| Objective: PD-1(Programmed death-1) is a sign of T-cell exhaustion and dysfunction. Overexpression of PD-1 on T lymphocytes may impair their proliferation and effector functions. We set out to detect the expression of PD-1 on peripheral blood mononuclear cells from cancer patients, analyze its relationship with the clinical data of cancer patients and verify its feasibility as an index evaluating immune status of cancer patients; Furhermore, we block the PD-1 expression on antigen specific T lymphocytes in vitro, investigate its influence on cytotoxicity effect of T lymphocytes, and analyze the probability of combining PD-1 monoclonal antibody therapy with adoptive antigen specific T cell immunotherapy.Methods: We analyzed PD-1 expression on PBMCs from 276 cancer patients and 58 healthy donors by flow cytometry to make a preliminary evaluation of the immune status of cancer patients. Meanwhile, PBMCs were isolated and IL-4, GM-CSF were used to culture dendritic cells while anti-CD3 m Ab, IFN- gamma, IL-2 were used to amplification T lymphocytes in vitro. The dendritic cells activated by tumor antigen were co-cultured with T lymphocytes after 8 days of culture. At the same time, PD-1 monoclonal antibody(10mg/m L) was added to block the PD-1 expression on T lymphocytes while the control group was established by adding Ig G1κ antibody(10mg/m L). Afterwards, T lymphocyte proliferation was measured and PD-1 expression rate was analyzed, while the secretion of cytokines such as IL-2, IL-4, IFN-gamma and TNF-a was detected by ELISA. Finally specific killing activity towards tumor cell lines HGC-27,SW480 and CNE-2 were detected by WST-1.Results:(1) The expression rate of PD-1 on the PBMC from cancer patients was significantly higher than that in healthy people(P<0.001), and the PD-1 expression was positively correlated with clinical stage of gastric cancer patients(P<0.05).(2) The expression rate of PD-1 was negatively related to the content of CD3+CD8+ cytotoxic lymphocyte and CD45RO+CD62Lˉ effector memory T cells(P<0.001).(3) Anti-PD-1 monoclonal antibody could effectively down-regulate the expression rate of PD-1 on T lymphocytes(P<0.001),while the proliferation rate of T lymphocytes stays constant(P>0.05).(4) ELISA showed that after blocking the expression of PD-1, the secretion of IL-2, IL-4, IFN-γ and TNF-a from T lymphocytes were significantly higher in the experimental group than those in the Ig G control group(P<0.05).(5) WST-1 showed that the T lymphocytes of PD-1 blocking group possess higher cytotoxic activity towards tumor cell lines HGC-27, SW480 and CNE-2, and the difference was statistically significant(P<0.05).Conclusions:(1) The average expression rate of PD-1 on PBMC from cancer patients was higher than that from healthy people, the high expression rate of PD-1 reflects T lymphocyte exhaustion. The PD-1 expression detected by flow cytometry on PBMC from cancer patients could serve as an effective predictive index for anti PD-1 monoclonal antibody.(2) The expression of PD-1 on PBMC from cancer patients was positively correlated to the prognosis of gastric cancer and could be used as an adjuvant prognosis index for gastric cancer.(3) Blocking expression of PD-1 expression on adoptive T cell in vitro could improve its cytotoxicity effectively, and the combination of anti PD-1 monoclonal antibody and adoptive T cell may be a new strategy for tumor immunotherapy. |
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