Flow Cytometric Measurement of Intracellular Cytokines Detects Immune Responses in MUC1 Immunotherapy

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-γ, and tumor necrosis factor α (TNF-α) 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 MUC1 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-α ( P < 0.038) and IFN-γ ( 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-α and IFN-γ 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-α and IFN-γ in activated CD69 + CD8 + T cells may be indicative of their immune status.