The Culture Of Tumor-reactive TGF-β Insensitive CTL And Its Application In Immunotherapy Of Prostate Cancer

ObjectivesCytotoxic T lymphocytes(CTL) are the most important effect cell in anti-tumor immune response in vivo. CTL 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 naive T cells but memory T cell. Cytotoxic T lymphocytes(CTL) can be induced by DCs which can provide co-stimulating signal that is indispensable to activation of T cells. Cancer cells produce large amounts of TGF-β, which is a potent immunosuppressant . The high levels of TGF-βproduced by cancer cells have a negative effect on surrounding cells, including the host immune cells. As a result, cancer cells are able to escape the host's immune surveillance program, leading to tumor progression and metastasis .Material and methodsThe present study involved isolation and generation of dendritic cells, CD4+ T cells, and CD8+ T cells from patients with prostate cancer. Next, We harvested tumor cells from patients with advanced prostate cancer undergoing radical prostatectomy to generate the primary cancer cell cultures but failed to subculture the cells. Because of the limited quantity of prostate tumor, we primed the CTL with PC3 cell lysates and dendritic cells. Tumor reactive CTL will be generated by isolate lymphocytes cocultured with dendritic cells in the presence of lysates of PC-3 cells and IL-2,IL-4,GM-CSF. Then, we will render these cells insensitive to TGF-βby transfection with the TβRⅡDN-tk construct. The control CTL will be transfected with the TRANSglytk vector. Then we performed adoptive transfer of autologous tumor-reactive TGF-βinsensitive CTL into these immuno-deficient mice bearing human prostate tumors. Finally, the effectiveness of tumor-reactive TGF-βinsensitive CTL in vivo were determined using FACS, immunofluorescence, western-blot, MTT and so on. ResultsThe mixed culture of lymphocytes and DCs made the autologous tumor-reacive CTL cells. 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. 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.ConclusionOur experiment made a foundation for further application of tumor-reactive TGF-βinsensitive CTL in adoptive immunotherapy of prostate tumors.