| Background: Improve the specificity and antitumor efficacy of the immune cells hasbecome an increasing important issue in adoptive cellular immunotherapy (ACI). Thetumor-associated antigen (TAA) is the basic of tumor specific immunotherapy. Mucin1(MUC1) is one of the TAAs, could induce specific cellular and humoral immunity, so itis an ideal target for tumor immunotherapy. At present the studies of MUC1in tumorimmunotherapy usually focused on lung cancer, breast cancer and so on. However,there are few studies of MUC1in ACI of gastric cancer. MUC1may induce specificcytotoxic T cells (CTL cells) in gastric cancer as the elevated levels. Testing andpreparation this CTL cells has important value in ACI of gastric cancer. MHC-peptidetetramer flow cytometry assay is the gold standard of quantitative detection of thespecific CTL. Furthermore, MHC-peptide pentamer is the latest and ideal method ofdetection of specific CTL as the higher sensitivity and specificity.The commonly usedtumor antigen in producing specific T cells included protein polypeptide and wholetumor cell lysate antigens. It has been reported that peptide-based immunotherapy relieson the identification of epitopes recognized by T-lymphocytes. However, the antitumoreffect of these immune cells is poor because of the high degree of polymorphism ofhuman leukocyte antigens and issues of tumor escape from the immune response. Theimmune cells induced by the whole tumor cell lysate antigens could target varietyantigens. However, these immune cells with low level of CTL cells targeting to specialtumor antigen. Therefore, the immune cells induced by using the whole tumor celllysate antigen combined with protein polypeptide could target both variety tumorantigens and the special tumor antigen and t may have better antitumor activity.Objective: To establish the method of detecting CTL cells using the MHC-MUC1pentamer and evaluate the pre-existing immune status in gastric cancer patient.Induction the MUC1specific CTL cells using the MUC1peptides combined the gastriccancer cell lysate and evaluate the antitumor effect of the CTL cellsMethods:1. The MUC1kit and the electrochemiluminescence immunoassay analyzerwere used to detect MUC1expression level in the peripheral blood of gastric cancer patients. The HLA-A2kit was used to screen the positive patient. The MUC1specificCTL cells, regular T lymphocytes (Treg) and central memory T lymphocytes (T_CM) inthe peripheral blood of gastric cancer patient were detected by flow cytometry. All theaspects were compared with the health people.2. The HLA-A2kit and MUC1Elisaassay were used to screen the gastric cancer cell line. The status of DC cells activatedby Mannatide/Pseudomonas aeruginosa preparation (BaiAn) was detected by flowcytometry. The KATOⅢ tumor cell lysate and two MUC1peptides were used toproduce different CTL cells and the specific CTL cells, T_CMcells, activated Tlymphocytes, Treg cells were detected by flow cytometry.3. The LDH assay was usedto appraise the cytotoxicity of different CTL cells in monolayer culture andthree-dimensional culture. The subcutaneous tumor model in nude mouse was used tocompare the antitumor effects of different CTL cells in vivo.Results:1. The expression level of MUC1, MUC1special CTL cells, CD8~+T_CMcells,CD4~+T_CMcells and Treg cells of gastric cancer patient were significantly higher thanhealth people (P<0.05).2. The gastric cell line KATOⅢ was HLA-A2positive andexpressed MUC1in high level.The antigen-presenting capacity and cell maturity of DCcells activated by BaiAn were higher than Mannatide. The MUC1special CTL cellsproportion, CD4~+/CD8~+T_CMcells proportion of combined group were significantlyhigher than other CTL cells(P＜0.05).The Treg cells proportion of combined group wassignificantly lower than other CTL cells(P＜0.05).3. In LDH release assay, thecytotoxicity of combined group was significantly higher than other CTL cells inmonolayer culture and three-dimensional culture(P＜0.05).The tumor growth inhibitionof combined group was significantly higher than other CTL cells in vivo (P<0.05).Conclusion:1. The MUC1special CTL cells in the peripheral blood of gastric cancerpatient was significantly higher than health people.2. There is pre-existing antitumoreffect in gastric cancer patient in vivo, but there are also significantlyimmunosuppressive factors in gastric cancer patient.3. The MUC1special CTL cellsproportion and the antitumor effect of the CTL cells induced by KATOⅢ tumor celllysate combined two MUC1peptides is higher than other CTLs. |
PDF Full Text Request