title: Quantitation of antigen-reactive T cells in peripheral blood by IFNgamma-ELISPOT assay and chromium-release assay: a four-centre comparative trial.
abstract: The ELISPOT assay is increasingly being used for the monitoring of the induction of antigen-reactive T cells in cancer vaccination trials. In order to evaluate the reliability of T cell frequency analysis with the ELISPOT assay, a comparative study was performed in four European laboratories. Six samples from healthy subjects were analyzed for the frequency of influenza-reactive CD8+ T cells in peripheral blood mononuclear cells (PBMC) by IFNgamma-ELISPOT assay. In addition, one laboratory determined cytotoxic T cell precursor (CTL) frequencies in these samples by limiting dilution chromium-release assay (LDA), and three laboratories performed a variant of the LDA, the multiple microculture assay (MMA). Consistent frequencies of influenza peptide-reactive T cells were obtained with the ELISPOT assay in all four laboratories. The numbers detected by ELISPOT assay correlated closely with those determined by LDA. In contrast, the frequencies obtained with the MMA differed considerably and showed little correlation with the other two assays. This study shows that it is possible to use the ELISPOT assay to determine with reliability antigen-reactive T cells in a multicenter setting. We suggest that this assay may be suitable for monitoring cancer vaccine trials.
authors.affiliation: Department of Medicine III, Hematology, Oncology and Transfusion Medicine, Klinikum Benjamin Franklin, Free University Berlin, Berlin, Germany. scheibenbogen@ukbf.fu-berlin.de
authors.forename: C
authors.initials: C
authors.lastname: Scheibenbogen
authors.forename: P
authors.initials: P
authors.lastname: Romero
authors.forename: L
authors.initials: L
authors.lastname: Rivoltini
authors.forename: W
authors.initials: W
authors.lastname: Herr
authors.forename: A
authors.initials: A
authors.lastname: Schmittel
authors.forename: J C
authors.initials: JC
authors.lastname: Cerottini
authors.forename: T
authors.initials: T
authors.lastname: Woelfel
authors.forename: A M
authors.initials: AM
authors.lastname: Eggermont
authors.forename: U
authors.initials: U
authors.lastname: Keilholz
mesh_terms: D000956:Antigens, Viral / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D002860:Chromium Radioisotopes; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D054303:HIV Reverse Transcriptase / Q000276:immunology; D015789:HLA-A2 Antigen / Q000276:immunology; D006801:Humans; D016056:Immunodominant Epitopes / Q000276:immunology; D007201:Indicator Dilution Techniques; D007371:Interferon-gamma / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014763:Viral Matrix Proteins / Q000276:immunology
chemical_list: D000956:Antigens, Viral; D002860:Chromium Radioisotopes; D018984:Epitopes, T-Lymphocyte; D015789:HLA-A2 Antigen; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D014763:Viral Matrix Proteins; C098952:influenza virus membrane protein (58-66); D007371:Interferon-gamma; D054303:HIV Reverse Transcriptase