title: A reproducible method for the enumeration of functional (cytokine producing) versus non-functional peptide-specific cytotoxic T lymphocytes in human peripheral blood.
abstract: One of the most difficult laboratory challenges in the field of therapeutic cancer vaccines has been the development of uncomplicated/reproducible methods for the quantification of vaccine immunization efficacy in peripheral blood of cancer patients. Existing methods are limited by lack of functional information (tetramers), difficulties with standardization/reproducibility [enzyme-linked immunosorbent spot (ELISPOT)] and reliance on endogenous (sample-specific) antigen presentation (cytokine flow cytometry). Herein we present a reproducible method utilizing an artificial antigen-presenting cell platform for flow cytometry-based quantification of the frequency and activation status of peptide-specific cytotoxic T lymphocytes. The methodology [currently presented for cytomegalovirus human leucocyte antigen (HLA)-A2 cognant peptide antigens] allows simultaneous ex vivo quantification of activated (cytokine-producing) and inactive tetramer-positive T cells following HLA class I/peptide/CD28 stimulation independent of endogenous antigen presentation. The simplicity and reliability of the assay provide for high-throughput applications and automation. The utility and application of this method are discussed.
authors.affiliation: Hematology/Oncology Department, Mayo Clinic, Rochester, MN 55905, USA. markovic.svetomir@mayo.edu
authors.forename: S N
authors.initials: SN
authors.lastname: Markovic
authors.forename: W K
authors.initials: WK
authors.lastname: Nevala
authors.forename: C B
authors.initials: CB
authors.lastname: Uhl
authors.forename: E
authors.initials: E
authors.lastname: Celis
authors.forename: D J
authors.initials: DJ
authors.lastname: McKean
mesh_terms: D000938:Antigen-Presenting Cells / Q000276:immunology*; D000956:Antigens, Viral / Q000276:immunology; D019496:Cancer Vaccines / Q000276:immunology*; D018929:Cell Culture Techniques; D003587:Cytomegalovirus / Q000276:immunology; D003601:Cytotoxicity Tests, Immunologic; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D008213:Lymphocyte Activation; D018655:Lymphocyte Count; D012680:Sensitivity and Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology* / Q000276:immunology
chemical_list: D000956:Antigens, Viral; D019496:Cancer Vaccines; D007371:Interferon-gamma