Intracellular cytokine staining for the characterization and quantitation of antigen-specific T lymphocyte responses

Standard proliferation assays used for analysis of T cell function have significant shortcomings, including limited sensitivity, lack of quantitative readouts, and considerable variability. Recently, flow cytometric methods have been developed to allow multiparametric detection of cell surface antigens and intracellular cytokine expression in response to polyclonal stimuli and antigen. We have optimized an intracellular cytokine staining assay in the non-human primate model of AIDS, which allows us to identify antigen-specific T lymphocytes at the single cell level with high sensitivity, while reducing background staining to a minimum. Central to our optimized protocol is the addition of cross-linked costimulatory anti-CD28 and anti-CD49d Mabs, a modification that results in up to 3-fold enhancement of the frequency of cytokine-secreting CD4+ T cells following superantigen or antigen-specific stimulation. Optimization of the antigen concentration and duration of antigenic stimulation resulted in a convenient and highly reproducible assay, which permits delineation of antigen-specific cells at the single cell level, thereby providing new insights into pathogen-specific immune responses and allowing detailed phenotypic analysis of extremely low frequency events.