Core Technology In Amplification And Preparation Of Antigen Specific T Cells In Colorectal Cancer

Objective:The incidence and mortality of colorectal cancer(CRC) is on its rising trend. Immunotherapy has earned impact status in colon cancer comprehensive therapy. Through a various ways, can the tumor antigens stimulate autologous lymphocytes to acquire antigen-specific cytotoxic cells. Antigen presenting cells(APCs) processed tumor antigens into antigen peptide followed by combination with major tissue compatibility complex(MHC) molecules to form a complex participating activation of tumor antigen specific cytotoxic T lymphocytes(CTL). Frame shift mutations of transformation growth factor beta type II receptor(TGF?R?) and O sites N-acetylglucosamine sugar based transfer enzyme(OGT) gene lead to two HLA-A2 restricted neo-antigens TGF?R?(RLSSCVPVA,131-139) and OGT (SLYKFSPFPL,28-37) respectively, which limited in 20-40% HLA-A2 of colon cancer patients and does not exist in normal and other cancers population. Tumors, in order to avoid recognition by APCs, downgrade the expression of MHC molecules. Besides, the decreased number of immune cells in some patients may not recognise the antigen, resulting in absence of T cells activation. In view of the present status, we synthesized antigen peptides and co-cultured with T cells in the presence of MHC, in order to improve the activation efficiency of peptide specific T cells. Next we compared the killing efficiency between peptiede stimulated dendritic cells(DC) induced killer T cell and peptide induced CTL, in order to obtain equal or better killing effect as well as reduce in vitro operation and reduce the incidence rate of adverse reactions. To sum up, this study aims to construct a simple and effective method for the preparation of neoantigen specific T cells, in order to achieve the purpose of enhancing specificity and reducing the safety.Methods:synthesis of TGF beta RII and OGT antigenic peptides followed by mass spectrometry analysis of purity identification. Achievement of the colon cancer cell lines with expression of TGF??(RLSSCVPVA,131-139), OGT (SLYKFSPFPL,28-37) and HLA-A2 restriction:DLD-1. Obtained the peptide stimulated CD8+T cells from peripheral blood mononuclear cells(PBMC) of HLA-A2 restricted colon cancer patients, flow cytometry analysis to identify the cell phenotype and the proportion; generation of antigen-specific CTL:stimulated CD8+T cells by antigen peptide. MTS assay determining the killing effect in DLD-1 among different ratio; Secondly, achievement of the HLA-A2 restricted mature DC loaded tumor antigen peptides to stimulate the corresponding healthy individual CTL, comparison the killing activity of DC induced CTL and direct the use of antigen peptide stimulated CTL.Results:We obtained the TGF beta RII(RLSSCVPVA,131-139) and OGT (SLYKFSPFPL,28-37) antigen peptide with purity of 99%-100%. By added directly to PBMC or via DC, we generated the antigen peptide specific CTL. MTS assay has confirmed the 3 days and 7 days killing effect of colon cancer patients and healthy control of peptide specific T cells to DLD-1 the colon cancer cell line at target ratio of 1/1,5/1,10/1,20/1.Under 5/1,10/1,20/1 condition, patient group achieved the toxicity of 48.54%,63.62% and 70.23% respectively, and in healthy control group, the killing rate were 66.32%,74.82% and 81.61%. The killing in the patients without antigen peptide treatment under 10/1 was better than that of healthy controls, which has shown that the antigen peptide stimulation could not improve the ability of autologous immune cells to kill DLD-1 in patients with colon cancer. Cells with DC load antigen peptide induced formation of tumor antigen peptide specific CTL compared to directly using the antigen peptide stimulated T cells from normal human PBMCs induced specific CTL can on DLD-1 obtained better killing effect. Compared with the DC vaccine, directly stimulating the specific cytotoxic T lymphocytes at day 14 in effect target ratio 5/1, DC load and antigen peptide 1(OGT) direct stimulation of CTL on DLD-1 killing rate was 26.71% and 65.84%(P=0.054), DC load and antigenic peptide 2(TGF?R?) direct stimulation of CTL on DLD-1 killing rate was 20.69% and 81.32%(P=0.039), respectively, DC load and antigenic peptide 1+2 direct stimulation of CTL on DLD-1 killing rates were 39.91% and 69.24%(P=0.048). When the effector to target ratio is 10/1, DC load and antigen peptide 1 direct stimulation of CTL on DLD-1 killing rate was 36.33% and 88.82%(P=0.038) DC load of antigenic peptides and direct stimulation of CTL on DLD-1 killing rates were 37.51% and 89.74%(P=0.022) DC load and antigenic peptide 1+2 direct thrust induced CTL on DLD-1 killing rate was 45.58% and 86.61%(P=0.043). The evaluation of IFN-? also proved the activation of CTL within different group in another hand.Conclusion:in this study, we managed to obtain better killing effect on the DLD-1 with the direct load of two colon cancer antigen peptides in healthy human cells. Two antigenic peptides may become new target for immunotherapy of colon cancer in the future.