observed common patterns of V-segment use, as well as variation in V-segment use between individuals, the relative contributions to variance from genetics, disease, age, and environment have remained unclear. Using high-throughput sequence analysis of monozygotic twins, we show that variation in naive V H and D H segment use is strongly determined by an individual's germ-line genetic background. The inherited segment-use profiles are resilient to differential environmental exposure, disease processes, and chronic lymphocyte depletion therapy. Signatures of the inherited profiles were observed in class switched germ-line use of each individual. However, despite heritable segment use, the rearranged complementarity-determining region-H3 repertoires remained highly specific to the individual. As it has been previously demonstrated that certain V-segments exhibit biased representation in autoimmunity, lymphoma, and viral infection, we anticipate our findings may provide a unique mechanism for stratifying individual risk profiles in specific diseases.heritable variation | next generation sequencing | V-gene S pecific biases in the antibody repertoire have been found in many diseases, from viral infections to cancers to autoimmune disorders (1-15). Although it is possible that heritable variation in the composition of the antibody repertoire could alter inherent risk to specific diseases, the diversity of the antibody repertoire has hindered direct characterization of heritable influences.Early twin studies provided some evidence of genetic variation affecting reactive titers from the antibody repertoire. Multiple studies observed both total Ig and antigen-specific titers to be more correlated in monozygotic twins than dizygotic twins or unrelated individuals (16)(17)(18). In some cases of monozygotic twins discordant for autoimmune diseases, the healthy twin often shared high autoantibody reactive titers with their affected twin (16,19,20).Early sequencing studies were able to identify some systematic biases in the antibody repertoire with limited sampling depth. The first sequencing studies to characterize V(D)J diversification mechanisms identified the gene segment recombination process, but also implied a repertoire too diverse to exhaustively interrogate by traditional sequencing technologies (21). Complete characterization of V-segment loci established ∼50 V H , 40 V κ , and 30 V λ segments in an individual, with a number of allelic variants for the majority of segments (22)(23)(24). Evaluation of use across individuals revealed biased V-gene representation that preceded selection (25)(26)(27). Quantitative PCR of V-gene families showed family use largely stable over time, with fluctuations in use correlated to antigen-specific responses (28). In the T-cell receptor (TCR) repertoire, TCRB-V use was more highly correlated in healthy monozygotic twins than unrelated individua...

Demyelination in MS disrupts nerve signals and contributes to axon degeneration. While remyelination promises to restore lost function, it remains unclear whether remyelination will prevent axonal loss. Inflammatory demyelination is accompanied by significant neuronal loss in the experimental autoimmune encephalomyelitis (EAE) mouse model and evidence for remyelination in this model is complicated by ongoing inflammation, degeneration and possible remyelination. Demonstrating the functional significance of remyelination necessitates selectively altering the timing of remyelination relative to inflammation and degeneration. We demonstrate accelerated remyelination after EAE induction by direct lineage analysis and hypothesize that newly formed myelin remains stable at the height of inflammation due in part to the absence of MOG expression in immature myelin. Oligodendroglial-specific genetic ablation of the M1 muscarinic receptor, a potent negative regulator of oligodendrocyte differentiation and myelination, results in accelerated remyelination, preventing axonal loss and improving functional recovery. Together our findings demonstrate that accelerated remyelination supports axonal integrity and neuronal function after inflammatory demyelination.DOI: http://dx.doi.org/10.7554/eLife.18246.001

In multiple sclerosis (MS), an exchange of lymphocytes, in particular B cells, between the central nervous system (CNS) and periphery is believed to be required for the maintenance of active disease. Therapeutic monoclonal antibodies that prevent lymphocytes from crossing the blood-brain barrier (BBB) or induce near-complete peripheral B cell depletion rapidly mitigate MS disease activity. Using next-generation sequencing technology, we recently found that clonally related B cells exist in the cerebrospinal fluid (CSF) and peripheral blood (PB) of MS patients, establishing the existence of an immune axis across the BBB. However, it remains unclear which subpopulations of the highly diverse peripheral B cell compartment share antigen-specificity with intrathecal B cell repertoires, and whether their antigen stimulation occurs on both sides of the BBB. To address these questions, we combined flow cytometry sorting of PB B cell subsets with deep immune repertoire sequencing of CSF and PB B cells. Immunoglobulin (IgM and IgG) heavy chain variable (VH) region repertoires of five PB B cell subsets from MS patients (n=8) were compared with their CSF Ig-VH transcriptomes. In 6 of 8 patients, we identified peripheral CD27+IgD−memory B cells, CD27hiCD38hi plasma cells/plasmablasts, or CD27−IgD− B cells providing an immune connection to the CNS compartment. Pinpointing Ig class-switched B cells as key component of the immune axis thought to contribute to ongoing MS disease activity strengthens the rationale of current therapeutic strategies and may lead to more targeted approaches.

Objective In multiple sclerosis (MS) cerebral gray matter (GM) atrophy correlates more strongly than white matter (WM) atrophy with disability. The corresponding relationships in the spinal cord (SC) are unknown due to technical limitations in assessing SCGM atrophy. Using phase sensitive inversion recovery (PSIR) MRI, we determined the association of the SCGM and SCWM areas with MS disability and disease type. Methods 113 MS patients and 20 healthy controls were examined at 3T with a PSIR sequence acquired at the C2/C3 disc level. Two independent, clinically-masked readers measured the cord WM and GM areas. Correlations between cord areas and Expanded Disability Status Score (EDSS) were determined. Differences in areas between groups were assessed with age and sex as covariates. Results Relapsing (R) MS patients showed smaller SCGM areas than age and sex matched controls (p=0.008) without significant differences in SCWM areas. Progressive MS patients showed smaller SCGM and SCWM areas compared to RMS patients (all p≤0.004). SCGM, SCWM, and whole cord areas inversely correlated with EDSS (rho: −0.60, −0.32, −0.42, respectively; all p≤0.001). SCGM area was the strongest correlate of disability in multivariate models including brain GM and WM volumes, FLAIR lesion load, T1-lesion load, SCWM area, number of spinal cord T2 lesions, age, sex, disease duration. Brain and spinal GM independently contributed to EDSS. Interpretation SCGM atrophy is detectable in-vivo in absence of WM atrophy in RMS. It is more pronounced in progressive than RMS and contributes more to patient disability than spinal cord WM or brain GM atrophy.

Myelin oligodendrocyte glycoprotein (MOG) is an integral membrane protein expressed in CNS oligodendrocytes and outermost myelin lamellae. Anti-MOG Abs cause myelin destruction (demyelination) in animal models of multiple sclerosis (MS); however, such pathogenic Abs have not yet been characterized in humans. Here, a method that specifically detects IgG binding to human MOG in its native, membrane-embedded conformation on MOG-transfected mammalian cells was used to evaluate the significance of these auto Abs. Compared with healthy controls, native MOGspecific IgGs were most frequently found in serum of clinically isolated syndromes (P < 0.001) and relapsing-remitting MS (P < 0.01), only marginally in secondary progressive MS (P < 0.05), and not at all in primary progressive MS. We demonstrate that epitopes exposed in this cell-based assay are different from those exposed on the refolded, extracellular domain of human recombinant MOG tested by solid-phase ELISA. In marmoset monkeys induced to develop MS-like CNS inflammatory demyelination, IgG reactivity against the native membrane-bound MOG is always detected before clinical onset of disease (P < 0.0001), unlike that against other myelin constituents. We conclude that (i) epitopes displayed on native, glycosylated MOG expressed in vivo are early targets for pathogenic Abs; (ii) these Abs, which are not detected in solidphase assays, might be the ones to play a pathogenic role in early MS with predominant inflammatory activity; and (iii) the cell-based assay provides a practical serologic marker for early detection of CNS autoimmune demyelination including its preclinical stage at least in the primate MS model. allergic encephalomyelitis ͉ autoimmunity ͉ clinically isolated syndrome ͉ demyelination