(C) CD21? cells as a proportion of na?ve B cells, stratified by anti-platelet antibody status. activity in immune thrombocytopenia patients regardless of treatment status. A populace of CD21-na?ve B cells was specifically expanded in autoantibody-positive immune thrombocytopenia patients. Furthermore, the B-cell maturation antigen, a receptor for B-cell activating factor, was consistently and strongly up-regulated on plasmablasts from immune thrombocytopenia patients. These observations have parallels in Mavoglurant other autoantibody-mediated diseases and suggest that loss of peripheral tolerance in na?ve B cells may be an important component of immune thrombocytopenia pathogenesis. Moreover, the B-cell maturation antigen represents a potential target for plasma cell directed therapies in immune thrombocytopenia. Introduction Primary immune thrombocytopenia (ITP) is usually a clinical diagnosis given to patients with an unexplained, prolonged isolated thrombocytopenia. ITP is usually a rare but chronic condition in adults and is associated with significant bleeding-related morbidity and mortality.1 The condition is characterized by both platelet destruction and impaired platelet production. A role for platelet-directed antibodies was established in the 1960s with transfer experiments showing that thrombocytopenia could be induced by transfer of the gamma-globulin fraction of ITP patient serum.2 Using the most sensitive assays, antibodies binding platelet membrane glycoproteins are present in approximately 50% of patients.3 Mavoglurant The mechanism by which B-cell tolerance is lost is a subject for debate, but an elevated serum level of B-cell Activating Factor (BAFF) is likely to be an important contributing factor.4 BAFF drives B-cell maturation, promotes B-cell survival and augments immunoglobulin production by binding three surface B-cell receptors: BAFF receptor (BAFF-R), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), and B-cell maturation antigen (BCMA).5 An expanded CD95 (Fas receptor) positive population of B cells has also been described in ITP and there are reports of fewer regulatory B cells, defined both as CD24hiCD38hi B cells and by IL-10 production.6,7 A modern view of ITP pathogenesis places these B-cell abnormalities within a complex network of abnormalities affecting multiple immune cell lineages. T cells, in particular, contribute to platelet destruction both by facilitating the production of class-switched, high affinity autoantibody and through B-cell impartial mechanisms such as cell-mediated cytotoxicity directed against platelets.8 The latter may be the primary mechanism of disease in a subset of patients with no detectable anti-platelet antibodies.9 High-affinity autoantibody production is facilitated by T follicular helper cells (TFH), a subset recently reported to be expanded proportional to germinal center and plasma cell numbers within the spleens of FLN ITP patients.10 This study sought to extend existing knowledge of immune dysregulation in ITP by performing detailed flow cytometry-based immunophenotyping of the B- and T-cell compartments. An interest in the therapeutic potential of belimumab, an anti-BAFF humanized monoclonal antibody, led us to focus on BAFF and its receptors in B cells. While recent studies of immune populations in splenectomy specimens from patients with ITP have by their nature enrolled patients with refractory disease receiving significant immunodulatory therapy, we chose to enroll a cross-section of ITP patients in order to make sure the broadest possible applicability of our findings. Therefore, autoantibody-positive and -unfavorable ITP patients were recruited across a range of platelet counts and prior treatments including Mavoglurant rituximab and splenectomy, despite the known effects of these therapies on B cells with the intention of identifying candidate biomarkers of relevance to future clinical trials. An initial analysis was performed comparing splenectomy- and rituximab-na?ve ITP patients with healthy volunteers, and significant results were evaluated in the larger cohort. Methods Patients and healthy volunteers A cross-sectional cohort of adult patients with a clinical diagnosis of chronic ITP was recruited from patients in the UK ITP registry visiting the outpatient clinic of the Royal London Hospital Department of Haematology (Table 1 and Online Supplementary Table S1). All patients able to give informed consent Mavoglurant were considered for inclusion; the only exclusion criterion was ongoing immunosuppressive or cytotoxic therapy for a non-ITP diagnosis (one renal transplant recipient). Recruitment was stratified to give approximately equal numbers of patients by anti-platelet antibody status. All participants provided one venous blood sample; a subset of patients provided Mavoglurant a second sample at a later time point. None of the patients had received a platelet transfusion in the ten days prior to venesection or intravenous immunoglobulin in the 21 days prior to venesection. Table 1. Baseline demographics, treatment received and autoantibody status for immune thrombocytopenia patients and healthy volunteers used in the B-cell analysis. Open in a separate window Age-(within 10 years) and sex-matched healthy volunteers (HV) were recruited.