The Role Of Endoplasmic Reticulum Stress In Activation–induced Cell Death Of T Cells

Objective: Endoplasmic reticulum stress（ER stress）is one of the apoptotic pathways,the cause of death associated with the pathogenesis of many diseases,such as diabetes,neurodegenerative diseases and kidney diseases.The activated-induced cell death（AICD）of T cells is the way to regulate the apoptosis of activated T cells after antigen removal,so as to avoid autoimmune diseases.Therefore,this study explores the effect of endoplasmic reticulum stress on AICD of T cells,and provides a preliminary theoretical basis for further understanding of the mechanism of endoplasmic reticulum stress in specific cellular immunity.Methods: 1.Regulation of endoplasmic reticulum stress on T cells activation induced apoptosis.（1）Mouse T lymphoma cell line EL-4 cells were induced AICD with Con-A in combination with IL-2 and CD3 antibody in vitro.（2）The spleen cells were obtained and erythrocytes were lysed by C57BL/6 mice.After counting,2×10⁵ cells were added to 96-well plates and AICD was induced with Con-A in combination with IL-2 and CD3 antibodies in vitro.（3）The spleen cells activated by Con-A and IL-2 were added with ER stress inducer dithiothreitol（DTT）or ER stress inhibitor 4-phenylbutyricacid（4-PBA）,then transferred to a 96-well plate coated with CD3 antibody,followed by incubation for 24 h.（4）The percentage of Annexin-V⁺7-AAD-cells and Annexin-V⁺7-AAD⁺ cells in CD4⁺T cells of spleen cells was analyzed by the flow cytometry to explore the regulation of endoplasmic reticulum stress on T cell AICD.2.The model of T cell-specific Sel1 L knockout mice.（1）To generate conditioned knockout mice by the Cre-Lox P recombination system,Sel1L^fl/fl homozygous mice and the Lck-Cre heterozygous mice were bred,and the Sel1 L gene loxp site and Cre recombinant enzyme identification were verificated by PCR and electrophoresis,screening to obtain the mice required for the experiments.（2）The expression levels of Sel1 L protein in spleen T lymphocytes in experimental mice were identified by Western Blotting.3.Endoplasmic reticulum stress regulates T cell AICD in T cell-specific Sel1 L knockout mice.（1）The spleen cells were prepared from Sel1 L knockout and wild type mice.Then,cells（2×10⁵）were added to 96-well plates and AICD was induced with Con-A in combination with IL-2 and CD3 antibody in vitro.（1）The percentage of Annexin-V⁺7-AAD-cells and Annexin-V⁺7-AAD⁺ cells in T cells in each of the two mice was analyzed by flow cytometry after DTT or 4-PBA added in the two kinds of T cells in vitro to inducting of AICD.Results:（1）After establishing the AICD model,compared with control group,the percentage of T cell apoptosis in the induction group increased significantly.It was also found that the proportion of AICD in EL-4 cells increased with the increase of CD3 antibody concentration.（2）Mice spleen cells induced AICD treated with DTT or 4-PBA,found that the activation of endoplasmic reticulum stress,could lead to the the increasing apoptosis or disappearance of CD4⁺ T cell.On the contrary,after inhibition of endoplasmic reticulum stress,the apoptotic CD4 ⁺T cells were significantly lower than the DTT and control group.（3）Lck^Cre x Sel1L^fl/fl homozygous mice of T cell-specific knockout Sel1 L gene were generated successfully and accurately identified.The protein expression of Sel1 L in spleen T cells of Lck^Cre x Sel1 Lfl / fl mice was significantly lower than that of Lck⁺ / ⁺ x Sel1 Lfl / fl wild type mice.（4）Sel1L knockout（KO）in mice and wild type mice（WT）of spleen cells were induced to AICD with addition of DTT or 4-PBA,endoplasmic reticulum stress did not alter AICD of T cells in Sel1 L knockout mice（KO）.While the T cell apoptosis in WT group was significantly increased.In contrast,after inhibiting endoplasmic reticulum stress,T cell apoptosis was remarkably lower in the DTT group than in the control group.Conclusions: In the process of T cell activation induced apoptosis,the addition of endoplasmic reticulum stress induction or inhibition,T cell apoptosis levels were increased or decreased.we can see that endoplasmic reticulum stress plays an important role in T cell AICD.The addition of endoplasmic reticulum induction or inhibition in induction of AICD in Sel1 L knockout and wild type mice,we found that Sel1L is an important molecule in regulation of endoplasmic reticulum stress on T cell AICD.