Study On Notch Pathway And Lymphocyte Development

T cells and B cells are the key components of the adaptive immunity. Their development involved the interplay of cell differentiation, proliferation, apoptosis and migration. Their abnormal development plays a critical role in the development of autoimmune disease. Research on their development has long been very attractive to immunologists. Many pathways including antigen receptor pathway, cytokine receptor pathway, Wnt pathway are all involved in lymphocyte development.Notch signaling pathway is a revolutionarily conserved, precisely regulated pathway that plays a critical role in the regulation of cell fate commitment. It is involved in lymphocyte development by regulating the differentiation of precursor cells, which has been verified in many differentiation system including T versus B cell fate commitment and the Marginal Zone B versus Follicular B cell fate commitment, while the molecular mechanism remains elusive.In order to further clarify the role of Notch/RBP-J pathway in T cell development and differentiation in the periphery, we generated Lck-Cre x RBP-J flox/flox mouse in which RBP-J, the key transcription factor of Notch pathway, can be specifically inactivated in T cells. We constructed the eukaryotic expression vector pLck-Cre, and generated twenty Lck-Cre mouse lines by microinjection of lineared pLck-cre vector. We identified two lck-cre mouse lines in which RBP-J can be completely inactivated in T cells by Southern Blot.Further phenotype analysis revealed that bothαβT development andγδT kept intact while there was an increase in the apoptosis of DP thymocytes in M19 Lck-cre x RBP-Jflox/flox. In addition, in the periphery, the proliferation of RBP-J deficient CD4 T cells decreased a lot in response to stimulation with anti CD3 or anti CD3 plus IL2. M5 Lck-cre x RBP-Jflox/flox mice had different phenotype. ItsαβT development was blocked while the development ofγδT cells was not affected.In order to further understand how Notch/RBP-J pathway regulates the deferentiation of B cells in the periphery, we compared gene expression profiles of FO and MZ B cells by DNA microarray using FACS-sorted mouse FO and MZ B cells. From 14,000 mouse genes, 1754 genes were expressed mainly in MZ B cells while 1226 genes expressed largely in FO B cells. Differentially expressed genes were classified into seven groups including membrane protein and receptors, signal transduction, transcriptional regulation, apoptosis and proliferation, structure protein and metabolism related. We found that several components of Notch pathway including Notch2, Mint, Deltex and Hes5 were differentially expressed in these two B cell subsets. Among them, Hes5, a member of bHLH transcriptional factor Hes family, was highly expressed in Marginal Zone B cells, but dimly expressed in Follicular B cells. It can be suggested as a potential target gene of Notch/RBP-J pathway. In addition, among all the differentially expressed genes, 27 membrane proteins were expressed mainly in MZ B cells while another 22 membrane proteins expressed largely in FO B cells. Using FACS analysis, we identified that CD36, CD68, and CD49e were expressed on MZ B cells but not on FOB cells. Among them,CD36, a type B scavenger receptor was highly expressed in Marginal Zone B cells, but dimly expressed in Follicular B cells. It can be viewed as a new surface marker for Marginal ZoneB cell due to its specific expression pattern.In addition, using semi-quantitative PCR, we found that the mRNA of CD131 were much more abundant in MZ B cells, which may be in line with the fact that Marginal Zone B cells can respond more rapidly to stimulation.Further we generated the polyclonal antibody of CXCR7, a chemokine orphan receptor differentially expressed in the two B cell subsets revealed by chip analysis, and analyzed its expression in lymphocytes. We found that CXCR7 was expressed on B cells from many lymphoid organs including bone marrow, spleen, lymph node and peritoneal cavity. We found CXCR7 was differentially expressed in Marginal Zone B cells and Follicular B cell. It also pocessed higher expression on B1 B cells than B2 B cells in the peritoneal cavity. The specific expression pattern of CXCR7 may be related with the special localization of Marginal Zone B cell and B1 B cells and also their role in the innate immunity.In summary, we constructed Lck-Cre RBP-J flox/flox mice, in which RBP-J can be specifically inactivated in T cells. We found that Notch/RBP-J pathway can inhibit the apoptosis of DP thmocytes in vivo, and also promoted the proliferation of CD4 T cells in the periphery. The results further clarified the role of Notch /RBP-J pathway in vivo. In addition, we compared the gene expression profile of Marginal Zone B cells and Follicular B cells, and found that Hes5 may be the potential target gene of Notch/RBP-J pathway in the differentiation of B cells in the periphery. We identified CD36 as a new surface marker of Marginal Zone B cells. We also found that CXCR7 was differentially expressed in Marginal Zone B cells and Follicular B cells, also B1 B and B2 B cells in the peritoneal cavity. These results revealed new phenotypic properties of MZ and FO B cells, and would facilitate further studies in the differentiation and functions of these mature B cells.