| Objective Cancer cells produce large amounts of TGF-β,which is a potent immunosuppressant.As a result,cancer cells are able to escape the host's immune surveillance program,leading to tumor progression and metastasis.Tumor immunotherapy has been focused largely on the enhancement of host immune responses against cancer.Tumor-derived TGF-β,however,is capable of inhibiting the host immune system,especially the cytotoxic T lymphocyte(CTL)at the site of tumor growth.Therefore,TGF-βis an attractive target for anti-cancer therapy.We render immune cells insensitive to TGF-β,these immune cells should be able to reject the tumor.On the other hand,CTLs are the most important effect cell in anti-tumor immune response in vivo.CTLs must be activated through the presentation of appropriate antigen by antigen-present cells(APC).Dendritic cells(DC)are the potentest APC.As we known,they can activate not only na(?)ve T cells but memory T cell.CTL can be induced by DCs which can provide co-stimulating signal that is indispensable to activation of T cells.Our hypothesis states that if we render CTLs insensitive to TGF-β,these immune cells should be able to reject the tumor.We are willing to developed a lentivirus mediated gene transfer program incorporating a herpes simplex virus thymidine kinase(HSV-tk)in our dominant negative TGFβtypeⅡreceptor(TβRⅡDNglytk)expression vector.Methods We developed a lentivirus mediated gene transfer program incorporating a herpes simplex virus thymidine kinase(HSV-tk)in our dominant negative TGF-βtypeⅡreceptor(TβRⅡDN)expression vector.First we construct the Lentiviral plenti6/v5-D-TOPO vector containing TβRⅡDnglytk.TβRⅡDN were obtained from the plasmid pAdTrack-CMV by PCR,then connected with HSV-tk by recombinant PCR.After the agarose gel electrophoresis was performed,the TβRⅡDNglytk was retrieved,and then were cloned into Lentiviral vector pLenti6/V5-TOPO by TOPO Cloning technique.The present study also involved isolation and generation of dendritic cells,T cells from patients with bladder cancer.We primed the CTL with tumor cell lysates and dendritic cells.Tumor specific CTL will be generated by isolate lymphocytes cocultured with dendritic cells in the presence of lysates of tumor cells and IL-2,IL-4,GM-CSF.These CTLs were rendered insensitive to TGF-βby introducing the dominant negative TGF-βtypeⅡreceptor with the herpes simplex virus thymidine kinase(HSV-tk)connected in the intra-cellular domains (TβRⅡDNglytk)in the lentiviral construct.The following tests will be carried out Western blot analysis for SMAD-2 phosphorylation was peformed with different types of CTLs to verify that the TβRⅡDNglytk(and TRANSglytk)transfected CTLs are insensitive to TGF-β.CTLs transfected with TRANSglytk will be used as the control.Results 1.The present experiment demonstrate a successful cloning of Tβ3RⅡDNglytk(TRANSglytk)into the Lentiviral vector pLenti6/V5-TOPO.The Sequencing map of plenti6/v5-D-TOPO-TβRⅡDNglytk vector accorded with theoretic analyses,and a successful construction Lentivirals contain TβRⅡDNglytk (TRANSglytk)by 293FT cells.2.Tumor specific CTLs were generated by isolate lymphocytes cocultured with dendritic cells in the presence of lysates of tumor cells and IL-2,IL-4,GM-CSF.3.Since Smad-2 phosphorylation is a direct result of activation of TGF-βactivation,an absence of Smad-2 phosphorylation following TGF-βtreatment of CTL will be an indication that the cells are insensitive to TGF-β.The effectiveness of tumor-specific TGF-βinsensitive CTL were determined using FACS, immunofluorescence,western-blot,MTT and so on.4.The transfection of Lentiviral containd TβRⅡDNglytk(TRANSglytk) were innocuity to the CTLs.CTLs which express TβRⅡDNglytk could be killed by GCV,and the control were still alive.Conclusion Our experiment made a foundation for further application of tumor-specific TGF-βinsensitive CTL in adoptive immunotherapy of tumors.These CTL will be used for adoptive transfer in the subsequent studies.
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