The Changes And Significance Of Thymic CD4~+ SP T Cells TCR Beta Chain CDR3 Repertoire In Endotoxin Tolerance

The project of this study aimed to explore the changes and significance of thymic CD4⁺SP T cells TCR beta Chain CDR3 repertoire in endotoxin tolerance.This study consists of two parts as listed below.ObjectivesPart One Endotoxin tolerance model was established by injecting 5mg/kg LPS i.p.into mice.And the attacking experiment were used to identify the endotoxin tolerance model.Then,CD4⁺SP cells were obtained by using magnetic bead sorting?MACS?.Furthermore,Illuma Solexa high-throughtput sequencing was performed to analysis the characteristics and changes of thymic CD4⁺SP T cells TCR beta Chain CDR3 repertoire.Part Two To observe the changes of thymic CD4⁺T cells in response to the specific antigen by using OVA transgenic mice in the condition of endotoxin tolerance.MethodsPart One C57BL/6J mice?6-8 weeks age?were injected with 5 mg/kg LPS i.p.to the establishment of endotoxin tolerance model.In order to identify the endotoxin model,the tolerance model mice were attacked with 10mg/kg LPS.After 24h,the survival rate were observed.HE staining was used to observe the histopathological changes of lung and liver.The expression level of cytokine?TNF-??IFN-??IL-4 and IL-10?of lung and liver were detected by RT-PCR.The thymic CD4⁺SP cells were separated by magnetic bead sorting?MACS?at 72 hours and 8 days of post LPS injection.Flow cytometry and gel electrophoresis were used to identify the cell purity and integrity of extracted genomic DNA in each group.The Illumina Solexa high-throughput sequencing was performed to evaluate TCR?CDR3 receptoire gene repertoire points individually with the help of Adaptive Biotechnologies immunoSEQ^TM Company.After HTS,the library of TCR?CDR3 was analyzed by Immuno SEQ software.Part Two Bone Marrow cells were obtained from female C57BL/6 mice?6-8weeks age?and cultured in vitro in the presence of GM-CSF?20ng/ml?for 7 days.The purify of Bone Marrow-derived Macrophages?BMDMs?was analyzed by FACS.Next,spleen CD4⁺SP cells were sorted by magnetic bead sorting?MACS?from OVA transgeniec mice?OT-II?.Then,4×10⁵CD4⁺SP cells were co-cultured with 1×10⁵ M?cells in the presence of OVA peptide.24 hours later,the expression level of activation molecular?CD62L and CD69?were analyzed by FACS.The BM cells were purified from OT-?mice and then adoptively tranfered into Rag-1^-/-mice through tail vein.After 24h,2×10⁶ M?cells and 2×10⁶ M?-OVA cells were also adoptively tranfered into Rag-1^-/-mice through tail vein respectively.24h later,the mice were injected with 0.5 mg/kg LPS i.p.to establish the endotoxin tolerance model.Then,the weight of thymus was observed at 72 hours and 8 days of after LPS injection.The proportion of the OVA-specific-CD4⁺SP and the expression of CD62L,CD69,CD44were analyzed by FACS.Next,we attacked the mice which were injected with 0.5mg/kg LPS after 72h at a high dose of 2mg/kg LPS i.p.24h later,the weight of spleen was detected.HE staining was used to observe the histopathological changes of lung and liver.The expression level of cytokine?IL-10?IL-4?IL-1??IFN-??TGF-?and TNF-??of lung and liver were detected by RT-PCR.The proportion of OVA-specific-CD4⁺T cells and the changes on the activation molecular?CD62L?CD69 and CD44?and functional molecular?IFN-?and IL-4?were detected by FACS.ResultsPart One The survival rate in ET group was 100%but all the mice were died in the attacked group.HE staining indicated that compared with ET group,the alveolar wall was markedly thickened and infiltration of inflammatory cells markedly increased,in addition,the structure of the hepatic lobule was disorganized and in the attacked group.Compared with attacked group,in addition,the expression level of pro-inflammatory factors TNF-?and IFN-?were obviously down-regulated?P<0.05?but the expression level of anti-inflammatory factors IL-10 was decreased?P<0.05?in lung.And the expression level of pro-inflammatory factors IFN-?was decreased significantly?P<0.05?and the expression level of anti-inflammatory factors IL-10 and IL-4 werre increased?P<0.05?in liver.Meanwhile the expression level of IL-4 was unchanged.High-throughtput sequencing results were analyzed as follow:1.The number of unique sequences and total productive sequences in each grouop:Control group:9993/23102;72h group:9992/21079;8d group:9995/27549?2.The 1/DS values of the thymic CD4⁺SP cells in each group respectively were:Control group:59112.08;72h group:37188.8;8d group:29921.15.3.The AA length of TCR?CDR3 sequences were Gaussian distribution which usage 12 amino acids in each group.And Serine?S?,Alanine?A?and Glycine?G?were used for high frequency.4.The top 10 clone sequences in each group were analyzed and the result showed that there are two common sequences which were CASSLGQYEQYF and CASSPGTGGYEQYF between control group and 8 days of after LPS injection.But no overlapping sequences were found in 72h of post LPS injection.5.The top 20 clone sequences in Control group were mostly few or no expression in 72h and 8d of post LPS injection.And the same phenomenon occurred in the 72 h and 8 d of post LPS injection.6.There were 208 overlap of TCR?CDR3 AA sequences in the three groups.The top 20 clone sequences of the common sequences in 72h of post LPS injection were mostly few expression in control and 8d of post LPS injection.7.The shared TRBV genes usage in the control?72h and 8d of post LPS injection were TRBV13-02,TRBV19-01,TRBV03-01.And the shared TRBJ genes usage in the control,72h and 8d of post LPS injection were TRBJ02-07,TRBJ02-05,TRBJ02-01.8.There were common dominant TRBV-TRBJ genes pair usage which were TRBV13-02-TRBJ02-07 in the 72h and 8d of post LPS injection.And it was same as the control group.In addition,some new TRBV-TRBJ gene pairing occurred in 72h and 8d of post LPS injection.Part Two FACS showed that the purity of macrophages was more than 90%in vitro.Compared with SP-OVA group,the cloned numbers were increased significantly in M?-SP?M?-OVA.And the proportion of CD69 were up-regulated?P<0.05?.Compared with the control group,the body weight of mice were attenuated in LPS-induced tolerance group at day 3 after LPS injection in each group.And then body weight gradually recovered to the normal level from day 4 to day 7.In addition,compared with the control group,the thymus weight of the mice in two group were both decreased?P<0.05?in the 72h of post LPS injection and gradually recovered to the normal level at 8 days of post-LPS i.p.FACS showed that compared with the control group,the proportion of OVA-specific-CD4⁺SP in thymus at 72h of post-LPS i.p.were marked increased?P<0.05?and markedly decreased at 8d of post-LPS i.p?P<0.05?in M?group.The same change occurred in the M?-OVA group.In addition,compared with M?group,the proportion of OVA-specific-CD4⁺SP in thymus at 72 hours of post-LPS i.p.was reduced?P<0.05?.In M?group,compared with the control,the expression level of CD69 and CD44 were no change at 72h and8d of post-LPS i.p.The proportion of CD62L was up-regulated?P<0.05?at 72 h and reduced at 8d?P<0.05?but still higher than control group?P<0.05?.In M?-OVA group,compared with the control,the expression level of CD69 was increased at 72h of post-LPS i.p?P<0.05?.The proportion of CD62L was up-regulated?P<0.05?at 72h and reduced at 8d?P<0.05?.But the expression of CD44 had no change.Compared with M?group,the proportion of CD69,CD62L and CD44 on OVA-specific-CD4⁺SP had no significance in M?-OVA group at 72h of post-LPS i.p.In attacking experiment,we found that the size and weight of spleen,compared with control group and M?-OVA group,were increased?P<0.05?in M?group.HE staining indicated that compared with M?-OVA group,the alveolar wall was markedly thickened and the number of lymphocyte infiltration was increased,in addition,the structure of the hepatic lobule was disorganized.Compared with M?group,the expression level of pro-inflammatory factors IL-1??IFN-??TNF-?were decreased significantly?P<0.05?and the expression level of anti-inflammatory factors IL-10 and TGF-?were increased obviously?P<0.01,P<0.05?in lung.But the expression of IL-4 had no obvious change.Moreover,the expression level of cytokines had no significant difference in liver.FACS showed that compared with the control group,the proportion of OVA-specific-CD4⁺T cells in spleen was obviously increased?P<0.05?.The proportion of OVA-specific-CD4⁺T cells in M?-OVA group was lower than its in the M?group?P<0.05?.The proportion of CD69 and CD44,compared with control group,were notably up-regulated?P<0.05?in M?group and M?-OVA group.Compared with M?group,the proportion of CD69 was increased significantly?P<0.05?in M?-OVA group.Compared with M?group,the proportion of IFN-?and IL-4 which related the function of T cells were obviously down-regulated.?P<0.05?.Conclusions1.There were changes in TCR beta Chain CDR3 repertoire of the thymic CD4⁺SP T cells underwent changes in the endotoxin tolerance model.2.In endotoxin tolerance,the recognition and response of OVA-specific-CD4⁺T cells to specific antigen were changed.