1.Over-expressing Akt In T Cells To Resist Tumor Immunosuppression And Increase Anti-tumor Activity2.Repeated Cycles Of 5-fluorouracil ChemotherapyImpaired Anti-tumor Functions Of Cytotoxic T Cells In ACT26 Tumor-bearing Mouse Model

BackgroundTumor microenvironment is heterogeneous and complex, accompanying with tumor formation. Various growth factors and cytokines are produced and different immune cells are recruited as part of tumor stromal. Tumor employs various means to escape immunosurveillance and inhibit immune attack. The efficacy of adoptive T cell therapy is reduced by tumor immunosuppression. Strategies have been developed to counteract the inhibitory signals. However, due to complex suppressive mechanisms in tumor microenvironment, blocking one or a few inhibitory signals has only limited effects on therapeutic efficacy. Instead of targeting tumor immunosuppression, we hypothesized that manipulating T cells to make them resist any known or unknown suppressive mechanism may be more effective for cancer treatment. Akt (also named as protein kinase B, PKB) is chose as it is the central node in the signaling of effector T cells and could help T cells to improve activity.MethodsWe analyzed the phosphorylation of Akt in T cells from tumor microenvironment. Then OT-1 cells were transduced with retro viruses encoding Akt and human peripheral blood lymphocytes (PBLs) were transduced with retroviruses encoding both Akt and a chimeric antigen receptor (CAR) specific for tumor antigen EpCAM to examine the effect of over-expressing Akt on tumor specific T cells in tumor environment.ResultsWe show that Akt activity of T cells in the tumor environment was inhibited, and over-expressing Akt in OT-1 cells increased the cytokine production and cell proliferation in the presence of B16-OVA tumor cells. What’s more, adoptive transfer of OT-1 cells over-expressing Akt inhibited B16-OVA tumor growth and prolonged mouse survival. To examine if over-expressing Akt could increase the anti-tumor activity of T cells in human cancer, PBLs co-expressing EpCAM specific CAR and Akt were cultured with EpCAM-expressing human prostate cancer cells PC3M, and less inhibition on cell proliferation and less apoptosis were observed. In addition, adoptive transfer of PC3M specific T cells over-expressing Akt resulted in more dramatic tumor inhibitory effects in PC3M bearing NOD/SCID mice.ConclusionThese data indicates that over-expressing Akt in tumor specific T cells increases T cell proliferation and activity in the tumor environment, and enhances anti-tumor effects of adoptively transferred T cells. Our study provides a new strategy to improve the efficacy of adoptive T cell therapy, and serves as an important foundation for clinical translation.BackgroundOne mechanism of tumor cells killed by chemotherapeutics is because of their unlimited proliferation which is different from normal cells. Chemotherapies are divided into cycles to minimize their toxicity to normal tissue. However, tumor cells are recovered as well as normal cells during the interval of chemotherapy. Chemoresistance, relapse and metastasis remain the problems after chemotherapy. Bone marrow suppression is a common toxicity of chemotherapy to the immune system. There are studies implied that chemotherapy in advanced cancer patients did not improve the quality of life and over survival. Recently, the immunostimulatory roles of chemotherapeutics have been increasingly revealed. Immunogenic cell death and immune reconstruction after chemotherapy could reactivate the anti-tumor immune response. The number and ratios of different immune subpopulations are analyzed after chemotherapy, but the details of immune status after each treatment cycle remain unclear. We speculated that different cycles of chemotherapy may have different effects on anti-tumor immune functions. Study on immune status during chemotherapy may help to develop precision medicine combining chemotherapy with immunotherapy to improve anti-tumor effect.MethodsWe treated CT26 tumor-bearing mice with one to four cycles of 5-fluorouracil (5-FU). Tumor growth suppression and mice survival were monitored. Immune statuses after one to three cycles of chemotherapy were analyzed. Data focused on one and three cycles of treatment was presented. Cell number and ratios of different immune subpopulations in spleens and tumors, as well as cytokines in the serum after one-and three-cycle chemotherapy were analyzed. In vitro proliferation, cytotoxicity and cytokines production of spleen cells were compared between treatment and control groups. Immunotherapies containing cytokine induced killer (CIK) cells and PD-L1 monoclonal antibodies were combined with one-cycle chemotherapy to improve antitumor efficacy.ResultsMore cycles of chemotherapy inhibited tumor growth to a greater degree than one-cycle treatment. But overall survivals of repeated cycles of chemotherapy were not better than one-cycle treatment. The absolute number of CD8+T cells and NK cells were not influenced significantly after each cycle, but proliferated CD8+T cells were decreased, and CT26-specific cytotoxicity and IFN-y secretion of spleen cells from three-cycle 5-FU treated mice were impaired in vitro. After one-cycle 5-FU treatment, a greater percentage of CD8+T cells infiltrated into tumors. In addition, more proliferated CD8+T cells, enhanced tumor-specific cytotoxicity as well as IFN-y secretion of spleen cells against CT26 in vitro were observed. Immunosuppressive factors including TGF-P, IL-10 and PD-L1 were increased after chemotherapy. CIK cells and PD-L1 monoclonal antibodies were administered after one-cycle 5-FU had better anti-tumor effect than chemotherapy or immunotherapy alone.ConclusionOne-cycle 5-FU treatment promoted anti-tumor immune response, whereas repeated chemotherapy cycles impaired anti-tumor immune functions. Though the amount of immune cells could recover after chemotherapy suspension, their anti-tumor functions were damaged after cycles of chemotherapy. Immediate implementation of immunotherapy is useful to improve anti-tumor effect. Our study provides guidelines for more correct combination of chemotherapy with immunotherapy in the progress of immunotherapy.