Dependence of IFN-γ production on histone acetylation. (A) Cultured iNKT cells were preexposed to CD1d⁺ APCs in the presence of vehicle alone or 11 µM HAT inhibitor dissolved in vehicle, then the APCs were removed, and the iNKT cells were washed and exposed to IL-12 and IL-18. IFN-γ secretion was measured by ELISA. The plot shows results from one representative experiment out of three; similar results were obtained in independent experiments using a different iNKT cell clone. Error bars indicate the standard deviations of the means. (B) Rested iNKT cells were left untreated (resting) or were exposed to CD1d⁺ APCs in the presence or absence of HAT inhibitor. The iNKT cells were fixed and lysed, and sheared chromatin was immunoprecipitated using an antibody against tetra-acetylated histone H4 (anti-H4Ac) or an isotype-matched negative control antibody (neg IgG), and the resulting purified DNA fragments were analyzed by quantitative PCR using primers that sit down in the region of the IFNG locus that is 18–25 kb downstream of the IFNG gene start site. Similar results were obtained with two additional primer pairs that localize to this region. (C) Compiled results from three independent experiments in which cultured iNKT cells were either preexposed to CD1d⁺ APCs or left untreated (resting). After removal of the APCs, the iNKT cells were either lysed immediately or after a 20-h incubation in culture medium. Immunoprecipitation was performed using an antibody against tetra-acetylated histone H4 (H4Ac) or with a negative control Ig, and PCR was performed using the same primers as in B. The PCR signals were normalized by the amount of input DNA for each condition, and the H4Ac enrichment was calculated by dividing the normalized signal from the H4Ac precipitate by that obtained from the negative control Ig precipitate. The plot shows the H4Ac enrichment from the indicated CD1d⁺ APC-pretreated iNKT cells as the fold over that obtained from the respective rested iNKT cells. Similar results were obtained in each case using a different set of primers that localize to this region. (D) Cultured iNKT cells were preexposed to CD1d-transfected APCs, then the APCs were removed, and the iNKT cells were incubated in the presence of medium alone or medium containing an HDAC inhibitor (TSA). After the indicated periods of time, IL-12 and IL-18 were added, and IFN-γ secretion was measured by ELISA. Results are expressed as the percentage of the amount produced when IL-12 and IL-18 were added immediately after removal of the APCs and are from one representative analysis out of three independent experiments. Similar results were obtained in three independent experiments on two additional iNKT cell clones. (E) Compiled results from four independent experiments in which rested iNKT cells were treated with TSA for 18 h or left untreated (control), and then the cells were washed and stimulated with IL-12 and IL-18, and secreted IFN-γ was quantitated by ELISA. The plot shows the amount of IFN-γ secreted by the indicated TSA-treated iNKT cells normalized by the amount secreted by the corresponding control iNKT cells. The horizontal bar indicates the mean. (F) Freshly isolated peripheral blood T cells depleted of CD1d⁺ cell populations were stimulated with IL-12, IL-18, and IFN-α and flow cytometrically analyzed for intracellular IFN-γ (dark gray histograms) compared with unstimulated cells (open histograms) or were incubated for 3 or 5 d in the presence or absence of the HDAC inhibitor TSA and then washed and similarly stimulated and analyzed for intracellular IFN-γ expression. Results are representative of four independent experiments performed using PBMCs isolated from two different donors.