title: A novel strategy for rapid and efficient isolation of human tumor-specific CD4(+) and CD8(+) T-cell clones.
abstract: Adoptive therapy with antigen-specific T cells is a promising approach for the treatment of infectious diseases and cancer. However, cloning of antigen-specific T cells by the traditional approach of limiting dilution is a time-consuming, laborious, and inefficient process. Here, we describe a novel flow cytometric strategy for rapid isolation of human tumor antigen-specific T-cell clones by using T-cell receptor (TCR) Vbeta antibodies in combination with carboxyfluorescein succinimidyl ester (CFSE)-based proliferation assay. The CFSE dilution following antigen stimulation identified proliferating antigen-specific T cells, and the TCRVbeta antibodies allowed distinguishing T cells at the clonal level from a heterogeneous T-cell population. This method of TCRVbeta/CFSE dilution was used for the isolation of four different human lymphoma and melanoma-specific CD4(+) and CD8(+) T-cell clones reactive against defined and undefined tumor antigens. Isolated tumor-specific T-cell clones could be expanded to large numbers ex vivo while maintaining phenotype, function, and tumor antigen specificity. The method was simple, efficient, and reproducible, and may have potential application for the development of adoptive immunotherapeutic strategies.
authors.affiliation: Department of Lymphoma and Myeloma, The University of Texas M D Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: Seung-Tae
authors.initials: ST
authors.lastname: Lee
authors.forename: Shujuan
authors.initials: S
authors.lastname: Liu
authors.forename: Laszlo
authors.initials: L
authors.lastname: Radvanyi
authors.forename: Pariya
authors.initials: P
authors.lastname: Sukhumalchandra
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Molldrem
authors.forename: Eric D
authors.initials: ED
authors.lastname: Wieder
authors.forename: Patrick
authors.initials: P
authors.lastname: Hwu
authors.forename: Yong-Jun
authors.initials: YJ
authors.lastname: Liu
authors.forename: Larry W
authors.initials: LW
authors.lastname: Kwak
authors.forename: Gregory
authors.initials: G
authors.lastname: Lizée
authors.forename: Sattva S
authors.initials: SS
authors.lastname: Neelapu
mesh_terms: D000951:Antigens, Neoplasm / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D002999:Clone Cells / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016219:Immunotherapy, Adoptive; D008223:Lymphoma / Q000276:immunology; D058965:MART-1 Antigen; D008545:Melanoma / Q000276:immunology; D009363:Neoplasm Proteins / Q000276:immunology; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000276:immunology*
chemical_list: D000951:Antigens, Neoplasm; D058965:MART-1 Antigen; C548667:MLANA protein, human; D009363:Neoplasm Proteins; D016693:Receptors, Antigen, T-Cell, alpha-beta