The Effect Of Sel1l In CD4~+T Cell Development

Objectives:T cells are the main immune cells in the body and play an important role in inflammation,autoimmune diseases,and tumor development.During T cells development,differentiation and activation stage,T cells are accompanied with cell activation and proliferation.In those stages,endoplasmic reticulum synthesizes large amounts of protein.In protein synthesis process,accompanied by the production of misfolded or unfolded proteins,the body manage these misfolded proteins to maintain the homeostasis of secreted proteins through endoplasmic reticulum-related degradation,autophagy-lysosomal pathways and unfolded protein reactions.The mammalian Lin-12 inhibitor/enhancer（Sel1L）is a linker protein of the E3-ubiquitin ligase family hydroxymethylglutaryl reductase degradation protein 1（Hrd1）in ERAD,which is essential for the maintenance of homeostasis.Therefore,this study focused on the role of the Sel1L gene in T cell development by constructing a mouse model of specific knockout of the Sel1L gene in T cells.Methods:1.The spleen na?ve CD4⁺T cells of C57BL/6 mice were sorted by magnetic beads and were activated by using CD3/CD28.Western blot was used to analyze the protein change about endoplasmic reticulum reactivity and autophagy-lysin level before and after T cell activation and proliferation.2.Prepare a mouse model of T cell-specific knockout of the Sel1L gene by breeding C57BL/6 Se1lL^f/f mice with Lck-Cre^+/-mice.Gene expression of offspring mice was identified by polymerase chain reaction（PCR）and agarose gel electrophoresis.Mice expressed Lck-Cre^+/-Sel1L^f/f are knockout mice with T cell-specific deletion of Sel1L gene,while littermate control mice expressed Lck-Cre^-/-Sel1L^f/f are wildtype mice.3.8-10 weeks old WT and KO mice were used for weighing and evaluating mouse thymus,spleen and mesenteric lymph node morphology.The CD4⁺T and CD8⁺T cells in thymus were analyzed by using flow cytometry.4.T cell developments in the thymus at different stages in T cell-specific gene knockout Sel1L mice were further analyzed.And the proportion of thymocytes proliferation and apoptosis were analyzed by flow cytometry.Using flow cytometry sorting techniques sort WT and KO mice thymus double-positive T cells,adding anti-CD2/TCR-βto induce double positive T cells to develop into single positive T cells in vitro to evaluate developmental efficacy.5.The ratio of WT and KO mice splenocytes at different stages of T cell activation were analyzed by using flow cytometry.Also,the proportion of activation and apoptosis of CD4⁺T cells were analyzed and the proportion of iTreg cells in peripheral immune organs were accessed.Results:1.Cell proliferation,endoplasmic reticulum reactivity and autophagy-lysosomal activity up-regulated after T cell activation.2.Successfully constructed a model of T cell-specific knockout mice and obtained the WT and KO mice.3.KO mice can grow normally,and compared with WT mice,the immune organ weight and the total number of cells were not affected.4.In Sel1L knockout mice,the DN thymocytes increased and T cells were blocked in development of DN3 stage.The proportion of DP and CD3⁺CD4⁺SP cells decreased.The maturity of thymocytes cells and the proportion of nTreg cells decreased,but the development ofγδT cells is not affected and the proportion ofγδT cells increased.The proliferation and apoptosis of DN phase thymocytes up-regulated,while the proliferation of DP cells decreased.However,the proliferation and apoptosis of TCR-β⁺CD4⁺SP cells did not change significantly.The proportion of DP cells induced into SP cells decreased in vitro culture.5.The CD4⁺effector cells in the spleen of KO mice increased,and the ratio of cell activation and apoptosis also increased,but the proportion of iTreg in peripheral immune organs was not significantly changed.Conclusions:With the specific knockout of Sel1L gene in T cells mice,thymic T cell development arrested in DN3 phase and the number of DN cells increased,while the proportion of DP cells and CD3⁺CD4⁺SP cells decreased,indicating that Sel1L gene is indispensable during early T cell development.