Gene regulation and chromatin remodeling by IL-12 and type I IFN in programming for CD8 T cell effector function and memory

Abstract

A third signal that can be provided by IL-12 or type I IFN is required for differentiation of naive CD8 T cells responding to Ag and costimulation. The cytokines program development of function and memory within 3 days of initial stimulation, and we show here that programming involves regulation of a common set of approximately 355 genes including T-bet and eomesodermin. Much of the gene regulation program is initiated in response to Ag and costimulation within 24 h but is then extinguished unless a cytokine signal is available. Histone deacetylase inhibitors mimic the effects of IL-12 or type I IFN signaling, indicating that the cytokines relieve repression and allow continued gene expression by promoting increased histone acetylation. In support of this, increased association of acetylated histones with the promoter loci of granzyme B and eomesodermin is shown to occur in response to IL-12, IFN-alpha, or histone deacetylase inhibitors. Thus, IL-12 and IFN-alpha/beta enforce in common a complex gene regulation program that involves, at least in part, chromatin remodeling to allow sustained expression of a large number of genes critical for CD8 T cell function and memory.

Figure 1. Programming of CD8 T cell differentiation requires IL-12 and/or IFNα along with Ag and costimulation

(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.

Figure 2. IL-12 and IFNα induce, enhance or sustain the expression of genes involved in effector functions

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).

Figure 3. IL-12- and IFNα-dependent regulation of T-bet and eomesodermin mRNA expression

(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.

Figure 4. Inhibition of histone deacetylation mimics signal 3 cytokine effects on CD8 T cell differentiation

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.

Figure 5. IL-12 and IFNα promote histone hyperacetylation at the grzB and Eomes gene loci during CD8 T cell differentiation

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).