Generation of Tumor-Specific CD8+ Cytotoxic T Lymphocytes and CD4+ Th1 Helper T Cells by Transfer of T-Cell Receptor Genes Isolated from a WT1-Specific CD8+ Cytotoxic T-Lymphocyte Clone.

Abstract

Background: Cytotoxic T lymphocytes (CTLs) and T-helper type 1 (Th1) cells undoubtedly play a crucial role in the eradication of tumors in vivo. However, the production of Th1 cytokines such as IL-2 and IFN-γ is markedly suppressed in the majority of tumor-bearing hosts. Such defects in Th1-mediated immunity in cancer patients have made it difficult to induce tumor-specific CTLs that promote tumor rejection. Adoptive transfer of tumor-specific CTLs and Th1 cells can overcome the difficulty to induce tumor-specific immune response in cancer patients; however, the generation and expansion of tumor-specific CTLs and Th1 cells in vitro are not easy. In the present study, to overcome this problem, we isolated TCR-α and -β chain genes from a WT1-specific CD8+ CTL clone, which had been shown to exert strong cytotoxicity against hematopoietic malignancies and solid tumors in an HLA-A24-restricted manner, and transduced them into nonspecifically activated human CD8+ and CD4+ T cells. Consequently, both CD8+ and CD4+ T cells appeared to acquire WT1-specific function in an HLA-A24-restricted manner.Methods: A WT1 peptide (CMTWNQMNL)-specific CD8+ CTL clone, TAK-1, was established as reported previously (Blood 95:286, 2000). TAK-1 exerts cytotoxicity against variety of tumor cells including leukemia, myeloma, and lung cancer cells but not against normal cells in an HLA-A24-restricted manner. cDNAs encoding TCR-α and -β chain genes were amplified from cDNA of TAK-1 by RT-PCR. TCR-α and -β chain cDNAs were inserted into the plasmid vector. Preparation of lentiviral vectors for transduction of TCR-α and -β chain cDNAs was performed as described previously (Cancer Res 64:1490, 2004). Peripheral blood CD4+ and CD8+ T cells isolated from healthy individuals were cultured with anti-CD3 mAb and retronectin and then infected twice with lentivirus vectors. The infected cells were expanded by culture in the presence of IL-2, IL-12, IFN-γ and anti-IL-4 mAb. Cytotoxicity of CTLs against WT1-peptide-loaded cells and various human tumor cells was examined by a standard 51Cr-release assay. Recognition of tumor cells by Th1 cells was examined by measuring IFN-γ production by ELISA.Results: CD4+ T-cell line (CD4-TCR) and CD8+ T cell line (CD8-TCR) expressing TCR-α and -β chains of TAK-1 were established. Both CD4-TCR and CD8-TCR cells exerted cytotoxicity against WT1 peptide-loaded HLA-A24-positive but not -negative cells. CD8-TCR cells appeared to be cytotoxic against human tumor cells including leukemia, myeloma, and lung cancer cells in an HLA-A24-restricted manner, but did not show any cytotoxicity against HLA-A24-positive normal cells. CD4-TCR cells produced IFN-γ in response to stimulation with HLA-A24-positive but not -negative leukemia cells.Conclusion: The present data demonstrate the functional reconstitution of CD4+ as well as CD8+ T cells by transfer of the αβ TCR complex of a WT1-specific CD8+ CTL clone. Since WT1 is a universal tumor-associated antigen, transfer of TCR genes of WT1-specific CTLs into CD4+ and CD8+ T cells would be useful for Th1-based immunotherapy of various malignancies.