Recognition And Function Research Of Tumor Antigen-specific T Cells In Gastrointrstinal Cancer

ObjectivesThe development of adoptive cell transfer therapy is mainly limited by the methods to identify tumor antigen specific T cells with long turnover cycle,high cost and low accuracy.In this study,we planned to probe gastrointestinal tumor antigen-specific T cells based on the chemical enzyme labeling method previously established by our research group.Then we identified and characterized TSA-reactive and bystander CD8+T cells in MC3 8 murine tumor model.In view of the absence of MHC-I on surface of pancreatic cancer cells,we captured CD4+tumor antigen-specific T cells of well-established "cold tumor" murine pancreatic cancer model and explored their biological functions.This study will promote the development of individualized tumor immunotherapy,and improve the prognosis of patients with gastrointestinal cancer.Methods1.GDP-Fuc-FT was synthesized,and installed onto cell surface of dendritic cells by chemoenzymatic approach.FT-modified DCs were primed with specific antigen before co-culturing with TILs,followed by the addition of GDP-Fuc-Biotin.FT labeled on DCs would transfer Biotin onto antigen specific T cells,then Biotin labeling T cells could be isolated with FACs.Specificity of this strategy was verified with OT-Ⅰ mouse model,the strategy was applied to recognize CD8+ tumor antigen-specific T cells in MC38 colorectal cancer mouse model.The possibility of using this system to identify CD4+ tumor antigen specific T cells was explored in OT-Ⅱ mouse model.The process was optimized and further used to detect CD4+tumor antigen-specific T cells in Pan02 pancreatic cancer mouse model.2.CD8+ tumor antigen specific TILs were isolated from MC38 tumors by flow cytometry and amplified in vitro.IFN-γ ELISpot and cell killing assay were used to verify the cytotoxicity of CD8+tumor antigen specific T cells in vitro,cell adoptive transfer was used to verify the antitumor effect in vivo.Then we characterized TCR clone characteristics and gene-expression of this subset with TCRβ sequencing and mRNA sequencing.3.CD4+and CD8+tumor antigen specific TILs were isolated from Pan02 tumors by flow cytometry.Auxiliary effect of CD4+tumor antigen specific T cells on CD8+T cells and phenotypes of CD8+tumor antigen specific T cells were analyzed by cell surface markers.Then we optimized in vitro amplification conditions of CD4+tumor antigen specific T cells.Tumor killing effect of CD4+tumor antigen specific T cells was verified by killing assay,and the adjuvant effect of CD4+T cells on CD8+tumor antigen specific T cells was investigated by IFN-y ELISpot.Results1.T cells receiving antigen presentation were successfully identified in OT-Ⅰ mouse splenocytes with strong antigen specificity.CD8+tumor antigen specific T cells and bystander T cells were successfully isolated and enriched in MC38 model TILs.The recognition process of CD4+tumor antigen-specific T cells was optimized in OT-Ⅱ mice.CD4+and CD8+tumor antigen-specific T cells were successfully detected and separated in Pan02 model TILs,as well as corresponding bystander T cells.2.In MC38 model TILs,TSA reactivity and tumor killing of CD8+tumor antigen specific T cells were significantly stronger than bystander T cells in vitro.In vivo experiments showed that the anti-tumor activity of CD8+tumor antigen specific T cells identified by this strategy was significantly higher than control group.Compared to bystander TILs,CD8+TSA-reactive TILs possessed a distinct T cell receptor repertoire and unique gene features.TCRβs in TSA-reactive TILs were significantly more oligoclonal than their counterparts in bystander subsets.TSA-reactive CD8+T cells were enriched in steroid biosynthesis and related metabolic pathways,although exhibited a dysfunctional phenotype,still possessed significant proliferative and tumor killing capacities.3.Intratumoral antigen specific CD4+T cells played bidirectional roles in the regulation of antitumor immunity of TSA-reactive CD8+TILs in Pan02 mouse model.There are more terminal exhaustion and effector memory T cells in CD8+tumor antigen-specific T cells.In vitro experiment confirmed weak killing effect of CD4+antigen specific T cells on tumor cells.CD4+tumor antigen specific T cells were divided into TSA-suppressive and TSA-reactive subsets.CD25-subset enhanced the secretion of IFN-γ,whereas CD25+subgroup significantly inhibited the immune response of CD8+tumor antigen specific T cells.Conclusions1.Our study successfully identified tumor antigen specific T cells in mouse models of colorectal cancer and pancreatic cancer based on selective label of direct interaction between TCR and pMHC.This stratage featured simple procedures,quick turnover cycle,and quite high economic benefits.It was proved to be a highly effective approach with really strong specificity to detect and enrich tumor antigen-specific T cells,laying methodological foundation for the development of individulized tumor immunotherapy.2.CD8+tumor antigen specific TILs isolated from MC38 tumor had undergone substantial TSA-driven clonal expansion,although displayed dysfunction gene signatures.The expanded TS A-reactive TILs exhibited significantly higher antitumor activities in vitro,and possessed markedly higher activities to control tumor growth vivo than other TILs that contained a large fraction of bystander T cells.3.TSA-suppressive and TS A-reactive CD4+TILs played opposite roles in regulating antitumor immunity of TS A-reactive CD8+TILs in a Pan02 tumor.Selectively boosting the TS A-reactive CD4+T cell subsetrather than the TSA-suppressive CD4+TILs in the tumor microenvironment may further potentiate antitumor immunity of TS A-reactive CD8+TILs.