Experimental Study Of Cytotoxic T Cells In Combination With PD-L1 MAb In Immunological Therapy Of Lung Cancer

PART 1 Experimental study of cytotoxic T cells in combination with PD-L1mAb on lung cancer in vitroObjective:Dendritic cells from healthy human peripheral blood were induced mature by pulsed with apoptotic tumor cells and CD40mAb ;and the cytotoxic T lymphocyte in combination with PD-L1 mAb against lung cancer cell activated by DCs were investigated in vitro .Methods:PBMCs were isolated from healthy human peripheral blood by density gradient centrifugation.While PBMCs were cultured in RPMI 1640 medium with 10% FCS for 2 hours,non-adherent cells were removed gently and adherent cells were cultured in mixed medium containing rhGM-CSF(100μg/L),rhIL-4(50μg/L) to generate DCs.After 6 days,immature DCs were cultured with apoptotic tumor cells and stimulated by CD40mAb(5mg/L) for another 2 days.Then the mature DCs were collected and cocultured with T cells at ratio of 1:50 for 3 days to activated antigen specific CTL.The phenotypes changes of CTL were analyzed by flow cytomety.MTT assays were used to determine the cytotoxicity of CTL in vitro and the cytokines such as IL-2 ,IFN-γin supernatant were quantified by enzyme-linked immunosorbent assays. Results:1. After DCs were cultured with apoptotic tumor cells and CD40mAb,the expression rates of CD1α,CD80,CD83,HLA-DR on the surface of DCs showed upregulated(P<0.05).2.The expression rates of CD8+Tcells were significant increased co-cultured with mature DCs(P<0.05).In addition,the cytotoxicity of CTL in combination with PD-L1mAb on lung cancer cell line( H1299) was higher than that of CTL groups:the apoptotic rate of H1299 in CTL group and CTL with PD-L1 mAb group were (41.9±9.8)%,(63.2±11.7)% ,respectively(P < 0.05),it indicates statistical difference exists between them.Meanwhile,the concentration of IL-2 in supernatant of CTL with PD-L1mAb group was(162.3±20)pg/ml higher than that of the CTL group(115.38±12.23)pg/ml (P<0.05). Conclusion:1.Compared with CTL group,CTL in combination with PD-L1mAb exhibit the higher cytotoxicity on H1299 cell.2. The apoptotic phenomenon of CTL cells in H1299 group was showed,and the apoptotic rates of CTL cells in H1299 with PD-L1 group was decreased.3. ELISA assays showed CTL in combination with PD-L1mAb release higher concentration IL-2 in supernatant comparing with CTL group.PARTⅡAntitumor effect of cytotoxic T cells with PD-L1mAb on Lung cancer in nude miceObjective:To investigate the effects of cytotoxic T cells alone and in combination with PD-L1mAb on the growth of human lung cancer transplanted subcutaneously in nude mice. Method:Human lung cancer cell line(H1299) transplanted subcutaneously in nude mice was established and divided into 4 groups:1.control group:normal saline group,2. PD-L1mAb group,3.CTL group,4. CTL with PD-L1mAb group.Tumor volume was measured and the cytokines such as IL-10,IL-2 in the mice serum were also quantified by ELISA assays.Results:1. The tumor volume of the NS group,PD-L1mAb group,CTL group and CTL in combination with PD-L1mAb group were (1.39±0.63)cm3,(1.53±0.71 )cm3,(0.63±0.26 )cm3,(0.16±0.03)cm3, respectively. CTL group and CTL in combination with PD-L1mAb group showed a significant inhibition of the growth of transplanted tumor cells in comparing with the NS group,PD-L1mAb group(P<0.05).2. IL-2 levels in NS group,PD-L1mAb group,CTL group and CTL in combination with PD-L1mAb group were(160.5±44.9)pg/ml,(133.4±46.2)pg/ml,(321.1±24.1)pg/ml,(469.4±8.5)pg/ml respectively;IL-2 levels in CTL group and CTL in combination with PD-L1mAb group were significantly higher than that of NS group,PD-L1mAb group(P<0.05) .IL-10 levels in NS group,PD-L1mAb group,CTL group and CTL in combination with PD-L1mAb group were (556.4±94.5)pg/ml,(485.5±33.4)pg/ml,(335.9±27.3)pg/ml,(288.5±33.4)pg/ml respectively. IL-10 in the serum of CTL group and CTL in with PD-L1mAb group were lower than other groups (P<0.05).Conclusion:CTL with PD-L1mAb can significantly inhibit the growth of the transplanted tumor cells in comparing with the other groups(P<0.05),and CTL with PD-L1mAb can decrease the IL-10 level and increase the level of IL-2,which may enhance the cytotoxicity of CTL cells .