The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4 + Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7β, 27-dihydroxycholesterol (7β, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ-or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7β, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7β, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4+ and γδ + T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.

RORγt and RORα are transcription factors of the RAR-related orphan nuclear receptor (ROR) family. They are expressed in Th17 cells and have been suggested to play a role in Th17 differentiation. Although RORγt signature genes have been characterized in mouse Th17 cells, detailed information on its transcriptional control in human Th17 cells is limited and even less is known about RORα signature genes which have not been reported in either human or mouse T cells. In this study, global gene expression of human CD4 T cells activated under Th17 skewing conditions was profiled by RNA sequencing. RORγt and RORα signature genes were identified in these Th17 cells treated with specific siRNAs to knock down RORγt or RORα expression. We have generated selective small molecule RORγt modulators and they were also utilized as pharmacological tools in RORγt signature gene identification. Our results showed that RORγt controlled the expression of a very selective number of genes in Th17 cells and most of them were regulated by RORα as well albeit a weaker influence. Key Th17 genes including IL-17A, IL-17F, IL-23R, CCL20 and CCR6 were shown to be regulated by both RORγt and RORα. Our results demonstrated an overlapping role of RORγt and RORα in human Th17 cell differentiation through regulation of a defined common set of Th17 genes. RORγt as a drug target for treatment of Th17 mediated autoimmune diseases such as psoriasis has been demonstrated recently in clinical trials. Our results suggest that RORα could be involved in same disease mechanisms and gene signatures identified in this report could be valuable biomarkers for tracking the pharmacodynamic effects of compounds that modulate RORγt or RORα activities in patients.

A prevalent observation in high-throughput screening and drug discovery programs is the inhibition of protein function by small-molecule compound aggregation. Here, we present the X-ray structural description of aggregation-based inhibition of a protein-protein interaction involving tumor necrosis factor α (TNFα). An ordered conglomerate of an aggregating small-molecule inhibitor (JNJ525) induces a quaternary structure switch of TNFα that inhibits the protein-protein interaction between TNFα and TNFα receptors. SPD-304 may employ a similar mechanism of inhibition.

ITK is organized in modular domains that play critical roles in its activation (47). Upon T-cell engagement, ITK colocalizes with the TCR, a process dependent on the pleckstrin homology (PH) domain of ITK and its interaction with PIP3 at the plasma membrane (11,19). Activation of ITK also requires interaction with adaptor proteins, such as SLP-76 and LAT (8, 10). The SH2 domain of ITK appears to be critical for its interaction with LAT, whereas both the SH2 and SH3 domains are required for interaction with 10). In vitro studies have demonstrated that the SH3 domain of ITK interacts with the proline-rich (PR) region of SLP-76, and it has been speculated that this interaction is critical for the activation of ITK (6,8). However, the biological significance of the interaction has not been demonstrated in live cells. In the present investigation, we used a cell-permeable peptide as a competitive inhibitor of the interaction between ITK and SLP-76. To this end, we synthesized a 12-amino-acid peptide, which represents the PR region of SLP-76 that binds to the ITK-SH3 domain, and rendered it cell permeable by the addition of nine arginines at its N-proximal end. Here, we show that this cellpermeable peptide, henceforth called R9-QQP, is readily taken up by both Jurkat T cells and murine splenocytes and disrupts events that are mediated by the engagement of the TCR. Thus, association of ITK and SLP-76, recruitment of ITK and actin polarization at the T-cell contact site, LCKmediated transphosphorylation of ITK on tyrosine 511, and production of Th2 cytokines are inhibited by R9-QQP in a dose-dependent and peptide-specific manner. The data presented here are novel and significant because they provide the first demonstration of the biological relevance of the specific interaction between the ITK-SH3 domain and the SLP-76 PR region in live cells. Furthermore, the data underscore the potential of cell-permeable peptides as useful probes for dissecting signal transduction pathways in live cells, and in view of the regulatory role that ITK plays in Th2 cytokine production, they have implications for the pharmacological manipulation of ITK in disease situations. MATERIALS AND METHODSCell lines, mice, antibodies, and other reagents. Wild-type Jurkat T cells (E6.1) were obtained from the American Type Culture Collection (ATCC). The SLP-76-deficient mutant, J14, was a kind gift from Art Weiss (University of California-San Francisco). The cells were cultured as previously described (10). Male C57BL/6 mice were purchased from Harlan Sprague Dawley or Jackson Laboratories and were used between the ages of 6 and 12 weeks. All experimental protocols using animals were approved by the

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