P38 Inhibitors Promote TCR-T Cell Function And Counteract The Immunosuppressive Function Of TGF-beta

Background: Adoptive cell therapy(ACT)is emerging immunotherapeutic in cancer treatment.Efforts to isolated tumor antigen-specific T cells from cancer patients or healthy donors,screening,expanding in vitro and refusing to the tumor patients.ACT has emerged as a potent strategy for treatment of advanced and refractory malignancies.Currently,ACT can be classified into three different types with each their own mechanism od action,namely tumor-infiltrating lymphocytes(TIL),T cell receptor-engineered T cells(TCR-T),and chimeric antigen receptor modified T cells(CAR-T).The efficacy of adoptive cell therapy(ACT)in a variety of tumors relies on the T cells long-term survival and the cytotoxic effects in vivo.The T cell culture is the key determinant of T cell status and the therapy efficacy.Objective: To improve the ACT therapeutic effectiveness and optimized the cell culture conditions,we compared the generation of T cells treating with p38 inhibitor either Doramapimod or SB202190 in either IL-2 or IL-7/IL-15,and finally selected the optimal cytokines and p38 inhibitor combination.Comparable results in capacity of T cells on proliferation,subsets of T cells,expression of CD62 L,and cytokines production even in high-dose TGF-β.Methods: 1、 In this study,the healthy donor’s venous blood was collected,and peripheral blood mononuclear cells(PBMCs)were isolated by density gradient centrifugation.2、 Cell counter was used to count the cell expansion;3、 multicolor flow cytometry was used to detect the CD8+T cell subsets,and CD45 RA and CCR7 were used to distinguish the cell subsets.And detected the expression of CD62 L in CD8+T cell,CD8+ TN and CD8+ TCM.4、 And IFN-γ and Granzyme-B production was evaluated by ELISA,and detected the IFN-γ and Granzyme-B production in TGF-β.5、 Melanoma tumor associated antigen MART-1 specific TCR was screened from healthy donors.The IL-7+IL-15 and Doramapimod combination was validated in MART-1 specific TCR-T.Result: 1、 Compared with veh group,there was no significant difference in T cell number on day 8,but the cell number was significantly increased on day 12(P<0.05).2、 The percentage of CD8+ TN cells increased significantly in p38 inhibitor group(P<0.05)on day 10,and the expression of CD62 L was significantly increased in total CD8+ T,CD8+ TN and CD8+ TCM in IL-7+IL-15(P<0.05),but the IL-2 was not.3、 The secretion of IFN-γ and Granzyme-B was significantly increased in p38 treated group(P<0.05).The production of IFN-γ was significantly decreased in nontreated group,and the IFN-γ was significantly increased in p38 inhibitor treated group in TGF-β environment(P<0.05).4、 Doramapimod and IL-7+IL-15 was used in MART-1 specific TCR-T 10 days.The expression of CD62 L was significantly increased in CD8+ TCR-T.And the secretion of IFN-γ and Granzyme-B was significantly decreased in nontreated group,but the production was significantly increased in treated group,in TGF-β environment after MART-1 specific antigen re-stimulated.Conclusion: We compared two p38 inhibitor in cell proliferation,T cell subsets and the function of secreting cytokines,we found there are no difference in two p38 inhibitor.However,the IL-7+IL-15 combinate with p38 inhibitor could increase the less differentiated T cell subsets.And p38 inhibitor could maintain the function of T cell in TGF-β.These results inform design of the combined Doramapimod and IL-7+IL-15 in T cell generation.