| BackgroundHepatocellular carcinoma(HCC)is a common cancer in China,and needs more effective treatment.Adoptive T cell immunotherapy has demonstrated a great potential for advanced HCC.AFP-specific TCR-T has been identified to recognize and kill HCC tumor cells.However,the limited persistence of TCR-T cells and immunosuppressive tumor microenvironment(TME)of solid tumors have challenged the effectiveness of the TCR-T cell therapy in clinical trials.One of the important reasons is lack of the stem cell-like memory T cells(Tscm)in solid tumors.IL-15 is fundamental to T-cell memory.In the study,we combined membrane-bound IL15(mbIL15)with TCR-T to enhance the TCR-T persistence and anti-cancer effect.Aims:Construct expression vectors of mbIL15 and generate mbIL15 TCR-T cells.Identify the effects of mbIL15 on TCR-T proliferation,cytokines secretion and memory phenotype.Identify the persistence and anti-cancer effect of mbIL15 TCR-T cells in vivo and in vitro,which can provide theoretical foundation for enhancing TCR-T treatment.Methods:1.The AFP-specific TCR-T cells were transferred into NSG mice bearing human HepG2 cells.Tumor growth was observed by measuring the tumor size with a caliper.TCR-T cells in mouse blood were monitored by immunological staining.2.To generate mbIL15,the full-length native IL-15 peptide was fused to the fulllength IL-15Ra sequence via the GS linker peptide.Send to the company for synthesis of mbIL15.Human T cells were transduced with lentivirus.Generate and identify mbIL 15 TCR-T cells.3.Detect antigen recognition and killing effect of T cells by LDH Cytotoxicity assay kit and fluorescence microscope in the co-incubation model of mbIL15 TCR-T cells and HepG2 cells.Detect the proliferation,memory and apoptosis biomarkers of T cells by immunological staining.4.NSG mice bearing human HepG2 cells were treated with human T cell adoptive cell transfer therapy.Tumor growth was observed by measuring the tumor size with a caliper.The alive cells of adoptive cell transfer therapy in mouse blood were monitored by flow cytometry.Results:1.The AFP-specific TCR-T cells can recognize HepG2 cells and inhibit tumor outgrowth in tumor-bearing mice.However,it was found that the persistence of TCRT cells was limited by dynamic monitoring of human T cells in mice.2.The soluble Interleukin 15(sIL-15)enhanced its anticancer function by inhibiting TCR-T apoptosis,and maintained the memory phenotype of Tscm-like cells.3.The expression vector of mbIL15 was constructed.Production of the mbIL15 lentiviral particles generated in 293T cells.The mbIL15 TCR-T cells were produced by transfection of human T cells with lentivirus mbIL15 and lentivirus TCR.The expression of exogenous TCR and mbIL l5 were detected by flow cytometry.4.Co-expression of mbIL15 helped TCR-T cells maintain the secretion of cytokines IFN-γ and IL-2.The mbIL15 enhanced the anti-apoptosis,proliferation and persistence of TCR-T cells,especially CD8~+TCR~+T cells by detection of Bcl-2,Annexin-V and CFSE with flow cytometry.The mbIL15 gave rise to the high proportion of CD8~+CD45RA~+CCR7~+Tscm-like cells,and enhanced the anti-cancer effect of TCR-T in vitro.4.The mbIL15 enhanced TCR-T persistence and improved the anti-cancer effect in NSG mice bearing human HepG2 cells.Conclusion:In this study,we identified that TCR-T cells targeting AFP can recognize tumor antigen and inhibit hepatocarcinogenesis in NSG mice bearing human HepG2 tumors.We generated mbIL15 TCR-T to improve the TCR-T persistence,and demonstrated that mbIL15 can promote long-term TCR-T proliferation and cytokines secretion,and retain CD8~+CD45RA~+CCR7~+Tscm-like cells.Our observations suggest that mbIL15 may provide durable immune surveillance and therapeutic potential through prolonging TCR-T persistence. |
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