The Study On The Expression Of Cancer-testis Antigens And Anti-tumor Function Influence Factors Of MAGE-A3 Specific T Cells In Patients With Esophageal Squamous Cell Carcinoma

Background and objective:The incidence and fatality rate of cancer were rising year by year, so cancer has become the major disease threatening human life and health. Esophageal cancer is one of the most common malignances. The regional differences survey showed that its incidence and mortality was highest in China and 90% of cases in China were esophageal squmous cell carcinoma. Despite the improvement of treatment, the 5-year survival rate was less than 10%. In order to further improve the survival and life quality of patients with esophageal cancer, it is urgent to explore novel prognostic markers and therapeutic targets for these patients. Recent years, cancer immunotherapy has become the most promising therapeutic strategy in treating solid tumors. Adoptive T cell therapy has gained more and more importance during the treatment of tumor. Tumor associated antigens as therapeutic targets of T cell therapy have been applied in clinical trials, such as vaccine therapy and T cell receptor(TCR) engineered T cell therapy. Cancer testis antigen(CTA) was highly expressed in tumor tissues and restricted in certain normal tissues(reproductive system, testis and placenta tissue). As the reproductive system is immune free organs, so cancer testis antigens become ideal targets for cancer immunotherapy. CTA expression was correlated with tumor growth, survival and disease recurrence in patients with malignant diseases. Downregulating the expression of CTA in vitro could change the biological characteristics of tumor cells, including reducing the adhesion, invasion and metastasis ability. The expression of CTA genes might be related to the poor prognosis of cancer, so it could be served as poor prognostic indicator for malignances. In recent years, some HLA class I restricted antigenic peptides have been identified. At the same time the function of antigen specific T cells play an important role in the process of disease development and recurrence. It has been reported that evaluating the antigen specific T cell response after vaccine with MAGE-A3/NY-ESO-1 peptide in patients with melanoma, they found that frequency and function of antigen specific T cells might be related to the prognosis of patients. Antigen specific T cells could significantly improve tumor remission rate and prolong the survival of patients with tumor. However, the expression of cancer testis antigen and T cell response to related peptide were rarely reported in esophageal cancer. In tumor microenvironment, the function of antigen specific T cell was limited. There are many factors which can affect antitumor function of T cells. In the process of T cells activation, a series of inhibitors such as programmed death receptor 1(PD-1) and cytotoxic T lymphocyte associated antigen 4(CTLA-4) were activated. Targeting the inhibitory molecules on the surface of the T cells to develop a series of monoclonal antibodies, especially targeting immune checkpoints PD-1/CTLA-4 in the treatment of various malignant tumors have been obtaining curative effects. Other factors influencing antineoplastic function of T cells such as reactive oxygen species(ROS) in the tumor microenvironment could also induce T cell apoptosis and inhibit T cell function. It is reported that suppressive immune cells inhibit T cell activation and function through producing ROS in the tumor microenvironment. Whether the application of combining reactive oxygen species scavenger with PD-1/PD-L1 blockade will influence the apoptosis of T cell and recover the function of PD-1+ T cell has not been reported. This study was to detect the expression of CTA genes and their encoded antigen in esophageal cancer tissue and analyze their correlation with clinical parameters. We found the expression of MAGE-A3 and MAGE-C2 in esophageal cancer tissues was associated with poor prognosis of esophageal cancer patients. We tested the frequency and function of MAGE-A3 specific CD8+T cells proportion from peripheral blood in patients with esophageal cancer, and analyzed their correlation with clinical parameters. Then we detected the expression of PD-1 in the responsive and nonresponsive MAGE-A3 specific T cells and compared the PD-1 level of CD8+ T cell from peripheral blood mononuclear cell(PBMC), tumor infiltrating lymphocyte(TIL) and paired normal tissue infiltrating lymphocyte(NIL), and found that PD-1/PD-L1 blockade could mildly increase the ratio and function of MAGE-A3 specific T cells. High level of ROS was found in the tumor-environment and PD-1 positive T cells. Finally we found that ROS scavenger glutathione(GSH) combined with PD-1/PD-L1 blockade could synergistically enhance the antitumor effect of T cells.Part 1 The expression of cancer testis antigens and their clinicalsignificances in esophageal squamous cell carcinoma Methods:1)The expression of MAGE-A3, MAGE-A4, MAGE-C2 and NY-ESO-1 in esophageal cancer tissue was detected by RT-PCR.2)Evaluate the correlation between the expression of these antigens and clinical characteristics of patients with esophageal cancer.3)Detect the protein expression of MAGE-A3 and MAGE-C2 by immunehistochemical in esophageal cancer tissues and explore the contact between the prognosis.4)The effects of MAGE-A3/C2 downregulation on ESCC cells were assessed in vitro. Results:1)Cancer testis antigens were frequently expressed in ESCC tumor samples.2)The expression of MAGE-A3 and MAGE-C2 was related to lymph node metastasis and late stage.3)MAGE-A3 positive patients had a poorer survival compared with negative patients. MAGE-A3 expression was an independent prognostic marker in ESCC patients.4)Downregulating MAGE-A3/C2 resulted in decreased colony-formation and migration ability of cancer cells. MAGE-A3/C2 played a role on ESCC metastasis through inducing epithelial-mesenchymal transition. SummaryCancer testis antigens were expressed with high frequencies in esophageal cancer tissues, the expression of MAGE-A3 could be served as an independent prognostic indicator. Affecting the progress of epithelial mesenchymal transformation on tumor cells was involved in invasion and metastasis of tumor cells. Cancer testis antigens could be used as potential targets for immunotherapy.Part 2 Clinical significance of the frequency and function andMAGE-A3 specific CD8+T cells in patients with esophageal squamous cell carcinoma Methods:1)Selected HLA-A2 and MAGE-A3 double positive patients with esophageal cancer and cultured MAGE-A3 specific CD8+T cells in vitro. MAGE-A3 specific CD8+T cells were detected using MAGE-A3 specific tetramer by flow cytometry.2)The specific lyses of tumor cells induced by antigen specific CD8+T cells was detected by PI-Annexiv assay and ELISPOT assay.3)Evaluated the presence of functionally reactive MAGE-A3 specific CD8+T cells and the correlation with clinical parameters. Results:1)In the patients with ESCC, MAGE-A3 tetramer positive CD8+ T cells were not observed in the early stage after obtaining the leukapheresis product(detection threshold <0.01%), however, after stimulations twice with DC loaded peptide, the tetramer positive T cells were at frequencies of >0.2%. These T cells could induce specific lyses of restricted HLA-A2+/MAGE-A3+ tumor cell lines(P<0.05).2)The frequency of tetramer positive CD8+T cells was higher in patients with elder age but lower in patients with lymph node metastasis and late tumor stage(P<0.05). The lower CD107 a expression level of CD8+T cells responding to MAGE-A3 peptide was significantly associated with poor outcome parameters, including advanced stage, tumor size and lymph node metastasis(P<0.05).3)In addition, functional T cells responding to MAGE-A3 peptide, according to CD107 a high expression, not the frequency of MAGE-A3 specific CD8+T cells, could be used as an independent prognostic factor in Cox regression analysis. SummaryOur results indicated that MAGE-A3 specific CD8+T cells could induce specific lyses of restricted HLA-A2+/MAGE-A3+ tumor cell lines, the presence of functional MAGE-A3 specific CD8+T cells had strong independent prognostic impact on survival in ESCC. In addition, our findings provided a rationale for pursuing MAGE-A3 based vaccination and T-cell transfer strategy.Part 3 PD-1 was highly expressed on non-responsive T cells in patients with esophageal cancer Methods:1)Detected the expression of PD-1 on antigen specific CD8+T cells by flow cytometry.2)Sorted out the CD107a+/IFN-g+ and CD107a-/IFN-g- MAGE-A3 specific CD8+T cells seperately and detect the differential PD-1 expression on these cells.3)Compared the expression of PD-1 on PBMC, TIL and NIL, analyzed the different function of PD-1 positive and negative CD8+T cells from TIL. Compared the expression of PD-L1 on the antigen presentation cells and epithelial cell from tumor tissue and paired normal tissue.4)Detected the influences of PD-1/PD-L1 blocking on proliferation and function of MAGE-A3 specific CD8+T cells. Results:1)PD-1 was highly expressed on non-responsive antigen specific CD8+T cells and tumor infiltrating lymphocytes.2)PD-L1 was highly expressed in tumor tissue and the function of PD-1+CD8+T cells from TIL was impaired.3)PD-1/PD-L1 blockade could mildly enhance the proliferation and function of MAGE-A3 antigen specific CD8+T cells. SummaryPD-1 was highly expressed on non-responsive antigen specific CD8+T cells and tumor infiltrating lymphocytes in patients with esophageal cancer. PD-1/PD-L1 blockade could mildly increase the proliferation and function of MAGE-A3 antigen specific CD8+T cells.Part 4 Combining GSH with PD-1/PD-L1 blockade enhance the antitumor efficacy of MAGE-A3 antigen specific CD8+T cells Methods:1)Compared the apoptosis rate, ROS and CD95 expression of PD-1 positive or negative CD8+ T cells.2)Detected how the ROS scavenger GSH influence the ROS level, apoptosis and function of CD8+T cells.3)Detected the influence of GSH combined with PD-1/PD-L1 blockade on CD8+T cells in vitro.4)Based on the adoptive T cell therapy, GSH and PD-1/PD-L1 blockade, detected the tumor growth of esophageal tumor-bearing mice in vivo.5)Isolated tumor infiltrating lymphocytes from tumor model mice treated with T cell therapy, detected the number and cytokine secretion of CD8+ T cells by flow cytometry. Results:1)PD-1 positive T cells have higher level of ROS and apoptosis rate than PD-1 negative CD8+T cells.2) GSH combined with PD-1/PD-L1 blockade synergistically reduced the expression of ROS and apoptosis of PD-1+ CD8+ T cells.3)GSH combined with PD-1/PD-L1 blockade synergistically reversed the cytokine secretion ability of PD-1+ CD8+ T cells.4)Antitumor ability of T cells could be improved after exposing to ROS scavenger combined with PD-1/PD-L1 blockade in vivo.5)GSH combined with PD-1/PD-L1 blockade increased the counts of infiltrated CD8+ T cells and enhanced the ability of cytokine secection. SummaryPD-1 positive T cells have higher level of ROS and apoptosis than PD-1 negative T cells. And the cytokine secretion of IFN-g, TNF-a was reduced compared with PD-1 negative T cells. GSH combined with PD-1 blockade synergistically reduced the expression of ROS and apoptosis of PD-1+ CD8+T cells and reversed the cytokine secretion ability of PD-1+ T cells. Based adoptive CD8+T cell therapy, combining GSH with PD-1/PD-L1 blockade synergistically inhibit the tumor growth of esophageal tumor animal model. Conclusions:1)Cancer testis antigen was frequently expressed in esophageal cancer tissues. MAGE-A3 expression was an independent prognostic marker of esophageal cancer.2)MAGE-A3 specific CD8+T cells could induce specific lysis of HLA-A2+/ MAGE-A3+ tumor cell line. The frequency of reactive MAGE-A3 specific CD8+T could predict the prognosis of patients with esophageal caner.3)PD-1 was highly expressed on MAGE-A3 specific CD8+T cells and tumor infiltrating lymphocytes in esophageal cancer. PD-1/PD-L1 blockade could mildly increase the ratio and function of MAGE-A3 antigen specific CD8+T cells.4) GSH combined with PD-1/PD-L1 blockade synergistically reduced the expression of ROS and apoptosis of PD-1+ T cells and reversed the cytokine secretion ability of PD-1+ T cells. Combining GSH with PD-1/PD-L1 blockade synergistically inhibited tumor growth in vivo.