Time courses for expression of selected genes regulated by IL-12 and IFNα are shown. Expression is shown as the fold change in signal intensities upon stimulation with Ag-B7 alone () or with IL-12 (ν) or IFNα (○) compared to the signal intensity for the gene in naïve cells (0 hr). (Note: different scales are used on y-axis for each gene, and scale breaks are included on the y-axis for IFNγ and CCR5).

Purified CD44^lo OT-1 CD8 T cells were stimulated for 3 days with Ag-B7 coated aAPC alone or with IL-12, IFNα or trichostatin A (TSA) (7.5ng/ml). TSA was dissolved in DMSO, and controls having Ag-B7 and DMSO were included.
(A-B) Expression of grzB and IFNγ by intracellular staining. Isotype controls were run for all samples (not shown), and gates set so that > 98% of events fell into the lower quadrant for the controls.
(C) Cytolytic activity measured by ⁵¹Cr release assay using E.G7 target cells.

(A) Total RNA was isolated from naïve (CD44^lo) OT-I CD8 T cells (0-h) or from purified OT-I CD8 T cells stimulated with Ag-B7 in presence of the indicated cytokines for 48 and 72h, and the mRNA expression level of T-bet and Eomes was determined by semi-quantitative RT-PCR. The linear range of amplification of transcripts was determined as described in Materials and Methods. The graphs represent mRNA quantification done by densitometry for three independent experiments. For each condition, the relative mRNA expression is shown as the mean ± S.D. Student's t-test was performed comparing IL-12 or IFNα with Ag-B7 alone and p-values are shown. (B) Naïve OT-I T cells were stimulated for 72hr with Ag-B7 alone or along with IL-12, as indicated, and the cells stained with anti-Tbet mAb or isotype control Ab and analyzed by flow cytometry.

Purified CD44^lo OT-I CD8 T cells were used as naïve control or were stimulated with Ag-B7 aAPC alone or with IL-12, IFNα or TSA (7.5ng/ml). Cells were harvested at 48h, processed, and examined by crosslinked-CHIP and qRT-PCR for acetylated histones H3 and H4. (A) Association of H3 and H4 histones with the grzB promoter, expressed as template copy numbers as a percent of whole genome copy number. Error bars show ranges for duplicate samples. (B-D) Association of H3 and H4 histones with the grzB promoter (B), grzB distal region (B) and the Eomes promoter (C) regions. Template copy numbers for each condition were internally normalized with their respective input control. Relative expression is calculated as the ratio of template copy numbers of each sample relative to the naïve control in each experiment, and is shown as mean with SEM of 2 or 3 independent experiments with duplicate samples for each. In comparison to naïve cells, activation with Ag-B7 resulted in significant increases of acetylated H3 and H4 histones with the promoter and exon regions of GrzB (p < 0.05), a significant decrease in acetylated H3 histone association with the Eomes promoter (p < 0.001), and no change in association of acetylated H4 with the Eomes promoter (p = 0.5). In comparison to cells stimulated with just Ag-B7, association of acetylated H3 and H4 histones was significantly greater when IL-12, IFNα or TSA was added (p < 0.03), with one exception. The exception was acetylated H4 association with the GrzB promoter, where the additions did not cause a significant increase (p > 0.05).

(A) Purified naïve (CD44^lo) OT-1 CD8 T cells were stimulated for 3 days in vitro with aAPC coated with H-2K^b / Ova_257-264 peptide and B7-1 either alone, or with IL-12 or IFNα. All cultures were supplemented with IL-2. Cytolytic activity was measured on day 3 by ⁵¹Cr release assay using E.G7 target cells.
(B-C) Cells stimulated as in (A) were analyzed by intracellular staining on days 1, 2 and 3 for expression of IFNγ and grzB.
(D) The upper bars (gray) show the numbers of genes regulated in common by IL-12 and IFNα at 24, 48 and 72h. The lower bars (colored) show the times during which Ag-B7 and IL-12 must be present for optimal clonal expansion and development of effector function (from ref. (16)).
(E) Expression patterns for 408 probe ids representing the 375 genes regulated by both IL-12 and IFNα. The stimuli used and time in culture for each sample are shown at the top. Red represents high expression (signal value) and blue represents low expression on a log₂ scale of 6.0 to 0.0 as shown at the bottom. Data not meeting the selection criterion was excluded and appears gray. The signal value was normalized to around 1.
(F) Hierarchical clustering linkage for cells treated with the various stimuli at 0, 24, 48 and 72h.