The Role Of Thymic B Cells In Regulatory T Cell Development And The Role Of Nk Cells In The Pathogenesis Of Primary Biliary Cirrhosis

First part:The role of thymic B cells in Treg cell development and the relevant mechanismsRegulatory T (Treg) cell is a subset of CD4+T cells that are critical for the maintenance of immunological tolerance and immune homeostasis. In general, Treg cells are defined by the expression of the specific transcriptional factor forkhead box protein3(Foxp3). Treg cells are directly involved in a wide range of diseases, such as autoimmune diseases, infection, allergic diseases, cancer and so on. The essential roles of Treg cells in immune responses are now generally accepted and Treg-cell targeting clinical therapies are under extensive research, making a comprehensive investigation of Treg cell development essential.FoxP3+Treg cells in the periphery can be divided into two subsets: periphery-derived Treg cells (p-Treg cells) and thymus-derived Treg cells (t-Treg cells). p-Treg cells develop from conventional CD4+T cells in the periphery, and t-Treg cells develop from CD4single positive thymocytes in the thymus. t-Treg cells constitute a great percentage of FoxP3+Treg cells in the periphery.The development of t-Treg cells takes place in the thymus in two sequential steps. In the first step, CD4single positive thymocytes undergo TCR-MHC Ⅱ and CD28-CD80/CD86dependent selection that give rise to the generation of thymic CD4+CD25+FoxP3-Treg cell precursors (pre-Treg cells). In the second step, an IL-2-dependent stimulation leads to the conversion of thymic pre-Treg cells into Foxp3+Treg cells. Several types of APCs including thymic DCs and thymic epithelial cells contribute to t-Treg cell development. Although B cells have been reported to be present within the thymus, there has not hitherto been a definition of their role in immune development and, in particular, whether or how they contribute to t-Treg cell development in the thymus. Therefore, in this study, we took the advantage of thymic B cells, to further deepen our understanding of Treg cell development. We obtained the results as following:1. Thymic B cells are required for the development of thymic Treg cells in the thymusWe found that both thymic B cells and thymic Treg cells localized to thymic medullary area. In μMT FoxP3-GFP mice, the absence of thymic B cells did not lead to any changes in the number of either CD4or CD8single positive thymocytes. However, the frequency and number of thymic Treg cells were significantly decreased. In vitro co-cultures of thymic B cells with CD4single positive thymocytes confirmed our observation.2. The ability of thymic B cells to contribute to the development of thymic Treg cells is dependent on cell-cell contactUsing an in vitro culture system, we found that thymic B cells contribute to the size of the thymic Treg compartment dependent on cell-cell contact, but not secreted cytokines. Moreover, we found that cell-cell contact between thymic B cells and thymic CD4single positive cells was regulated by MHC II and costimulatory molecules, such as CD40, CD80, and CD86.3. Thymic B cells promote the development of thymic Treg cells from CD4single positive thymocytes to pre-Treg cells onlyWe found that thymic B cells did not contribute to the conversion of thymic pre-Treg cells to mature Foxp3+Treg cells in vitro by co-culturing thymic B cells with thymic pre-Treg cells. On the contrary, thymic B cells had strong capacity to promote the generation of thymic pre-Treg cells from CD4single positive thymocytes in vitro in their co-culture experiment.4. Thymic B cells contribute to the proliferation of thymic Treg cellsIn μMT FoxP3-GFP mice, the absence of thymic B cells resulted in decreased frequency of proliferating thymic Treg cells. Meanwhile, in in vtro Treg proliferation experiments, we also found that adding of thymic B cells promoted the proliferation of thymic Treg cells.5. Differences in the roles of MHC II and CD40/CD80/86for thymic Treg proliferationAfter blocking cell-cell contact with blocking antibodies to some surface molecules, we found that MHC II contact provided by thymic B cells participated in thymic Treg cell proliferation, whereas CD40/CD80/CD86co-stimulation provided by thymic B cells were not involved in the proliferation of thymic Treg cells.In summary, we assessed the function of thymic B cells on thymic Treg cell compartment. We demonstrated that thymic B cells contributed to the size of thymic Treg cell compartment through two pathways. In the first, thymic B cells promoted thymic Treg cell generation. Second, thymic B cells directly enhanced thymic Treg cell proliferation. These findings provided new insights into the development of t-Treg cells. Second part:The role of NK cells in the pathogenesis of Primary biliary cirrhosis and the relevant mechanismsPrimary biliary cirrhosis (PBC) is a chronic cholestatic liver disease derived from attacking of intrahepatic biliary epithelial cells by the immune system. PBC is characterized by destruction of small and middle bile duct, lymphocytic infiltration in portal tracts. The immune cells infiltrating near the portal tracts include B cells, CD4+T cells, CD8+T cells, dendritic cells and macrophages, as well as natural killer (NK) cells. The role of the adaptive immune cells in disease process of PBC has been widely and productively studied. However, less attention was focused on the role of innate immune cells, especially NK cells which are enriched in the liver, accounting for25%-40%of human hepatic lymphocytes and10%-20%mouse hepatic lymphocytes.Previous research has identified that the frequency and absolute number of NK cells from both liver and peripheral blood are markedly increased in PBC patients. Meanwhile, the cytotoxic activity and perforin expression of NK cells are increased but the cytokines secreted by NK cells are decreased. Although this study indicated that the phenotype of NK cells in PBC patients was obviously changed, the role of NK cells in the pathogenesis of PBC was not fully understood, and the relationship between NK cell and other effector cells involved in PBC was still unknown. In this study, we sought to address the mechanism of NK cells participating in immunopathogenesis in the murine model of PBC (dnTGF-βRⅡ mice). We got some results as follows:1. Hepatic NK cells inhibit autoimmune cholangitis in dnTGF-βRⅡ miceBy Linear regression analysis of hepatic data to describe the relationship between NK cells and liver inflammation, we found that the number of hepatic mononuclear cells was negatively correlated with the frequency of liver NK cells. Furthermore, the deletion of NK cell in dnTGF-βRⅡmice aggravated lymphocytes infiltrating around portal tracts and worsened bile duct damage.2. Hepatic NK cells suppress liver inflammation through inhibiting CD4+T cell response.We found that the deletion of NK cell led to significant increase in the number of hepatic CD4+T cells in NK-/-dnTGF-(βRII mice. Moreover, we took the advantage of adoptive transfer cholangitis model to co-transfer splenic CD4+T cells and CD8+T cells into Rag1-/-or NK-/-Rag1-/-mice. We found that CD4+T cells but not CD8+T cells remarkably increased in NK-/-Ragl-/-mice compared to Rag1-/-mice.3. Hepatic DX5" NK cells, but not DX5+NK cells served as a negative regulator to CD4+T cells.Using proliferation suppressive assay in vitro, we determined that the hepatic DX5-NK cells could inhibit CD4+T cells proliferation while hepatic DX5+NK cells could promote CD4+T cells proliferation. Meanwhile, we found that both the number of hepatic mononuclear cells and the frequency of hepatic CD4+T cells were negatively correlated with the frequency of hepatic DX5" NK cells.In summary, we revealed the regulatory role of hepatic NK cells in liver inflammation of dnTGF-βRⅡ mice through inhibiting CD4+T cells. Moreover, we demonstrated that the two different NK cell subsets played opposite roles in the pathogenesis of biliary disease in dnTGF-βRⅡ mouse model. These findings provided new insights into the pathogenesis of PBC.