title: Flow cytometric measurement of intracellular cytokines detects immune responses in MUC1 immunotherapy.
abstract: The detection of tumor-specific T cells in immunized cancer patients usually relies on lengthy and difficult CTL assays; we now report on flow cytometry to detect the intracellular cytokines interleukin 2 (IL-2), IL-4, IFN-gamma, and tumor necrosis factor alpha (TNF-alpha) produced by CD4+CD69+ and CD8+CD69+ activated T cells after MUC1 antigen stimulation. Peripheral blood mononuclear cells were obtained from 12 patients with adenocarcinoma injected with mannan-MUC1; cells were exposed in vitro for 18 h to MUCI peptide in the presence of CD28 monoclonal antibody and Brefeldin; permeabilized cells were used for the expression of cytokines. After stimulation in vitro with MUC1-variable number of tandem repeats peptides, CD8+CD69+ T cells from all immunized patients generated 3-9 times higher levels of TNF-alpha(P < 0.038) and IFN-gamma (P <0.010) than did cells from 12 normal subjects; minor increases in IL-4 occurred. By contrast, CD4+CD69+ cells showed no overall alteration in TNF-alpha and IFN-gamma cytokine production, although in some patients, their measurement was informative; the measurement of IL-2 was not useful in either CD4+CD69+ or CD8+CD69+ cells. We conclude that in MUC1-immunized patients, the measurement of TNF-alpha and IFN-gamma in activated CD69+CD8+ T cells may be indicative of their immune status.
authors.affiliation: Immunology and Vaccine Laboratory, The Austin Research Institute, Victoria, Australia.
authors.forename: V
authors.initials: V
authors.lastname: Karanikas
authors.forename: J
authors.initials: J
authors.lastname: Lodding
authors.forename: V C
authors.initials: VC
authors.lastname: Maino
authors.forename: I F
authors.initials: IF
authors.lastname: McKenzie
mesh_terms: D000230:Adenocarcinoma / Q000628:therapy; D016207:Cytokines / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D007114:Immunization; D007167:Immunotherapy; D007252:Influenza Vaccines / Q000494:pharmacology; D007371:Interferon-gamma / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D008351:Mannans / Q000276:immunology; D018396:Mucin-1 / Q000235:genetics / Q000276:immunology*; D010835:Phytohemagglutinins / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D013745:Tetanus Toxoid / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D016207:Cytokines; D007252:Influenza Vaccines; D007376:Interleukin-2; D008351:Mannans; D018396:Mucin-1; D010835:Phytohemagglutinins; D011993:Recombinant Fusion Proteins; D013745:Tetanus Toxoid; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma