The Therapeutic Effect Of Anti-PD-L1 Monoclonal Antibody And CTL On BALB/c Nude Mice Bearing Lung Cancer

Programmed death-1 ligand (PD-L1) is one of the important members of B7/CD28 costimulatory superfamily. Its'receptor is PD-1. PD-1/PD-L1 pathway functions as a negative co-inhibitor in T cells proliferation and plays a pivotal role in the evasion of tumor immunity. The purposes of our experiment are to establish stable H460/PD-L1 cell line and study on its regulatory effect on T cells. The effect of PD-L1 on the growth of tumor was observed, meanwhile the antitumor activity of PD-L1mAb and antigen specific CTL in vivo was evaluated in BALB/c nude mice bearing lung cancer.PartⅠ: Establishment of lung cancer cell line transfected with human PD-L1 gene and preliminary study on its regulatory effect on T cellsObjective: To establish H460 cells expressing human PD-L1 gene and analyze its regulatory effect on T cells, so that it could lay the foundation to study the role of PD-L1 in the immune evasion and the immunotherapy of lung cancer.Methods: pIRES2-EGFP/PD-L1 was transfected into H460 cell by lipofectamine AMINE. After screening culture by G418, stable transfected H460 cell line was established, and the expression of PD-L1 was identified by FCM, RT-PCR. Effect of H460/PD-L1 cells on T cells proliferation and cytokine production in vitro was investigated by means of MTT and ELISA.Results: Stable transfected H460 cell line was established, and PD-L1 gene was expressed successfully. FCM analysis identified that the expression level of green fluorescence protein(EGFP)report gene in the transfected cells was high(≥90%). The expression of PD-L1 protein on the surface of H460/PD-L1 transfected cells detected by commercialized mouse anti-human PD-L1 mAb was 99.4%. Transcription of PD-L1 gene identified by RT-PCR indicated that recombinant pIRES2-EGFP/PD-L1 had been successfully inserted into eukaryotic genome. In vitro, compared with H460/mock cells, H460/PD-L1 cells could obviously suppress the proliferation of T cells stimulated by anti-CD3 mAb[(0.703±0.014)vs(0.665±0.014), P<0.01] , while down-regulated the production of IFN-γmeasured by ELISA[(403.9±4.7)pg/ml vs(118.7±7.4)pg/ml, P<0.01]. Anti-PD-L1 monoclonal antibody could block these inhibiting effects. The proliferation of T cells was reconverted [(0.665±0.014)vs(0.690±0.017), P<0.05] and the production of IFN-γwas up-regulated[(118.7±7.4)pg/ml vs(401.0±10.7)pg/ml, P<0.01].Conclusion: A cell line H460/PD-L1 stably expressing the human PD-L1 gene has been established successfully, it can prominently suppress the proliferation of T cells and cytokine production in vitro and anti-PD-L1 monoclonal antibody could block these inhibiting effects.PartⅡ: The combination of anti-PD-L1 monoclonal antibody and CTL in the treatment of BALB/c nude mice bearing H460/PD-L1 lung cancer Objective: Evaluate the antitumor activity of the combination of PD-L1mAb and antigen specific CTL in BALB/c nude mice bearing lung cancer.Methods: DCs were induced from healthy human peripheral blood monocytes(PBMC) by rhGM-CSF and rhIL-4 and loaded with apoptotic tumor cells. The immature DCs were activated by CD40mAb. The mature DCs were co-cultured with autologous T cells to obtain CTL. The phenotypes of CTL were analyzed by flow cytometry. The antitumor activity of PD-L1mAb combined with CTL in vivo was evaluated in BALB/c nude mice bearing lung cancer.Results: The expressions of CD1a, CD80, CD83, HLA-DR were up-regulated after DCs were activated by CD40mAb combined with apoptotic tumor cells. The CD8 +T cells were markedly up-regulated after co-culture of mature DCs and autologous T cells. In vivo experiments, in NS group, compared with H460/mock group, the tumor volumns are larger in H460/PD-L1 group (P<0.05), and metastasis in skin and livers, these nude mice all died in 5 weeks, its survival time was shorter(P<0.01). CTL significantly inhibited the growth of transplanted tumors in nude mice, the killing effect was stronger in H460/mock group than H460/PD-L1 group(P<0.01); in transplanted H460/PD-L1 tumors in nude mice, compared with NS group, CTL group, PD-L1mAb group and PD-L1 mAb combined with CTL group induced a substantial antitumor effect(P<0.01). The combination of PD-L1mAb and CTL showed stronger antitumor activity than CTL and PD-L1mAb(P<0.05), and the survival time of nude mice was longer(P<0.05).Conclusion: Anti-PD-L1 monoclonal antibody combined with CTL exhibited a substantial antitumor effect , it could make the nude mice live longer, and provide the rationale for developing a novel therapy of targeting the PD-L1/PD-1 pathway against lung cancer.