The Effect Of Loss Of ERAD Function On CD8~+T Cell Response After Listeria Infection

Objective:CD8⁺T cells are activated as cytotoxic T cells(CTL).They have cytotoxic effects and can specifically kill target cells.As an important part of cellular immune response,CD8⁺T cells are helpful to clear intracellular pathogens.During the immune response of CD8⁺T cells,Endoplasmic reticulum(ER)is needed to synthesise a large number of proteins,and some misfolded and unfolded proteins need the strict quality control system of ER associated degradation(ERAD)to effectively recognize,transport and degradation.The accumulation of misfolded proteins may affect the proliferation,differentiation and function of CD8⁺T cells when ERAD is dysfunctional.This article uses the Cre-lox P recombinase system to knock out the Sel1L molecule of ERAD to explore the effect of ERAD dysfunctional on the effect/memory CD8⁺T subgroup and function.Methods:1.Preparation of Sel1L deficient mouse model.The Cre-lox P recombinase system was used to construct an ERAD-deficient mouse model in which the Sel1L gene was specifically knocked out in T cells:Se1l L^f/f mice and Lck-Cre^+/-mice were bred to the third generation,so that they could stably generate Lck-Cre^+/-Sel1L^f/f mice(knockout,KO)and Lck-Cre^-/-Sel1L^f/fmice(wildtype,WT),using Polymerase Chain Reaction(PCR)technology and Agarose Gel Electrophoresis Technology to identify the genotype of the offspring.2.To evaluate the expression of Sel1L in CD8⁺T cells.CD8⁺T cells in spleen of WT mice were sorted by magnetic beads and its purity was detected.The purity of cell above 90%was required for the experiment.Anti-CD3/CD28 antibody was used to activate CD8⁺T cells,and the expression of Sel1L molecules was analyzed by Western blot and q PCR.3.Flow cytometry was used to analyze the effect of Sel1L deletion on CD8⁺T cells.Splenic single cell suspension was prepared.CD8⁺T cells were activated by anti-CD3/CD28 antibody or Con A.The effects of Sel1L molecule deletion on the proportion,activation,proliferation,apoptosis and surface molecules of CD8⁺T cells were analyzed by flow cytometry.4.The effect of Sel1L deletion on the killing function of CD8⁺T cells.6-10 age weeks old mice was divided into four groups(WT+PBS,WT+OVA group and KO+PBS,KO+OVA).WT and KO mice in the immune OVA group were treated with Freund's complete adjuvant+OVA protein for 7 days in advance.Spleens of C57BL/6 mice were taken out on the 7th day and divided into two parts,with high concentration of CFSE(5?M)CFSE staining and low concentration(0.5?M)dyeing,OVA protein antigen was added to the high-concentration CFSE stained cells(CFSE-H),while the low-concentration CFSE cells(CFSE-L)were not added.The cells were cultured in an incubator at 37?and 5%CO₂ for 6 hours,and the CFSE-H cells and CFSE-L cells were mixed 1:1.Each of the 4 groups of mice had 1×10⁷ mixed cells in the tail vein.After 48 hours,the mice were sacrificed by neck severing,and the splenic single-cell suspension was prepared.Flow cytometry was used to detect the changes in the proportion of high and low concentrations of CFSE in the 4 groups of mice.The killing ability of CD8⁺T cells in WT and KO mice was compared according to the killing ability formula(4.2.4).5.The effect of Sel1L deletion on CD8⁺T cell subsets and memory T cell phenotypes.WT and KO mice were infected with the recombinant OVA_257-264 peptide attenuated intracellular listeria monocytogenes(LM-OVA),using flow cytometry to detect the resting period(0 days),effector phase(7 days),memory(50 days)of CD8⁺T cells in mice spleen related indicators of change,such as killing function,IFN-?⁺CD8⁺T ratio,Granzyme B,perforin,memory cell phenotypes.The proportions of na(?)ve T cells,TCM,TEFF and TRM in the CD8⁺T cell population were determined.Results:1.PCR and Western blot showed that ERAD-deficient mice were successfully constructed.2.The results of WB and q PCR showed that the expression of Sel1L was increased when CD8⁺T cells were activated.3.Sel1L molecule deletion down-regulates the expression of CD8⁺T activation markers CD69 and CD25,promotes the proliferation and apoptosis of CD8⁺T cells,and promotes the secretion of intracellular factor granulease and perforin.4.The OVA immune model and LM-OVA intracellular infection model showed that CD8⁺T cells of KO mice were stronger than WT mice.5.The proportion of na(?)ve T in KO mice in resting period is lower than WT and higher than TCM.TEFF had no significant changes in the resting period,response period and memory period.In the memory period,there was no significant difference between TCM in WT and KO,but the proportion of TRM in KO mice lung tissue decreased than WT.Conclusion:The deficiency of Sel1L in CD8⁺T cells promoted the proliferation and apoptosis of CD8⁺T cells,promoted the secretion and killing function of effector factors,and had an effect on the memory cell population and phenotypes.