The Effect Of CTLA-4Blockade On CTLs Treatment On Hepatocarcinoma

Tumor immunity gets widely attention from oncologists in the past more than a century,and in the past several decades immunotherapy gradually wins its place in tumor treatment.Tumor immunotherapy refers to the stimulation of the immune system against cancer cellsthrough the introduction of vaccine, cytokines, antibodies or transferred immune cellsthemselves. Adoptive cell therapy (ACT) is a kind of tumor immunotherapy. ACT refer toculture specific anti-tumor immune cells in vitro, and then transfer them back to tumorpatients, so as to achieve the effect of anti-tumor.T lymphocyte can be categorized into three main groups, CD4+T helper cell (Th), CD8+cytotoxic T lymphocyte (CTLs), the third group is a group of specific T cell, calledregulatory T cells (T reg), which was found to have immune suppressive function. Amongthe three group cells, CD8+cytotoxic T lymphocyte is considered to play a vital part infighting against tumor. In recent years, many investigations found regulatory T cellsinfiltrate in hepatocarcinoma, melanoma, ovarian cancer and other tumors, and researchershave also demonstrated high frequency regulatory T cells in patient peripheral bloodcorrelating with poor prognosis, and regulatory T cells have the ability of inhibitingantitumor immunity. Also, studies have found that in the process of CD8+cytotoxic Tlymphocyte activation and proliferation in vitro, regulatory T cells have a negative effect.Immune checkpoints are inhibitory pathways in the immune system, these pathways playan important role in immune tolerance, and have the ability to adjust the peripheral tissuesphysiological immune response in order to reduce the damage to the surrounding tissues.These molecules include CTLA-4, PD-1, LAG-3, TIM-3and so on. Studies have foundhigh expression of CTLA-4on the surface of regulatory T cells, and the expression ofCTLA-4and the inhibitory function of regulatory T cells have a close correlation. At thesame time, the immune checkpoint molecules express on the activated T cells. In thisexperiment, we directly add the anti-CTLA-4antibody to the T cell culture system andstudy if blockade of the immune checkpoint molecule can improve the activation of the Tcells and reduce the regulatory T cells.This experiment mainly consists the following three parts:1.CTLs targeting MUP53activation in vitroUse the cytokines to induce the peripheral blood mononuclear cells (PBMCs) to maturedendritic cells (DCs), and then use the adenovirus loading the MUP53gene to infect theDCs. Stimulate the T lymphocyte with DCs to obtain targeting MUP53specific T cells,and test the activation of CTLs(PD-1, IFN-γ)，and demonstrate that DCs and T cells co-culture can get the specific T cells2.The activation of specific T cells after removal of regulatory T cells Remove the regulatory T cells before T lymphocyte activation, and use the same way to getspecific T cells. Then test find that the CD8+cells ratio increase, T cell activation makerPD-1, and the secretion of IFN-γ increase.3.The activation of specific T cells after add anti-CTLA-4antibodyDirectly add the anti-CTLA-4antibody to the T cell culture system and culture specific Tcell in the same way with mature DCs. Then test result shows tha the the proliferation ofcells is weak, CD8+/CD4+ratio don not change, T cell activation maker PD-1, and thesecretion of IFN-γ increase. Also, test the number of regulatory T cells and its inhibitionfunction, and the killing effect of activated T cells on liver cancer cell HepGII.Conclusion: Regulatory T cells have negative effect on activation of T cell in vitro, addanti-h-CTLA4antibody can reduce the number of regulatory T cells, andrestrain its suppressive effect, so as to enhance the effect of T cell activation,and anti-tumor function.