title: Ex vivo generation and expansion of anti-tumor cytotoxic T-cell lines derived from patients or their HLA-identical sibling.
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-lines 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.
authors.affiliation: Immunology Laboratory and Pediatric Hematology/Oncology, IRCCS Policlinico San Matteo, University of Pavia, Piazzale Golgi, 27100 Pavia, Italy. d.montagna@smatteo.pv.it
authors.forename: Daniela
authors.initials: D
authors.lastname: Montagna
authors.forename: Roberta
authors.initials: R
authors.lastname: Schiavo
authors.forename: Nadia
authors.initials: N
authors.lastname: Gibelli
authors.forename: Paolo
authors.initials: P
authors.lastname: Pedrazzoli
authors.forename: Roberto
authors.initials: R
authors.lastname: Tonelli
authors.forename: Sara
authors.initials: S
authors.lastname: Pagani
authors.forename: Elisa
authors.initials: E
authors.lastname: Assirelli
authors.forename: Franco
authors.initials: F
authors.lastname: Locatelli
authors.forename: Andrea
authors.initials: A
authors.lastname: Pession
authors.forename: Vittorio
authors.initials: V
authors.lastname: Fregoni
authors.forename: Elisa
authors.initials: E
authors.lastname: Montini
authors.forename: Gian Antonio
authors.initials: GA
authors.lastname: Da Prada
authors.forename: Salvatore
authors.initials: S
authors.lastname: Siena
authors.forename: Rita
authors.initials: R
authors.lastname: Maccario
mesh_terms: D000293:Adolescent; D000328:Adult; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D005260:Female; D006650:Histocompatibility Testing; D006801:Humans; D008297:Male; D008875:Middle Aged; D009369:Neoplasms / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*