The Experimental Study On The Influence Of ShRNA Interference In Helios Expression Of Tregs And Anti-tumor Activity Of TIL

Background:With the potent tumor antigen specificity,the adoptive cell immunotherapy of TIL is regarded as one of the most promising immunotherapeutic methods.Although many breakthroughs of the adoptive cell immunotherapy for kinds of tumor have been made,the phenomenon of "immune escape" of tumor greatly affected the treatment effects,which is still an obstacle in the immunotherapy of tumor.Tregs is a subset of T cells with the function of immunomodulation,which is capable of suppressing anti-tumor response and thus impair the effects of immunotherapy.Therefore,how to eliminate the effect of immunosuppression of Tregs has always been the focus and difficulty in immunotherapy researches.Helios(IKZF2),a member of the Ikaros transcription factor family,is found closely related to FoxP3 transcription factor which controls the growth and function of Tregs.Simultaneously,it was found that within Tregs,a high level Helios protein expression is able to maintain its immunosuppression function,while lack of Helios can cause instability of Treg cell phenotype,leading to down regulation of FoxP3 expression,which then results in inability of providing immunosuppressive effect.Therefore,we designed shRNA lentivirus vector that target on Helios gene.With the help of RNA interfere technique mediated by lentivirus vector to silence Helios gene,immunosuppressive factors can be eliminated and thus strengthen the anti-tumor activity of TIL.Objectives:To establish the method of separating TIL and tumor cells from malignant ascites effectively,design shRNA lentivirus vector that target on Helios gene,and explore the influence of interfering Helios expression on specific anti-tumor activity of TIL in vitro and in vivo,utilizing shHelios lentivirus to transfect TIL cell,and interfering the number and function of Treg cell.Methods:1)PBMC of healthy donors and gastric cancer patients,separate TIL and autologous tumor cells were obtained from malignant ascites,and then carry on activation and expansion in vitro.To explore and compare the subsets characteristics of PBMC and TIL by flow cytometry.Detect and compare the cellular phenotypes of TIL before and after activation and expansion in vitro.To explore its anti-tumor activity against autologous and xenogenous tumor cells.2)design shRNA lentivirus vector targeted on Helios gene,explore and comfirm the best MOI value for transfecting TIL and detect the best shHelios lentivirus vector with the effect of silencing Helios.3)applies fibronectin-centrifugation approach to transfect TIL,flow cytometry to detect TIL phenotype,ELISA to detect IFN-γ,TGF-β,IL-10 and IL-35 that TIL secretes and LDH release assay to detect anti-tumor activity of blank-TIL,negative-TIL and shHelios-TIL on autologous tumor cells in vitro.4)establish chmice subcutaneous tumor model,inject different groups of TIL cells through vena caudalis,compare and analyze the influence of shHelios lentivirus transfection on the anti-tumor activity of TIL in vivo.Results:1)this experiment successfully established the technical method of effectively separating TIL and autologous tumor cells from malignant ascites as well as culturing,activating and expanding them in vitro.The proportion of CD4+ T cell and Treg cell were imporved and the CD8+ T cell/Treg ratio were decresed(both the P<0.05).TIL has an apparent specific anti-tumor activity on autologous tumor cells.2)the interfering segment we deigned and synthesized was successfully connected into the specific site of double-enzyme-digestion linearized expression vector,which was correct according to the sequencing result,and lentivirus prepared after transfecting it into 293T cell corresponded to experimental titer;the best MOI of transfecting TIL was confirmed to be 150;the best lentivirus with silencing effect was comfirmed and named pLKD-eGFP-shHelios.3)the expression of Helios and FoxP3 mRNA expression of shHelios-TIL group decreased eminently(both the P<0.05);the ratio Helios+ in CD3+CD4+FoxP3+T cells reduced significantly(P<0.05);the percentage of CD8+T cells was increased in the shHelios-TIL group(P<0.05);the percentage of CD3+CD4+FoxP3+T cells was reduced in the shHelios-TIL group(P<0.05);quantity of IFN-y in shHelios-TIL group was improved,while that of TGF-β and IL-10 were reduced(both the P<0.05);LDH result showed that specific anti-tumor activity on autologous tumor cells was enhanced in shHelios-TIL group(P<0.05);this experiment successfully established chmice subcutaneous tumor model,and the result showed that the anti-tumor effect of injection through vena caudalis was the strongest in shHelios-TIL group,and could inhibit tumor growth more significantly compared to control group of blank-TIL and negative-TIL(P<0.05).Conclusions:1)Relative to peripheral blood,there are more effector T cells activated with HLA-DR+ in TIL separated from malignant ascites,in the meantime,the ratio of Treg increased while the ratio of CD8+ T cell/Treg reduced,showing that there was significant immunosuppressive factors.2)TIL has a significantly specific anti-tumor activity on autologous tumor cells;3)Lentivirus pLKD-eGFP-shHelios transfected TIL using fibronectin-centrifugation approach,which could effectively interfere expression of Helios gene,intervene number and function of Treg,and strengthen specific anti-tumor activity of TIL in vitro and in vivo.This can act as a new strategy to treat terminal gastric caner with malignant ascites.