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Flow Cytometric Measurement of Intracellular Cytokines Detects Immune Responses in MUC1 Immunotherapy¹

Immunology and Vaccine Laboratory, The Austin Research Institute, Heidelberg 3084, Victoria, Australia [V. K., J. L., I. F. C. M.], and Becton Dickinson Immunocytometry Systems, San Jose, California 95131 [V. C. M.]

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

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