T-bet Down-Modulation in Tolerized Th1 Effector CD4 Cells Confers a TCR-Distal Signaling Defect That Selectively Impairs IFN-γ Expression

Abstract

When Th1 effector CD4 cells encounter tolerizing Ag in vivo, their capacity to express the effector cytokines IFN-γ and TNF-α is lost more rapidly than noneffector functions such as IL-2 production and proliferation. To localize the relevant intracellular signaling defects, cytokine expression was compared following restimulation with Ag vs agents that bypass TCR-proximal signaling. IFN-γ and TNF-α expression were both partially rescued when TCR-proximal signaling was bypassed, indicating that both TCR-proximal and -distal signaling defects impair the expression of these two effector cytokines. In contrast, bypassing TCR-proximal signaling fully rescued IL-2 expression. T-bet, a transcription and chromatin remodeling factor that is required to direct the differentiation of naive CD4 cells into IFN-γ-expressing Th1 effectors, was partially down-modulated in tolerized Th1 effectors. Enforcing T-bet expression during tolerization selectively rescued the ability to express IFN-γ, but not TNF-α. Conversely, expression of a dominant-negative T-bet in Th1 effectors selectively impaired the ability to express IFN-γ, but not TNF-α. Analysis of histone acetylation at the IFN-γ promoter further suggested that down-modulation of T-bet expression during Th1 effector CD4 cell tolerization does not impair IFN-γ expression potential through alterations in chromatin structure.

Histone Acetylation at theIfngPromoter in Tolerized CD4 Cells Is Associated with Increased IFN-γ Expression during Subsequent Immunization to the Same Antigen

“…Thus, to directly confirm that the ability of self-Ag-tolerized clonotypic CD4 cells to express IFN-␥ in response to PMA plus ionomycin stimulation does in fact indicate that the chromatin structure of the Ifng locus can open during tolerization, the extent of histone H3 acetylation at the proximal promoter was measured using a real-time PCR-based ChIP assay (27) (Fig. 5A).…”

“…The functional response of 6.5 and TEa clonotypic CD4 cells were analyzed by FACS following recovery from spleens of the indicated adoptive transfer recipients as previously described (13,26,27). In short, clonotypic CD4 cells (identified as CD4 ϩ Thy1.1 ϩ ) were analyzed for frequency and dilution of CFSE directly ex vivo.…”

“…Clonotypic CD4 cells were isolated from recipient spleens either by positively selecting Thy1.1 ϩ cells using MACS columns as previously described (27) high and viral-HA counterparts isolated by the different methods were comparable (data not shown). Clonotypic CD4 cell samples were analyzed directly ex vivo (i.e., without in vitro restimulation), and each sample was divided and analyzed by real-time PCR-based RT-PCR and ChIP assays to measure T-bet and IL-12R␤2 mRNA and histone H3 acetylation at the Ifng and Tnfa promoters, respectively, using our previously established protocols and PCR primers (27).…”

“…Clonotypic CD4 cell samples were analyzed directly ex vivo (i.e., without in vitro restimulation), and each sample was divided and analyzed by real-time PCR-based RT-PCR and ChIP assays to measure T-bet and IL-12R␤2 mRNA and histone H3 acetylation at the Ifng and Tnfa promoters, respectively, using our previously established protocols and PCR primers (27). In short, after isolating RNA and reverse transcribing cDNA, T-bet and IL-12R␤2 mRNAs were quantified by first normalizing for input sample amounts by calculating the difference in threshold cycle, C T , values relative to HPRT message, and then using the ⌬⌬ threshold cycle method to calculate the ratio between experimental samples and a representative naive CD4 cell sample.…”

“…Notably, PMA plus ionomycin elicited increased IFN-␥ expression in self-HA high -tolerized CD4 cells relative to expression observed in peptide restimulated counterparts ( p ϭ 0.0005) that reached about one-third the level observed in viral-HA counterparts. Because naive clonotypic CD4 cells are unable to express appreciable levels of IFN-␥ in response to PMA plus ionomycin stimulation (27), and because the chromatin structure of the Ifng gene locus exists in a condensed configuration that is inaccessible to the transcriptional machinery (7,17,28), this result suggested that self-Ag causes the Ifng gene locus in cognate naive CD4 cells to undergo some degree of chromatin/epigenetic modification, but that these tolerized cells are nevertheless unable to express substantial levels of IFN-␥ in response to antigenic stimulation due to a blockage in signaling steps that are proximal to the TCR.…”