Development Of Adoptive Transfer Of Lentivirus Mediated TGF-β Insensitive Tumor Specific CTL In A Xenograft System With Human Prostate Carcinoma Grown In Immunodeficient Mice

With the development of tumor immunology,the role of the human immune system in the pathogenesis and advance is being payed more and more attention by scientists.Since 1950s,Thomas studied the evolutionary mechanism of body cellular immunity,and found that the lower expression of the tumor-antigen in cancer cells or damaged body cellular immunity played an important role in tumorigenesis. Whereafter,Burnet established immune surveillance theory,and according to this theory,immune system can find and eliminate abnormal cells expressing new antigens so as to maintain immune system homeostasis.But there are still a certain proportion of primary neoplasms have the capacity of escaping the surveillance of immune system and are apt to metastasis and recurrence.Among many factors that are related to tumor immune escape,TGF-βsecreted by tumor cell is one of the most strong and immunosuppressive molecules.TGF-βis a family of pleiotropic growth factors with diverse functions.A common property of advanced cancer is the over-production of TGF-β.The high levels of TGF-βproduced by cancer cells have a negative effect on surrounding cells,including the host immune cells.TGF-βis a potent tumor-induced immuno-suppression.It can suppress the differentiation of CTL and decrease the production of immunoregulation cytokines,including the important cell factor IL-12 secreted by monocytes.Therefore,we hypothesize that the use of TGF-βinsensitive immune cells may be considered as a strategy for cancer therapy.In 2005,We start the study and collaborated with professor Lee CH and Zhang Q whose animal studies had showed that adoptive transfer of tumor-reactive TGF-β-insensitive CD8+ T cells to tumor-bearing mice was able to completely reject established mouse tumors with no apparent toxicity to the hosts.We divided the research into 4 steps.At first,we cloned dominant negative TGF-βtypeⅡreceptor gene(TβRⅡDN) and herpes simplex virus thymidine kinase(HSV-tk)gene from pMIG-TβRIIDN (MSCV-RIIDN-IRES-GFP)and pAdTrack-CMV-TK plasmid using PCR respectively. Then we connected these two genes using recombinant PCR technique,so we got the fusion gene containing TβRⅡDN and HSV-tk.In 2006,we cloned a lentiviral vector (pLenti6/V5-D-TOPO(?)-TβRⅡDNglytk)which contained a dominant negative receptor for TGF-βtypeⅡreceptor(TβRⅡDN)and herpes simplex virus thymidine kinase(HSV-tk).At the same time,we constructed the control lentiviral vector containing TRANSglytk gene.The lentiviral construct containing the TβRⅡDN-tk vector is a method of choice for our future gene therapy program aimed at the blockade of TGF-βsignaling in ummune cells.Secondly,we established tumor xenogrsfts from patients with PCa.At the time of radical retropubic prostatectomy due to PCa,we collected fresh PCa specimens and established xenografts PCa tumors in immuno-deficient(BALB/C-nu/nu)mice. When the size of xenografts reach 1.5cm,remove the xenografts and do the implantation again.We had done this tissue mass passages in mice for 3 generations and obtained 14 mice with xenografts.Total 12 of them were divided into 2 group and used as the immunotherapy target in vivo,and the other two were used in primary culture and extraction of tumor antigen stimulating the CTL.Thirdly,Generation of autologous tumor-reacive CTL cells:Peripheral blood will be collected through the vein.Peripheral blood mononuclear cells(PBMCs)were obtained from the diluted blood by Ficoll-Paque Plus gradient centrifugation.Freshly isolated monocytes were differentiated into dendritic cells by plating them in flasks cultured in RPMI-1640 with 10%BSA.After 2 hours,non-adherent cells will be removed and cultured as lymphocytes.The adherent monocytes willl be treated with GM-CFS(500ng/ml)IL-4(500IU/ml)in RPMI-1640 supplemented with 10%BSA for 4 days.Then the DCs were stimulated by the protein extracts tumor tissue.48 hours later,the stimulated DCs were added to the cultured lymphocytes.Then the mixed culture of lymphocytes and DCs made the autologous tumor-reacive CTL cells.Fourthly,Establish the efficacy of transfection using the lentivirus-based gene transfer method to generate TGF-βinsensitive CTL cells:Tumor reactive CTL were infected with the lentiviral particle containing TβRⅡDNglytk and TRANSglytk. Infection efficiency were assessed for V5-epitope using anti-V5-FITC.Western blot analysis for SMAD-2/3 phosphorylation were performed with different types of CTL. HSV-tk function was tested using MTT after GCV being added.The last step was to perform adoptive transfer of tumor-reactive TGF-β-insensitive CTL in immunodeficient mice bearing xenograft tumor from human PCa. When the subcutaneous xenograft tumors reached approximately 1.0 cm in diameter, we divided 12 mice bearing xenograft tumor into 2 groups randomly.They received adoptive transfer of the two kinds of CTL respectively via the tail veins.Thirty days after the adoptive transfer,mice were sacrificed by cervical dislocation.The tumor tissue were isolated,weigthed,and taken photos.The results showed that adoptive transfer of tumor-reactive TGF-β-insensitive CTL in immunodeficient mice bearing xenograft tumor from human Prostate carcinoma can significantly suppress the growth of the xenograft tumor.In vitro, GCV can eliminate the infected CTL that expressed TβRⅡDNglytk or TRANSglytk genes.