Cytokine Regulation Of Regulatory T Cell Development And The Pathogenesis Of Autoimmune Myelofibrosis

First part: Cytokine regulation of regulatory T cell developmentDefining the factors that lead to loss of tolerance is critical for understanding the development of autoimmunity. Regulatory T cells are important immunosuppressive cells that control excessive host response and prevente autoimmunity. Increasing studies have reported that Treg cells participate in many diseases such as bacteria and virus infection, tumor, organ transplantation and tissue repair. Thus, investigation about the regulation of Treg cell development can lead to better understanding of their functions, which facilitates targeting Treg cells for many diseases.Treg cells develop either from thymus or from peripheral induction, and their development and differentiation are regulated by cytokines. dnTGF-?RII mice, which express a dominant-negative form of the TGF-? receptor type II under the control of the murine CD4 promoter, develop inflammatory infiltration due to T cell hyperactivation and decreased Treg suppressive function. Il2ra-/- mice develop autoimmune disorders due to dysregulated Treg cells and polyclonal T and B cell expansion. Thus in this study, we take advantage of Il2ra-/-Tg mice to investigate the synergetic role of TGF-? and IL-2 signaling in Treg cell development.We found that Il2ra-/-Tg mice develop multi-organ lymphocytic infiltration and pathological lesion that mimic scurfy mice, which lack Treg cells. Il2ra-/-Tg mice develop lymphadenopathy with increased cell number, activation and IFN-y secreting ability of T cells. We found increased Tfh cells, GC B cells and plasma cells in lymph nodes ofIl2ra-/-Tg mice. We also found plasma cell infiltration in non-lymphoid tissues.Treg cell percentage decreased in periphery but increased in thymus in Il2ra-/-Tg mice.They were in a more activated status, indicated by higher expression of activation marker CD69 and Treg functional molecules. They also demonstrated increased expression of Thl chemokine receptor CXCR3 and transcription factor Eomes. Treg cells from Il2ra-/-Tg mice had increased IFN-? secreting ability and demethylation of TSDR.Further, we found that Nrp-1 and PD-1 expression on both thymus and peripheral Treg cells from Il2ra-/-Tg mice decreased significantly, with disappeared Tfr cells.Increased Tfh cells and enhanced germinal center response in Il2ra-/-Tg mice were suppressed in bone marrow chimera experiment which supplemented with WT Tfr cells,indicating that defective Tfr cell development was the main reason of lymphadenopathy and enhanced germinal center response.In summary, we found that TGF-(3 and IL-2 signaling act synergistically to regulate Nrp-1+ Treg and follicular regulatory T cell development, and may provide a new therapeutic target for germinal center dependent autoantibody mediated autoimmune diseases.Second part: The pathogenesis of autoimmune myelofibrosisMyelofibrosis is a myeloproliferative neoplasm (MPN) characterized by stem cell-derived clonal myeloproliferation, bone marrow fibrosis induced by myofibroblast proliferation and reticulin fiber distribution, anemia, splenomegaly, extramedullary hematopoiesis (EMH). In most cases, myelofibrosis is not considered as an autoimmune disease. However, myelofibrosis can accompany well-defined autoimmune diseases in some cases, especially systemic lupus erythematosus, which is called autoimmune myelofibrosis (AIMF). AIMF can also accompany primary biliary cholangitis (PBC),but the mechanism remains unclear. Our previous work reported that p40-/-IL-2R?-/-mice can be a good mouse model for PBC, which spontaneously develop liver fibrosis.Further, we found symptoms that mimic clinical features of myelofibrosis such as splenomegaly and anemia. So we investigated the possibility of using p40-/-IL-2R?-/-mice as a mouse model for AIMF, and further the pathogenesis of AIMF.Different from control mice, spleens from p40-/-IL-2R?-/- mice demonstrated colonies similar to those generated in colony-forming unit-spleen (CFU-S) experiments with megakaryocyte infiltration. Hematopoietic foci were also found in liver of p40-/-IL-2R?-/- mice. LSK cells were found in peripheral blood, liver and spleen of p40-/-IL-2R?-/- mice, suggesting the presence of EMH. These increased LSK cells positively correlated with spleen weight and liver inflammation. Red blood cell count, hemoglobin,hematocrit and white blood cell count decreased in peripheral blood of p40-/-IL-2R?-/-mice compared with control mice, indicating the presence of anemia. H&E staining demonstrated increased bone marrow fibroblasts, and reticulin staining showed apparent bone marrow reticular fibers in p40 -/-IL-2R?-/- mice, indicating the presence of bone marrow fibrosis. We found that the percentage and number of bone marrow LSK cells were much higher, while their ability of hematopoiesis was decreased in p40-/-IL-2R?-/- mice compared with control mice. We found increased activated CD4+ and CD8+ T cell infiltration in bone marrow of p40-/-IL-2R?-/- mice, with increased IFN-y secreting ability. The percentage of infiltrated T cells positively correlated with the percentage of bone marrow LSK cells.Taken advantage of knockout mice, we found that knockout of CD8a or IFN-y but not CD4 could abrogate the development of AIMF, as indicated by disappear of EMH,myofibroblasts and reticulin fiber, normal percentage and function of LSK cells.Importantly, depletion of CD8+ T cells by anti-CD8a antibody after disease onset could alleviate autoimmune myelofibrosis significantly.In summary, we found that CD8+ T cells and IFN-y are critical in the pathogenesis of autoimmune myelofibrosis. Depletion of CD8+ T cells using anti-CD8? antibody could cure AIMF even after disease onset, which provide a new strategy for clinical treatment of AIMF.