Abstract
Abstract 381
Immune thrombocytopenic purpura (ITP) is a T cell-mediated autoimmune disorder, in which IgG autoantibodies to platelet surface glycoproteins promote platelet clearance in the reticuloendthelial system. Since CD4+CD25+ regulatory T cells (Tregs) are known to play a crucial role in the maintenance of immune homeostasis to self-antigens, it has been believed that Treg dysfunction contributes to the development of a various forms of human autoimmune disorders. Several lines of recent evidence have shown that Tregs are decreased in number and are functionally impaired in patients with ITP. However, it remains unclear how Treg alteration is involved in the pathophysiology of ITP.
Recently, we have found that a group of Treg-deficient mice develop autoantibody-mediated thrombocytopenia. For preparation of Treg-deficient mice, Treg-depleted T cells were prepared from BALB/c splenocytes by serial purification steps consisting of a positive selection of CD4+ T cells and a negative selection of CD25+ cells using magnetic bead-based cell sorting, and were transferred into syngeneic T cell-deficient nude mice via tail vein. Treg-depleted T cell fraction transferred contained >99% CD4+CD25− cells, and was confirmed to lack expression of Foxp3, a typical Treg marker. Three weeks after transfer, approximately one third of the recipient mice spontaneously developed thrombocytopenia, which sustained for > 20 weeks. Thrombocytopenic mice represented elevated platelet-associated IgG and increased proportion of reticulated platelets, but non-thrombocytopenic mice did not. In addition, platelets eluates and culture supernatants of splenocytes prepared from thrombocytopenic mice contained IgG antibodies capable of binding to intact platelets, which were not detected in non-thrombocytopenic mice. The presence of anti-platelet antibodies and increased platelet turnover observed in thrombocytopenic Treg-deficient mice are analogous to ITP patients. Treg-deficient mice prepared by transfer of a less number of Treg-depleted T cells resulted in reduced prevalence of thrombocytopenia, suggesting that onset of thrombocytopenia depends on the number of conventional T cells transferred. Treg-deficient mice are known to frequently develop autoimmune gastritis, another autoimmune disease mediated by IgG anti-parietal cell antibodies, but anti-parietal cell antibodies were almost equally detected in plasma from thrombocytopenic and non-thrombocytopenic mice (70% versus 60%). Transplantation of Tregs together with Treg-depleted T cells completely prevented the onset of thrombocytopenia, but Treg transplantation was not effective as a treatment once thrombocytopenia occurred. To further investigate how Tregs exert the regulatory function, Treg-depleted T cells and Tregs were simultaneously transferred in the presence of antibodies that blocked engagement of cytotoxic T lymphocyte-associated antigen 4 (CTLA4). This treatment cancelled Treg function and resulted in development of thrombocytopenia in recipient nude mice, while mock treatment with control antibodies had no effect.
In summary, these results together indicate that CD4+CD25+Foxp3+ Tregs play a critical role in preventing the development of murine autoantibody-mediated thrombocytopenia, in part, through CTLA4 engagement.
Disclosures:
No relevant conflicts of interest to declare.
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