Aberrant expression of IFN-γ in Th2 cells from Th2 LCR-deficient mice

The Th2 locus control region (LCR) has been shown to be a crucial cis-acting element for Th2 cytokine expression and Th2 cell differentiation. To study the role of Th2 LCR in ifng locus regulation, we examined the expression of IFN-γ in Th2 cells from Th2 LCR-deficient mice. We found IFN-γ to be aberrantly up-regulated. In addition, histone 3(H3)-acetylation and histone 3 lysine 4 (H3-K4)-methylation greatly increased at the ifng locus of the Th2 cells. GATA-3 and STAT6 bound to the ifng promoter in Th2 cells from the wild type but not from the Th2 LCR-deficient mice, and they directly repressed ifng expression in transient reporter assay. Moreover, ectopic expression of GATA-3 and STAT6-VT repressed the aberrant expression of the ifng gene and restored repressive chromatin state at the ifng locus in Th2 cells from Th2 LCR-deficient mice. These results suggest that expression of the ifng gene and chromatin remodeling of the ifng locus are under the control of a Th2 LCR-mediated Th2 differentiation program.     

Primary Murine CD4+ T Cells Fail to Acquire the Ability to Produce Effector Cytokines When Active Ras Is Present during Th1/Th2 Differentiation

Constitutive Ras signaling has been shown to augment IL-2 production, reverse anergy, and functionally replace many aspects of CD28 co-stimulation in CD4⁺ T cells. These data raise the possibility that introduction of active Ras into primary T cells might result in improved functionality in pathologic situations of T cell dysfunction, such as cancer or chronic viral infection. To test the biologic effects of active Ras in primary T cells, CD4⁺ T cells from Coxsackie-Adenovirus Receptor Transgenic mice were transduced with an adenovirus encoding active Ras. As expected, active Ras augmented IL-2 production in naive CD4⁺ T cells. However, when cells were cultured for 4 days under conditions to promote effector cell differentiation, active Ras inhibited the ability of CD4⁺ T cells to acquire a Th1 or Th2 effector cytokine profile. This differentiation defect was not due to deficient STAT4 or STAT6 activation by IL-12 or IL-4, respectively, nor was it associated with deficient induction of T-bet and GATA-3 expression. Impaired effector cytokine production in active Ras-transduced cells was associated with deficient demethylation of the IL-4 gene locus. Our results indicate that, despite augmenting acute activation of naïve T cells, constitutive Ras signaling inhibits the ability of CD4⁺ T cells to properly differentiate into Th1/Th2 effector cytokine-producing cells, in part by interfering with epigenetic modification of effector gene loci. Alternative strategies to potentiate Ras pathway signaling in T cells in a more regulated fashion should be considered as a therapeutic approach to improve immune responses in vivo.     

Cutting edge: chromatin remodeling at the IL-4/IL-13 intergenic regulatory region for Th2-specific cytokine gene cluster

During the differentiation of naive Th cells into Th2 effector cells, the entire IL-4/IL-13 locus is remodeled into an accessible chromatin conformation. Here we show that ectopic expression and activation of Stat6 or GATA-3 in Th cells developing under Th1-polarizing conditions lead to the induction of chromatin remodeling not only at the flanking regions of the IL-4 and IL-13 genes but also at the IL-4/IL-13 intergenic regulatory region for the IL-4/IL-13/IL-5 gene cluster. Furthermore, we demonstrate that GATA-3 and another Th2-specific, inducible protein complex interact with the IL-4/IL-13 intergenic DNase I hypersensitive region specifically in Th2 cells.