The Effect Of Sel1l Gene On The Function Of CD4~+T Cell

Objective:Epidemiology shows that the prevalence of autoimmune diseases have increased year by year and there is no good treatment.CD4⁺T cells are closely related to the occurrence and development of a variety of autoimmune diseases in which Th1and Th17 cells are the main lymphoid groups,but the specific pathogenesis is not clear.The studies have shown that the unfolded protein response?UPR?caused by endoplasmic reticulum stress?ERS?plays an important role in the occurrence and development of autoimmune diseases.The endoplasmic reticulum related protein degradation?ERAD?complex,composed of Lin-12 like inhibitor/enhancer?Sel1L?,plays an indispensable role in maintaining endoplasmic reticulum homeostasis and removing unfolded and erroneous folding proteins.Therefore the mouse model of specific Sel1L knockout in T cell has been established by Cre-lox P system to explore the regulation effects of Sel1L on CD4⁺T cell activation,proliferation and differentiation and reveal the potential role of Sel1L in the pathogenetic process of autoimmune diseases.Methods:1.Establishment of the mouse model of specific Sel1L knockout in T cell.According to the theory of cre-loxP system,the Lck-Cre^+/-mice and Se1lL^f/f/f mice has been crossbred to the third generation and the recombinant enzyme cre gene and Se1lL floxed site were identified by polymerase chain reaction?PCR?and agarose gel electrophoresis.2.Effects of Sel1L on proliferation and apoptosis of activated T lymphocytes.?1?The splenic single cell suspension from WT and KO mice has been prepared and the splenocytes or CD4⁺T cells enriched by MACS were and activated with anti-CD3/anti-CD28 or Concanavalin A?ConA?for 72-96h in 37?and 5%CO₂environment.In addition,the CFSE staining for proliferation was finished before activation.?2?The cells were collected and analyzed by FACS.The activation level was evaluated with CD69 and CD25.The proliferation was detected with CFSE and Ki67.The apoptosis level was assessed with Annexin V.3.Effects of Sel1L on the differentiation of CD4⁺T cell.?1?Splenocytes of WT and KO mice was activated with anti-CD3/anti-CD28 or ConA for 72-96h in 37?and 5%CO₂ environment.Th1,Th2,Th17 and Treg proportion was detected by FACS.?2?Splenic purified CD4⁺T cells of WT and KO mice were activated with anti-CD3/anti-CD28 for 72-96h in 37?and 5%CO₂ environment.The expression level of Th1/Th17 key transcription factor T-bet and ROR-?t was detected by FACS.?3?Naive CD4⁺T cells of WT and KO mice were enriched by MACS and activated with anti-CD3/anti-CD28 for 4 days.IL-2,IL-12,anti-IL-4 were added for Th1polarization.In addition,TGF-?,IL-6,IL-23,anti-IL-4 and anti-IFN-?were added for Th17 polarization.Th1 and Th17 percentages were detected by FACS.?4?The expression level of CD3 and TCR on lymphocyte surface in WT and KO groups was analyzed by FACS and the expression level of lymphocyte P-Lat was analyzed by immunoblotting after anti-CD3/antiCD28 activation.4.Expression analysis of UPR related molecules in CD4⁺T lymphocytes.?1?CD4⁺T cells of WT and KO mice were enriched for total protein extraction.The expression level of Sel1L,Bi P,eIF2?,P-eIF2?,JNK and P-JNK was detected by western blotting.?2?CD4⁺T cells of WT and KO mice were enriched by MACS and activated with anti-CD3/anti-CD28 for 72h in 37?and 5%CO₂ environment.Then total protein of CD4⁺T cells was extracted and the expression level of BiP,eIF2?,P-eIF2?and PCNA was detected by western blotting.Results:?1?The mouse model of specific Sel1L knockout in T cell has been established.The third generation mouse Sel1L floxd loci were all homozygous.KO represented the genotype of Lck-Cre^+/-,Se1lL^f/f/f and WT represented the genotype of Lck-Cre^-/-,Se1lL^f/f.?2?After activation with anti-CD3/anti-CD28 or ConA,there was no obvious difference between WT and KO group of CD69 and CD25.But the cell colony of KO group was obviously larger than that of group and the proliferation activity of KO mice was higher.In addition,the T cell of KO mice was more susceptible to apoptosis.?3?After activation with anti-CD3/anti-CD28 or ConA,the proportion of Th1,Th2and Th17 cells increased while the proportion of Treg decreased in KO group compared with WT group.?4?After activation with anti-CD3/anti-CD28,the proportion of Th1/Th17 specific transcription factor T-bet and ROR-?t increased in KO mice.?5?After Th1 and Th17 polarization,the naive CD4⁺T of KO mice has a higher potential for differentiation to Th1 and Th17.?6?The expression level of CD3 and TCR on lymphocyte surface in group KO was higher and the expression of P-Lat increased significantly after anti-CD3/anti-CD28activation.?7?The Sel1L protein of CD4⁺T cells from KO mice was obviously deficient.The expression level of BiP and P-elf2a in CD4⁺T cells from KO mice was up-regulated compared with WT mice but there was no obvious change of P-JNK between two groups.?8?After activation with anti-CD3/anti-CD28,the expression level of PCNA,BiP and P-elf2a in CD4⁺T cells from KO mice was all up-regulated.Conclusions:Sel1L,an important composition of ERAD complex,is involved in regulating the activation and differentiation of CD4⁺T cells by affecting the UPR signaling pathway,indicating that the integrity of ERAD structure and function is indispensable to the function of CD4⁺T cells.