title: A robust human T-cell culture method suitable for monitoring CD8+ and CD4+ T-cell responses from cancer clinical trial samples.
abstract: Many tumor antigenic determinants have been identified and included in cancer clinical trials. Due to low T-cell frequencies even after vaccination, few T-cell responses can be revealed ex vivo without in vitro stimulation. Various expansion protocols have been employed for this purpose and the outcomes tend to be quite variable, partly due to the high complexity involved in the protocols. Here we systematically studied various common culture conditions including sera, cytokines and feeders and describe a reliable "bulk" culture method that is robust, simpler and more economical. We demonstrated that fetal calf serum (FCS) supported T-cell proliferation better than multiple commercially available pooled human AB sera. IL-2 is critical in our cultures, but IL-7, IL-15 and anti-CTLA-4 in combination with IL-2 did not further enhance T-cell expansion. We typically achieve more than a 40-fold expansion within a 10-day culture period for antigen-specific T cells measured by HLA-peptide tetramer before and after culture. This method was not only validated by multiple operators as a standard operating procedure for monitoring T-cell responses but was also successfully used for discovering novel CD8+ and CD4+ T cells specific to previously unknown epitopes from the NY-ESO-1 tumor antigen.
authors.affiliation: Ludwig Institute for Cancer Research, Melbourne Branch, Austin Health, Heidelberg VIC 3084, Australia.
authors.forename: Heather M
authors.initials: HM
authors.lastname: Jackson
authors.forename: Nektaria
authors.initials: N
authors.lastname: Dimopoulos
authors.forename: Qiyuan
authors.initials: Q
authors.lastname: Chen
authors.forename: Tina
authors.initials: T
authors.lastname: Luke
authors.forename: Tsin
authors.initials: T
authors.lastname: Yee Tai
authors.forename: Eugene
authors.initials: E
authors.lastname: Maraskovsky
authors.forename: Lloyd J
authors.initials: LJ
authors.lastname: Old
authors.forename: Ian D
authors.initials: ID
authors.lastname: Davis
authors.forename: Jonathan
authors.initials: J
authors.lastname: Cebon
authors.forename: Weisan
authors.initials: W
authors.lastname: Chen
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D000938:Antigen-Presenting Cells / Q000166:cytology / Q000276:immunology; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology*; D060908:CTLA-4 Antigen; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002986:Clinical Trials as Topic; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D019409:Interleukin-15 / Q000494:pharmacology; D015851:Interleukin-7 / Q000494:pharmacology; D007700:Kinetics; D009369:Neoplasms / Q000097:blood / Q000276:immunology*; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D015203:Reproducibility of Results; D044967:Serum
chemical_list: D000906:Antibodies; D015703:Antigens, CD; D000943:Antigens, Differentiation; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D000939:Epitopes; D019409:Interleukin-15; D015851:Interleukin-7; D010446:Peptide Fragments