Histone acetylation at the Ifng promoter in tolerized CD4 cells is associated with increased IFN-gamma expression during subsequent immunization to the same antigen

Abstract

When naive CD4(+) Th cells encounter cognate pathogen-derived Ags they expand and develop the capacity to express the appropriate effector cytokines for neutralizing the pathogen. Central to this differentiation process are epigenetic modifications within the effector cytokine genes that allow accessibility to the transcriptional machinery. In contrast, when mature self-reactive CD4 cells encounter their cognate epitopes in the periphery they generally undergo a process of tolerization in which they become hyporesponsive/anergic to antigenic stimulation. In the current study, we used a TCR transgenic adoptive transfer system to demonstrate that in a dose-dependent manner parenchymal self-Ag programs cognate naive CD4 cells to acetylate histones bound to the promoter region of the Ifng gene (which encodes the signature Th1 effector cytokine) during peripheral tolerization. Although the Ifng gene gains transcriptional competence, these tolerized CD4 cells fail to express substantial amounts of IFN-gamma in response to antigenic stimulation apparently because a blockage in TCR-mediated signaling also develops. Nevertheless, responsiveness to antigenic stimulation is partially restored when self-Ag-tolerized CD4 cells are retransferred into mice infected with a virus expressing the same Ag. Additionally, there is preferential boosting in the ability of these CD4 cells to express IFN-gamma relative to other cytokines with expression that also becomes impaired. Taken together, these results suggest that epigenetic modification of the Ifng locus during peripheral CD4 cell tolerization might allow for preferential expression of IFN-gamma during recovery from tolerance.

FIGURE 1

Functional response and properties of naive clonotypic CD4 cells exposed to cognate viral or self-Ag. CFSE-labeled naive Thy1.1 ⁺ HA-specific clonotypic CD4 cells were adoptive transferred into Thy1.2⁺ viral-HA (V), self-HA^low (S^L), or self-HA^high (S^H) recipients and recovered from spleens 6 days later for analysis. A, In vivo proliferation of clonotypic CD4 cells. Representative CFSE dilution histograms with the percentage of clonotypic CD4 cells displaying diluted CFSE fluorescence indicated. B, The frequency of clonotypic CD4 cells is expressed as a percentage within the total splenocyte population. C, The ability of clonotypic CD4 cells exposed to cognate viral and self-Ag to express cytokines in response to in vitro restimulation. Plots representative of intracellular IFN-γ vs TNF-α expression following in vitro restimulation with HA peptide or PMA plus ionomycin (PMA + I), with the percentage of clonotypic CD4 cells expressing each cytokine and the corresponding mean fluorescence intensity (MFI) value indicated. D, Quantitation of cytokine expression shown in C. Total intracellular IFN-γ, TNF-α, and IL-2 is expressed in arbitrary units, and was calculated by multiplying the percentage of cytokine-expressing clonotypic CD4 cells by the corresponding mean fluorescence intensity. Data are expressed as the mean ± SEM for n= 4 recipients per group.

FIGURE 2

Splenic APCs recovered from both viral-HA and self-HA adoptive transfer recipients are equivalent in their abilities to stimulate clonotypic CD4 cells to express intracellular cytokines in vitro. Spleens recovered from viral-HA, self-HA^low, and self-HA^high recipients 6 days after receiving adoptive transfers of naive clonotypic CD4 cells were fractionated into T cell (depleted of APCs using magnetic beads) and APC (depleted of T cells using magnetic beads) fractions, remixed in the nine possible APC to T cell combinations at a 1:1 ratio, and incubated with HA peptide followed by intracellular cytokine staining. Data shown are representative of two independent experiments.

FIGURE 3

Self-HA^low and self-HA^high transgenic mice do not differ in their intrinsic CD4 cell priming/tolerization capacity. Naive CFSE-labeled TEa clonotypic CD4 cells on the B6 (H-2^b) Thy1.1⁺ background that are specific for an I-E^d-derived peptide (Eα) presented by I-A^b were adoptively transferred into self-HA^low, self-HA^high, or viral-HA recipients that had been backcrossed to the B6 Thy1.2⁺ background. These adoptive transfer recipients were then challenged with a bolus of soluble Eα peptide, and recovered from spleens 6 days later for analysis. FACS plots from n= 3 recipients per group represent CFSE dilution (A) and intracellular IFN-γ vs TNF-α expression following in vitro restimulation with Eα peptide or PMA plus ionomycin (PMA + I) (B). C, Quantification of cytokine expression in B is total intracellular cytokines expressed in arbitrary units and calculated as described in Fig. 1.

FIGURE 4

The ability of clonotypic CD4 cells exposed to cognate self-Ag to express IFN-γ is not influenced by the number of adoptively transferred naive clonotypic CD4 cells. The indicated number of naive clonotypic CD4 cells were adoptively transferred into viral-HA or self-HA^high recipients, and recovered from spleens 6 days later for analysis. A, Frequency of clonotypic CD4 cells is expressed as the percentage of total splenocytes. FACS plots (B) and quantification (C) representative of intracellular IFN-γ expression following in vitro restimulation with HA peptide or PMA plus ionomycin from n= 3 recipients per group.

FIGURE 5

CD4 cells that have undergone peripheral tolerization in response to cognate self-Ag display acetylated histone H3 bound to the Ifng promoter region and also exhibit other characteristics normally associated with Th1 effectors. Clonotypic CD4 cell samples isolated from viral-HA (n = 9 recipients), self-HA^low (self-HA^L, n = 3), each determination deriving from three pooled recipient samples), and self-HA^high (self-HA^H, n = 11 recipients) were divided and analyzed by quantitative real-time PCR-based ChIP to measure histone H3 acetylation at the Ifng (A) and Tnfa (B) proximal promoters. Quantitative real-time RT-PCR to measure T-bet (C) and IL-12Rβ2 (D) mRNAs. Data are expressed as a ratio relative to naive clonotypic CD4 cells.

FIGURE 6

Viral priming preferentially boosts the ability of tolerized CD4 cells to express IFN-γ. Viral-HA-primed clonotypic CD4 cells (primary effectors or 1°E) and self-HA^high-tolerized (T) clonotypic CD4 cells (both pooled from the samples shown in Fig. 1) were relabeled with CFSE and retransferred into viral-HA-infected secondary recipients (1 × 10⁶ clonotypic CD4 cells per secondary recipient) to generate secondary effector (2°E) and recovered tolerized (RT) clonotypic CD4 cells, respectively. A, Diagram of the retransfer experimental schema. B, Plots of representative CFSE dilution and intracellular IFN-γ vs TNF-α expression. C, Total intracellular IFN-γ, TNF-α, and IL-2 expression is expressed in arbitrary units and was calculated as described in Fig. 1. Note that data corresponding to 1°E and tolerized clonotypic CD4 cells are the same as in Fig. 1 for n= 4 recipients per group.