Regulation of the Ifng locus in the context of T-lineage specification and plasticity.

Study of the development of distinct CD4(+) T-cell subsets from naive precursors continues to provide excellent opportunities for dissection of mechanisms that control lineage-specific gene expression or repression. Whereas it had been thought that the induction of transcription networks that control T-lineage commitment were highly stable, reinforced by epigenetic processes that confer heritability of functional phenotypes by the progeny of mature T cells, recent findings support a more dynamic view of T-lineage commitment. Here, we highlight advances in the mapping and functional characterization of cis elements in the Ifng locus that have provided new insights into the control of the chromatin structure and transcriptional activity of this signature T-helper 1 cell gene. We also examine epigenetic features of the Ifng locus that have evolved to enable its reprogramming for expression by other T-cell subsets, particularly T-helper 17 cells, and contrast features of the Ifng locus with those of the Il17a-Il17f locus, which appears less promiscuous.

Epigenetic instability of cytokine and transcription factor gene loci underlies plasticity of the T helper 17 cell lineage.

Phenotypic plasticity of T helper 17 (Th17) cells suggests instability of chromatin structure of key genes of this lineage. We identified epigenetic modifications across the clustered Il17a and Il17f and the Ifng loci before and after differential IL-12 or TGF-beta cytokine signaling, which induce divergent fates of Th17 cell precursors. We found that Th17 cell precursors had substantial remodeling of the Ifng locus, but underwent critical additional modifications to enable high expression when stimulated by IL-12. Permissive modifications across the Il17a-Il17f locus were amplified by TGF-beta signaling in Th17 cells, but were rapidly reversed downstream of IL-12-induced silencing of the Rorc gene by the transcription factors STAT4 and T-bet. These findings reveal substantial chromatin instability of key transcription factor and cytokine genes of Th17 cells and support a model of Th17 cell lineage plasticity in which cell-extrinsic factors modulate Th17 cell fates through differential effects on the epigenetic status of Th17 cell lineage factors.

The AP-1 transcription factor Batf controls T(H)17 differentiation.

Activator protein 1 (AP-1, also known as JUN) transcription factors are dimers of JUN, FOS, MAF and activating transcription factor (ATF) family proteins characterized by basic region and leucine zipper domains. Many AP-1 proteins contain defined transcriptional activation domains, but BATF and the closely related BATF3 (refs 2, 3) contain only a basic region and leucine zipper, and are considered to be inhibitors of AP-1 activity. Here we show that Batf is required for the differentiation of IL17-producing T helper (T(H)17) cells. T(H)17 cells comprise a CD4(+) T-cell subset that coordinates inflammatory responses in host defence but is pathogenic in autoimmunity. Batf(-/-) mice have normal T(H)1 and T(H)2 differentiation, but show a defect in T(H)17 differentiation, and are resistant to experimental autoimmune encephalomyelitis. Batf(-/-) T cells fail to induce known factors required for T(H)17 differentiation, such as RORgamma t (encoded by Rorc) and the cytokine IL21 (refs 14-17). Neither the addition of IL21 nor the overexpression of RORgamma t fully restores IL17 production in Batf(-/-) T cells. The Il17 promoter is BATF-responsive, and after T(H)17 differentiation, BATF binds conserved intergenic elements in the Il17a-Il17f locus and to the Il17, Il21 and Il22 (ref. 18) promoters. These results demonstrate that the AP-1 protein BATF has a critical role in T(H)17 differentiation.

Developmental plasticity of Th17 and Treg cells.

The emergence of Th17 cells as a distinct subset of effector CD4 T cells has led to a revised model of the adaptive immune system. Whereas the Th1-Th2 paradigm revolutionized our understanding of adaptive immunity by introducing the concept of alternative developmental pathways for naïve CD4 T cells induced by distinct cytokine cues from microbe-activated innate immune cells, delineation of Th17 cell differentiation has extended this concept and has led to a greater appreciation of the developmental plasticity of CD4 T cells. In contrast to Th1 and Th2 cells, which have been thought to represent terminal products of their respective developmental programs, recent studies suggest that Th17 cells are less rigid. In addition to early developmental links to induced regulatory T cells (Tregs) reflected in the shared requirement for TGF-beta, it is now apparent that there is substantial plasticity late in the Th17 program, which allows committed Th17 cells to transition from effectors that produce predominantly IL-17A and IL-17F, to effectors that produce predominantly IFNgamma. Tregs appear to have similar plasticity. This promises new insights into strategies for balancing antimicrobial defense with restraints on immune-mediated tissue injury, and raises new questions regarding the stability of epigenetic modifications that accompany induction of cytokine gene expression during T cell lineage development.

Rapid receptor-proximal signaling assays for FcR gamma-containing receptors.

Novel, cell-based assays, based on bioluminescence resonance energy transfer, have been developed for FcepsilonRI- and GPVI-FcRgamma complex-mediated signaling at receptor-proximal steps. In a stable transfectant of the HEK-293 cell line expressing human FcepsilonRIalpha, FcepsilonRIbeta, and FcRgamma-GFP2 and Syk(1-265)-Rluc fusion proteins, FcepsilonRI cross-linking markedly increased BRET2 ratios, which are the ratios of GFP2 emission to Rluc emission. These ratios reflect the FcRgamma-GFP2-Syk(1-265)-Rluc interaction in living cells. The signals are specifically inhibited by the Src-family kinase inhibitor PP2. Separately, in transient transfectants expressing GPVI, FcRgamma-GFP2, and Syk(1-265)-Rluc, the GPVI-specific ligand convulxin induced a two-fold increase in the BRET2 ratio and this increase was also inhibited by PP2. Finally, a differential assay was developed which permits the measurement of FcepsilonRI- and GPVI-FcRgamma complex-mediated signaling in the same cell. These assays provide useful methods for monitoring FcRgamma-Syk interaction in real time in living cells and may contribute to the understanding of signal regulation through FcRgamma-containing receptors.