A Human ROR?t Mutant Enhances Th17-Mediated Inflammation via Increased HIF-1a Recruitment
T helper 17 (Th17) cells play a central role in mucosal defense and autoimmune pathology, with their differentiation and effector function critically controlled by the transcription factor ROR?t. While genome-wide association studies (GWAS) have identified variants in the RORC locus associated with inflammatory diseases, the functional impact of these variants remains largely unexplored. Here, we report the identification and characterization of a hyperactive ROR?t point mutation, N277D (mouse homolog N275D), which enhances Th17 inflammatory potential through increased cooperation with the hypoxia-inducible factor HIF-1a. Using a combination of computational modeling, retroviral expression, and CRISPR-engineered knock-in mice, we demonstrate that ROR?tN275D promotes IL-17A?IFN-?? Th17 cell differentiation and exacerbates colitis in a T cell transfer model without affecting T cell development or homeostasis. Transcriptomic and metabolomic profiling revealed upregulation of glycolytic and hypoxia-associated pathways in mutant Th17 cells. Consistent with enhanced ROR?tN275D-HIF-1a interaction, ROR?tN275D was recruited to HIF-1a binding site at the Pdk1 locus, which is required for the upregulation of IFN-? production in mutant Th17 cells. These findings reveal a novel regulatory axis between ROR?t and HIF-1a that links transcriptional and metabolic programming in pathogenic Th17 cells and suggest a framework for functionally interrogating autoimmune risk variants to uncover therapeutic targets. Overall design: To gain mechanistic insight into how the ROR?tN275D mutation enhances Th17 pathogenicity, we performed transcriptomic profiling of in vitro-differentiated Th17 cells. Our previous in vivo studies revealed that ROR?tN275D does not affect the development or homeostasis of steady-state Th17 cells, such as those in the intestinal lamina propria, but instead promotes the emergence of highly inflammatory, IL-17A?IFN-?? Th17 cells under disease conditions. This suggested a mutation-specific effect under inflammatory, but not homeostatic, cues. Therefore, to mimic the inflammatory environment and uncover a broader range of transcriptional changes, we polarized naïve CD4? T cells from WT and ROR?tN275D mice using IL-6, IL-1ß, and IL-23. This cytokine combination is known to promote the differentiation of highly inflammatory, pathogenic Th17 cells implicated in autoimmunity and tissue inflammation, and it aligns with the cytokine milieu used in our in vivo colitis mode