Flow cytometric measurement of intracellular cytokines detects immune responses in MUC1 immunotherapy

The detection of tumor-specific T cells in immunized cancer patients usuall y relies on lengthy and difficult CTL assays; we now report on flow cytomet ry to detect the intracellular cytokines interleukin 2 (IL-2), IL-4, IFN-ga mma, and tumor necrosis factor alpha (TNF-alpha) produced by CD4(+)CD69(+) and CD8(+)CD69(+). activated T cells after MUC1 antigen stimulation. Periph eral blood mononuclear cells were obtained from 12 patients,vith adenocarci noma injected with mannan-MUC1; cells were exposed in vitro for 18 h to MUC 1 peptide in the presence of CD28 monoclonal antibody and Brefeldin; permea bilized 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 TNP-alpha (P < 0.038) and IFN-gamma (P < 0.010) than did cells from 12 norm al subjects; minor increases in IL-4 occurred. By contrast, CD4(+)CD69(+) c ells showed no overall alteration in TNF-alpha and IFN-gamma cytokine produ ction, although in some patients, their measurement was informative; the me asurement of IL-2 was not useful in either CD4(+)CD69(+) or CD8(+)CD69(+) c ells. We conclude that in MUC1-immunized patients, the measurement of TNF-a lpha and IFN-gamma in activated CD69(+)CD8(+) T cells may be indicative of their immune status.