Phenotypic Characterization And Anticancer Capacity Of CD8+T Cells In CIK Culture After Antigen-induced Expansion

Background Cytokine-induced killer cells(CIK)are a type of polyclonal killer T cells activated and induced by interferon-garnma(IFN-y)and CD3 antibody.They have been used for adoptive cell therapy in malignant tumors.But there are individual differences in the CIK cell preparations and anti-cancer therapeutic effects.Objectives To eliminate the individual difference of therapeutic effects in the CIK adoptive cell therapy and obtain the highly effective anti-cancer CIK cells(CD8+CIK)by means of the improvement of CIK cell preparation,and to study the anti-cancer mechanism of CD8+CIK cells.Methods The study was designed in 3 parts.In Part 1,Magnetic Activated Cell Sorting(MACS)was used to select and collect CD8+CIK cells from CIK cells of 25 healthy cell donors.The CD8+CIK cells in 17 donors were cultured and expanded by IL-2 for 2 weeks for the evaluation of the purity and expanding capacity of CD8+CIK cells.And the CD8+CIK cells in other 8 donors were cultured and expanded for the study of their phenotypic characterization by flow cytometry(FCM).In Part 2,by means of detecting CD107 a expression in CD8+CIK cells with their NKG2 D receptors activated by HLA-MICA/B+ K562,the NK-like killing capacity of CD8+CIK cells was studied.The antigen polypeptides of 2 pluripotent transcription factors(PTF)of cancer stem cell(CSC)--OCT4 and Sox2 were used to stimulate CD8+CIK cells in culture for evaluation of immune response ability by TCR.In Part 3,the gene expression of NKG2 D ligands in the ovarian cancer cell lines(3AO,A2780,HO8910,SKOV3 and OVCAR3)was detected,and the HLA-I expression also detected by HLA-ABC specific antibody,and OCT4 and Sox2 gene expression detected by quantitative-PCR.The anti-cancer capacity of CD8+CIK against 5 ovarian cancer lines and K256 was detected by in vitro experiment.Results: In Part 1,the CD8+CIK cells collected from CIK cells increased by over 90 times after 2 weeks' culture and stimulation with IL-2.Almost all CD8+CIK cells expressed CD3+CD8+ and NKG2 D receptors.The CD3+CD56+ T cells(NK-like cells)accounted for 32.4% of all CD8+CIK cells,and 2 kinds of memory T-lymphocytes--CD45+CD28+T cells and CD45RA+CD27+ T cells respectively accounted for 23.6% and 50.5%.In Part 2,CD8+CIK cells proved,by detect of e CD107 a xpression,to be activated by HLA-MICA/B+K562 cells through NKG2 D receptors,and also to be specifically activated by OCT4 and Sox2 through TCR.In Part 3,ULBP4 gene expression was detected in all 5 cancer cells and was significantly higher in 3AO,A2780 and HO8910 than in K562.ULBP4 would be the main NKG2 D ligands in ovarian cancer cells.The killing capacity of CD8+CIK against K562 and 5 cancer cells was significantly far stronger than that of CIK cells(p<0.001),which suggested that CD8+CIK cells could markedly kill cancer cells.OVCAR3 and SKOV3 were weak at binding NKG2D-Fc recombinant protein,but could be also markedly destroyed by CD8+CIK.That may be because OVCAR3 and SKOV3 express HLA-I and OCT4 / Sox2,and could be recognized by TCR of CD8+CIK and then killed.Conclusions 1.The high purity CD8+CIK cells could be collected from CIK cells by MACS,and cultured and expanded massively.The expanded CD8+CIK cells were almost CD3+CD 8+T cells and CD8+NKG2D+T cells,and contained some memory T-lymphocytes.Therefore this could overcome the problem of individual difference of CIK cells in CIK therapeutic preparation.2.CD8+CIK cells could be activated not only through NKG2 D receptor but also through specific TCR of OCT4 and Sox2,which showed CD8+CIK was bifunctional in immune response activity.Since OCT4 and Sox2 were fully accepted as the specific CSC markers,our results showed that there were CSC specific T cells in CD8+CIK cells,and anti-OCT4 T cells in CD8+CIK have not been reported.3.CD8+CIK cells possessed significantly far stronger killing capacity against K562 and ovarian cancer cells than CIK cells.Their bifunctional anti-cnacer capacity made an even wider anti-cancer spectrum.That suggested that CD8+CIK cells could provide a highly-effective way of adoptive cell immunotherapy for ovarian cancers.