| Objectives:Rituximab targets the CD20antigen and efficiently depletes pre-B cells and mature B cells, all of which express CD20. After B cell depletion, repopulation follows subsequently. Therefore, rituximab therapy provides a unique opportunity to study the kinetics of repopulation of subsets of B cells in MG. B-cell IL-10can regulate immune responses in both health and disease. TLR-mediated IL-10induction represents one such context. It is likely that B-cell-derived regulatory IL-10has evolved to serve distinct immune functions, and that these functions are in turn carefully regulated in a cell-subset and context dependent manner. Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction in which autoantibodies to the acetylcholine receptor (AChR) or muscle specific kinase (MuSK) impair neuromuscular transmission and cause muscle weakness. Anti-AChR or MuSK autoantibodies are produced by B cells with the help of T cells and other lymphocytes. In contrast to the conventional role of effector B cells, i.e. antibody production and priming of immune response, regulatory B cells suppress inflammatory and autoimmune responses via their capacity to produce IL-10. The frequencies and functional properties of IL-10-producing B cells (referred to as B10cells hereafter) have been reported in patients with autoimmune and inflammatory diseases, including rheumatoid arthritis, Graves disease and systemic lupus erythematosus. With notable exceptions, most studies have found that B10cells exhibit reduced frequency and impaired function in these disorders. However, this novel B cell subpopulation has not been characterized in MG patients, rituximab, has several advantages in controlling the symptoms of MG, particularly in patients who are refractory to conventional therapies. Here we investigated the presence and dynamics of B10cells in patients with MG compared to healthy subjects. We sought a correlation between clinical severity as rated by Myasthenia Gravis Association of America (MGFA) status and the frequency of B10cells. As patients underwent therapy with rituximab, we studied the clinical response to treatment and looked for an association with different patterns of B10cell populations. The goal was to identify a possible marker for disease activity and responsiveness to immune therapy in MG.Methods:A total of48consecutive patients who met diagnostic criteria were recruited for the study. Refractory was defined as having an unsatisfactory response to2immunomodulatory agents, at least1of which was prednisone. Patients with antibodies to either acetylcholine receptors (AChRs) or muscle specific receptor tyrosine (MuSK) were included. Each patient received4infusions of IV rituximab at a dosage of375mg/m2, administered once per week. Twenty healthy subjects were recruited as controls and were matched with the MG patients for age, gender, and demography. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation on a Ficoll-Hypaque density gradient. MG patients are stratified by Myasthenia Gravis foundation of America(MGFA) clinical classification. Frequencies of B10cells from MG patients and healthy controls were monitored by fluorescence-activated cell sorting (FACS). PBMCs were stained with the following anti-human primary mAbs (BD Pharmingen, San Diego, CA):CD19(H1B19), CD5(L17F12), CD1d (CD1d42), CD24(ML5), CD27(M-T271), and CD38(HB7). They were conjugated with1of the following fluorescent tags:FITC, PE, PeCy5, or allophycocyanin (APC),Results:MG patients had fewer B10cells than controls, which was associated with more severe disease status. Moreover, patients who responded well to rituximab therapy exhibited rapid repopulation of B10cells, whereas in patients who did not respond well to rituximab, B10cell repopulation was delayed. The kinetics of B10cells were related to the responsiveness to rituximab in MG.Conclusion:We have characterized a specific subset of B10cells in MG patients which may serve as a marker for MG activity and responsiveness to immune therapy... |
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