Differentiation-dependent functional and epigenetic landscapes for cytokine genes in virus-specific CD8+ T cells

Abstract

Although the simultaneous engagement of multiple effector mechanisms is thought to characterize optimal CD8(+) T-cell immunity and facilitate pathogen clearance, the differentiation pathways leading to the acquisition and maintenance of such polyfunctional activity are not well understood. Division-dependent profiles of effector molecule expression for virus-specific T cells are analyzed here by using a combination of carboxyfluorescein succinimidyl ester dilution and intracellular cytokine staining subsequent to T-cell receptor ligation. The experiments show that, although the majority of naive CD8(+) T-cell precursors are preprogrammed to produce TNF-α soon after stimulation and a proportion make both TNF-α and IL-2, the progressive acquisition of IFN-γ expression depends on continued lymphocyte proliferation. Furthermore, the extensive division characteristic of differentiation to peak effector activity is associated with the progressive dominance of IFN-γ and the concomitant loss of polyfunctional cytokine production, although this is not apparent for long-term CD8(+) T-cell memory. Such proliferation-dependent variation in cytokine production appears tied to the epigenetic signatures within the ifnG and tnfA proximal promoters. Specifically, those cytokine gene loci that are rapidly expressed following antigen stimulation at different stages of T-cell differentiation can be shown (by ChIP) to have permissive epigenetic and RNA polymerase II docking signatures. Thus, the dynamic changes in cytokine profiles for naive, effector, and memory T cells are underpinned by specific epigenetic landscapes that regulate responsiveness following T-cell receptor ligation.

Fig. 1.

Naive OT-I T cells rapidly express TNF-α and IL-2, but not IFN-γ, after peptide stimulation. (A and B) Naive (CD44^loCD62L^hi) OT-I T cells were taken 24 h after adoptive transfer into uninfected B6 recipients and stimulated with 1 μM SIINFEKL peptide for 5 h in the presence of 5 μg/mL brefeldin. Coexpression of intracellular cytokines is illustrated for TNF-α and IFN-γ (A) or TNF-α and IL-2 (B). Data are representative of five independent experiments. (C and D) IFN-γ and TNF-α mRNA levels within resting naive (CD44^loCD62L^hi) OT-I CTLs (C) or after 5 h of peptide stimulation (D) by quantitative real-time PCR. Shown is the relative increase in mRNA levels for TNF-α compared with IFN-γ mRNA levels in naive resting OT-I CTLs.

Fig. 2.

Gain of IFN-γ production in vivo is tightly coupled to division. CFSE-labeled Ly5.1⁺OT-I cells (10⁶) were injected into B6 mice 2 d after infection with 10⁴ pfu of x31-OVA. Profiles of CFSE dilution and cytokine production following peptide stimulation for IFN-γ (A) or TNF-α (B) are shown for splenic OT-I cells 60 h after transfer. Cellular division was measured by CFSE dilution (C) and the coexpression of IFN-γ and TNF-α was determined for CTLs from the spleen and compared with that for naive OT-I cells from uninfected recipients (D). Data shown are mean ± SEM (n = 3) and are representative of three independent experiments.

Fig. 3.

Development of the TNF-α⁻IFN-γ⁺ phenotype is dependent on in vivo expansion. Ly5.1⁺OT-I cells (10²–10⁶) were adoptively transferred into B6 mice 1 d before i.n. infection with 10⁴ pfu x31-OVA. Ten days later, splenic OT-I cells were analyzed for expansion (A), the coexpression of IFN-γ and TNF-α within the cytokine-producing population (B), and the mean fluorescence intensity (MFI) of cytokine staining (C) for OT-I cells derived from the MLN and spleen. Data shown are mean ± SEM (n = 3–7) and are representative of three independent experiments. Significance tested with one-way ANOVA test with Tukey posttest (***P < 0.001) (A) or Student t test (*P < 0.05 and **P < 0.01) (C). OT-I expansion and effector function are influenced by antigen dose in vivo. Ly5.1⁺OT-I cells (10⁴ or 10⁶) were transferred into naive B6 mice and infected i.n. with 10⁴ pfu x31-OVA or 10² pfu x31-OVA (in x31-WT) 24 h later. The OT-I cells were isolated 60 h after transfer, and the fold expansion (D) and coexpression of IFN-γ and TNF-α (E) determined. Data are shown as mean ± SEM (n = 4).

Fig. 4.

Histone modifications present at the ifnG and tnfA promoters in memory OT-I cells. Sort-purified naive effector (5–10 pooled mice, day 10 after infection) and memory (20–30 pooled mice, day > 70 after infection) OT-I cells were fixed, sonicated, and subjected to ChIP with antibodies directed against H3 (A), H3K9ac (n = 2), H3K4me3 (n = 3), or H3K27me3 (n = 3) (B and C). The CHiP enrichment ratio is expressed as the extent of enrichment vs. input DNA (A) or enrichment vs. H3 ChIP (B and C) for the ifnG and tnfA set C primers only (data for n = 3).

Fig. 5.

Docking of RNA pol II at the TSS of effector genes. ChIP for RNA pol II binding at the proximal promoter of the ifnG (white bars) or tnfA (black bars) genes was performed as in Fig. 4. The ChIP enrichment ratio is expressed as enrichment vs. input DNA for the ifnG and tnfA set C primers only. Data shown are for n = 3.