These data suggest, CD8+ T cells may play an overall regulatory role in the context of immune thrombocytopenia and enhance the therapeutic effect of DEX treatment


These data suggest, CD8+ T cells may play an overall regulatory role in the context of immune thrombocytopenia and enhance the therapeutic effect of DEX treatment. dexamethasone (DEX) treatment selectively expanded CD8+ Tregs while decreasing CTLs. In vitro coculture studies revealed CD8+ Tregs suppressed CD4+ and CD19+ proliferation, platelet-associated immunoglobulin G generation, CTL cytotoxicity, platelet apoptosis, and clearance. Furthermore, we found increased production of anti-inflammatory interleukin-10 in coculture studies and in vivo after steroid treatment. Thus, we uncovered subsets of CD8+ Tregs and demonstrated their potent immunosuppressive and protective roles in experimentally induced thrombocytopenia. The data further elucidate mechanisms of steroid treatment and suggest therapeutic potential for CD8+ HOE 32021 Tregs in immune thrombocytopenia. Introduction Immune thrombocytopenia or autoimmune thrombocytopenia (ITP) is a common bleeding disorder characterized by autoantibody-mediated destruction of platelets.1,2 Fatal intracranial hemorrhage can occur in severe cases (2% of patients).3,4 Autoantibodies targeting platelet glycoproteins (GPs) are considered to be the major mechanism behind platelet destruction by the reticuloendothelial system.5 The primary platelet surface antigen targets are GPIIbIIIa (IIb3 integrin, 70-80% of patients).2,6,7 Approximately 10% to 20% of patients do not respond to first-line therapies including steroids,8,9 intravenous immunoglobulin G,10,11 and anti-Rh(D).4,10 Approximately 14% do not respond to splenectomy, and 20% of responders relapse within weeks to years.10,12,13 Reasons for refractoriness to therapy are unknown, and unfortunately, there are currently no reliable predictive factors to anticipate the success of any therapeutic regimen. Development of more effective treatments is restricted by the current limited understanding of the immunopathogenesis of ITP.12,14-16 In addition to autoantibodies, other mechanisms of platelet destruction have been studied, the most significant of which is CD8+ cytotoxic T-lymphocyte (CTL)-mediated platelet destruction.17,18 Platelet-specific CTLs have been found to be elevated in patients with active ITP.19 In addition, CTL-mediated cytotoxicity of autologous platelets has been shown in vitro, whereby CTLs from ITP patients can cause MUC12 direct platelet apoptosis or lysis.17,18 Further, in a murine model of ITP, transfusion of CD19+ B cell-depleted splenocytes, which contain CD8+ T cells, into SCID mice led to development of thrombocytopenia.20 CD8+ CTLs may also attack megakaryocytes in the bone marrow20 and affect platelet production. Thus, CD8+ T cells may play a significant pathogenic role in ITP.17,21,22 Immune dysregulation is central to the antiplatelet response.23 T-regulatory cells (Tregs), particularly CD4+CD25+FOXP3+ Tregs, play important roles in maintaining peripheral tolerance and have been shown to be dysfunctional or decreased in ITP patients.24-29 Recent studies suggest that successful therapies that raise platelet counts do so through normalization of CD4+ Tregs.30-32 In contrast to CD4+ Tregs, the study of CD8+ Tregs or T-suppressive cells, following their discovery in the 1970s,33-35 has fallen to the wayside. Technical limitations and conflicting evidence have led to difficulties with investigations into the functional properties of CD8+ Tregs. Recently, there has been a reemergence of the study of CD8+ Tregs and their roles in autoimmune diseases36-39; however, the role of CD8+ Tregs in ITP has not been investigated. In this study, we developed 2 murine models of steroid treatment of ITP, which we termed experimentally induced thrombocytopenia (EIT). We found that, contrary to the current prevailing views of their pathogenic role (ie, CTL activity) in ITP, CD8+ T cells predominantly suppressed the pathogenesis and were required for effective steroid therapy in EIT. HOE 32021 These results could potentially indicate that refractory patients may have a quantitative or functional deficit of CD8+ Tregs, which may serve as an indicative biomarker for response. Importantly, our study suggests that transfusion of CD8+ Treg cells may have therapeutic potential in ITP and deserves further investigation in human patients. Methods Animals 3 integrin knockout (3?/?) mice were kindly provided by Dr Richard O. Hynes (Massachusetts Institute of Technology, Boston. MA)40 and backcrossed 10 times to the BALB/c background as previously described.20,41-45 Syngeneic wild-type (WT) BALB/c mice (6-8 weeks) were purchased from Charles River Laboratories (Montreal, QC, Canada). All procedures were approved by the Animal Care Committee at St. Michaels Hospital, Toronto, ON, Canada. Passive ITP model of steroid therapy As was previously described,46,47 WT BALB/c mice were injected intravenously via the tail vein on day 0 with 20 L sera/mouse from immunized 3?/? mice or with anti-3 monoclonal HOE 32021 antibodies (mAbs) developed in our laboratory48,49 (Table 1) at a.