Successful ex-vivo priming and long-term maintenance of anti-tumor cytotoxic T-cell (CTL) lines are preliminary conditions for their use in approaches of adoptive immunotherapy for patients with cancer. We describe the results of a novel procedure for generating in vitro anti-tumor CTL using CD8-enriched peripheral blood mononuclear cells (PBMC) and dendritic cells (DC), pulsed with irradiated tumor cells (TC) as source of tumor antigen. Eight patients were enrolled in our study: 4 sarcoma, 2 renal cell carcinoma, 1 ovarian carcinoma and 1 breast carcinoma. Ten anti-tumor CTL-lines cytotoxic towards patient TC were generated. Five CTL-lines were obtained using both DC and PBMC from the patients (autologous setting). For 5 CTL-lines, DC derived from an HLA-identical sibling were employed (allogeneic setting): patients or siblings PBMC were used to generate CTL-Iines in 2 and 3 cases, respectively,. After tumor-specific rounds of stimulation, followed by antigen-independent cycle of expansion, CTL-lines obtained in both autologous and allogeneic setting showed an expansion of the absolute number of cultured cells. In 6 of 10 CTL-lines, the majority of effector cells (>70%) were CD3+/CD8+, while in the remaining 4, 40-70% of effector cells were CD3+/CD4+. Both CD8+ and CD4+ T cells displayed anti-tumor cytotoxic activity. Spectratyping analysis of the TCR-Vbeta subfamilies revealed a preferential expansion of oligoclonal populations in 18 of 24Vbeta subfamily. Altogether these results demonstrate that our experimental approach is suitable for efficiently generating and expanding anti-solid tumor CTL to be used for adoptive immunotherapy. (C) 2004 Wiley-Liss, Inc.
Montagna, D., Schiavo, R., Gibelli, N., Pedrazzoli, P., Tonelli, R., Pagani, S., Assirelli, E., Locatelli, F., Pession, A., Fregoni, V., Montini, E., Da Prada, G. A., Siena, S., Maccario, R., Ex vivo generation and expansion of anti-tumor cytotoxic T-cell lines derived from patients or their HLA-identical sibling, <<INTERNATIONAL JOURNAL OF CANCER>>, 2004; 110 (1): 76-86. [doi:10.1002/ijc.20081] [https://hdl.handle.net/10807/260392]
Ex vivo generation and expansion of anti-tumor cytotoxic T-cell lines derived from patients or their HLA-identical sibling
Locatelli, Franco;
2004
Abstract
Successful ex-vivo priming and long-term maintenance of anti-tumor cytotoxic T-cell (CTL) lines are preliminary conditions for their use in approaches of adoptive immunotherapy for patients with cancer. We describe the results of a novel procedure for generating in vitro anti-tumor CTL using CD8-enriched peripheral blood mononuclear cells (PBMC) and dendritic cells (DC), pulsed with irradiated tumor cells (TC) as source of tumor antigen. Eight patients were enrolled in our study: 4 sarcoma, 2 renal cell carcinoma, 1 ovarian carcinoma and 1 breast carcinoma. Ten anti-tumor CTL-lines cytotoxic towards patient TC were generated. Five CTL-lines were obtained using both DC and PBMC from the patients (autologous setting). For 5 CTL-lines, DC derived from an HLA-identical sibling were employed (allogeneic setting): patients or siblings PBMC were used to generate CTL-Iines in 2 and 3 cases, respectively,. After tumor-specific rounds of stimulation, followed by antigen-independent cycle of expansion, CTL-lines obtained in both autologous and allogeneic setting showed an expansion of the absolute number of cultured cells. In 6 of 10 CTL-lines, the majority of effector cells (>70%) were CD3+/CD8+, while in the remaining 4, 40-70% of effector cells were CD3+/CD4+. Both CD8+ and CD4+ T cells displayed anti-tumor cytotoxic activity. Spectratyping analysis of the TCR-Vbeta subfamilies revealed a preferential expansion of oligoclonal populations in 18 of 24Vbeta subfamily. Altogether these results demonstrate that our experimental approach is suitable for efficiently generating and expanding anti-solid tumor CTL to be used for adoptive immunotherapy. (C) 2004 Wiley-Liss, Inc.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.