T cell functional differentiation is mediated by lineage-specific transcription factors. T helper 17 (Th17) has been recently identified as a distinct Th lineage mediating tissue inflammation. Retinoic acid receptor-related orphan receptor gamma (ROR gamma) was shown to regulate Th17 differentiation; ROR gamma deficiency, however, did not completely abolish Th17 cytokine expression. Here, we report Th17 cells highly expressed another related nuclear receptor, ROR alpha, induced by transforming growth factor-beta and interleukin-6 (IL-6), which is dependent on signal transducer and activator of transcription 3. Overexpression of ROR alpha promoted Th17 differentiation, possibly through the conserved noncoding sequence 2 in Il17-Il17f locus. ROR alpha deficiency resulted in reduced IL-17 expression in vitro and in vivo. Furthermore, ROR alpha and ROR gamma coexpression synergistically led to greater Th17 differentiation. Double deficiencies in ROR alpha and ROR gamma globally impaired Th17 generation and completely protected mice against experimental autoimmune encephalomyelitis. Therefore, Th17 differentiation is directed by two lineage-specific nuclear receptors, ROR alpha and ROR gamma.

Regulatory T (Treg) and T helper 17 (Th17) cells were recently proposed to be reciprocally regulated during differentiation. To understand the underlying mechanisms, we utilized a Th17 reporter mouse with a red fluorescent protein (RFP) sequence inserted into the interleukin-17F (IL-17F) gene. Using IL-17F-RFP together with a Foxp3 reporter, we found that the development of Th17 and Foxp3(+) Treg cells was associated in immune responses. Although TGF-beta receptor I signaling was required for both Foxp3 and IL-17 induction, SMAD4 was only involved in Foxp3 upregulation. Foxp3 inhibited Th17 differentiation by antagonizing the function of the transcription factors RORgammat and ROR*. In contrast, IL-6 overcame this suppressive effect of Foxp3 and, together with IL-1, induced genetic reprogramming in Foxp3(+) Treg cells. STAT3 regulated Foxp3 downregulation, whereas STAT3, RORgamma, and ROR* were required for IL-17 expression in Treg cells. Our data demonstrate molecular antagonism and plasticity of Treg and Th17 cell programs.

Th17 and regulatory T (Treg) cells play opposite roles in autoimmune diseases. However, the mechanisms underlying their proper migration to inflammatory tissues are unclear. In this study, we report that these two T cell subsets both express CCR6. CCR6 expression in Th17 cells is regulated by TGF-β and requires two nuclear receptors, RORα and RORγ. Th17 cells also express the CCR6 ligand CCL20, which is induced synergistically by TGF-β and IL-6, which requires STAT3, RORγ and IL-21. Th17 cells, by producing CCL20, promote migration of Th17 and Treg cells in vitro in a CCR6-dependent manner. Lack of CCR6 in Th17 cells reduces the severity of experimental autoimmune encephalomyelitis and Th17 and Treg recruitment into inflammatory tissues. Similarly, CCR6 on Treg cells is also important for their recruitment into inflammatory tissues. Our data indicate an important role of CCR6 in Treg and Th17 cell migration.

CARMA1 mediates T cell receptor (TCR)-induced NF-kappaB activation. However, how TCR links to CARMA1 in the signaling pathway is not clear. Here, we show that CARMA1 is inducibly phosphorylated after TCR-CD28 costimulation. This phosphorylation is likely induced by PKCtheta, since PKCtheta induces phosphorylation of CARMA1 in vitro and in vivo. Our results indicate that the PKCtheta-induced phosphorylation of CARMA1 likely occurs on Ser552 on the Linker region of CARMA1. Importantly, expression of CARMA1 mutant, in which Ser552 is mutated, fails to mediate TCR-induced NF-kappaB activation in CARMA1-deficient T cells. The functional defect of this CARMA1 mutant is likely due to the fact that this mutant cannot be phosphorylated at the critical residue, thereby failing to recruit the downstream signaling components into the immunological synapse. Together, our studies provide the first genetic evidence that the phosphorylation of CARMA1 plays a critical role in the TCR signaling pathway.