Fig 2: RORγtS182A mice have exacerbated diseases in DSS-induced colitis(A) Weight changes of RORγtWT and RORγtS182A cohoused littermates challenged with 2% DSS in drinking water for 7 days and monitored for another 3 days. Each bar represents the sample mean. Each dot represents the result from 1 mouse. * p < 0.05 (multiple t test).(B) Left: Representative bright-field images of colons from (A). Right: Summarized colonic lengths of DSS-treated RORγtWT (n = 5) and RORγtS182A (n = 6) mice harvested on day 10. * p < 0.05 (t test).(C) Left: Representative colonic sections from (B). Right: Summarized score of colonic inflammatory infiltrates in DSS-treated RORγtWT (n = 4) and RORγtS182A (n = 5) mice. Each bar represents the sample mean. * p < 0.05 (t test).(D) Proportion and absolute number of IL-17A+ producing Th17 (T cell receptor [TCR]β+RORγt+Foxp3−), RORγt+ Treg (TCRβ+RORγt+Foxp3+), and conventional Tregs (TCRβ+RORγt−Foxp3+) in colons from DSS-challenged RORγtWT and RORγtS182A mice. Each dot represents the result from 1 mouse. Each bar represents the sample mean. *p < 0.05; n.s., not significant (paired t test).(E) Representative flow cytometry analysis of colonic CD4+ T subsets and IL-17A and IL-10 production capacities in Th17 cells from DSS-treated mice.(F) Linear regression analysis of Il17a and Il1r1 average expressions in colonic Th17 subsets from the indicated conditions as determined by scRNA-seq.(G) IL-1Rhigh proportion and IL-1R geometric mean fluorescence (gMFI) in colonic RORγtWT and RORγtS182A Th17 cells from DSS-challenged mice. Each dot represents the result from 1 mouse. Each bar represents the sample mean. * p < 0.05 (paired t test).(H) Representative flow cytometry analysis of IL-1R in colonic Th17 cells from DSS-challenged mice harvested on day 10. Forward scatter (FSC).

Fig 3: Transfer of RORγtS182A CD4+ T cells promote exacerbated wasting in Rag1−/− mice(A) Weight changes of Rag1−/− mice receiving RORγtWT (n = 6) or RORγtS182A (n = 6) naive CD4+ T cells. Each dot represents the result from 1 mouse. Each bar represents the sample mean. **p < 0.01 and ***p < 0.001 (multiple t test).(B) RNA expression level of Il17a and Il10 in total cLP cell lysate from Rag1−/− mice in (A). Each dot represents the result from 1 mouse. Each bar represents the sample mean. *p < 0.05; n.s., not significant (t test).(C) Right: Activated RORγtWT (transduced with GFP expressing pMIG construct) or RORγtS182A (transduced with Thy1.1 expressing MSCV construct) CD4+ T cells were mixed in a 1:1 ratio and injected into Rag1−/− mice. Left: Summarized proportion of IL-17A+ and IL-10+ colonic Th17 and RORγt+ Treg cells from Rag−/− recipients 33 days post-transfer (n = 5). Each line represents results from same Rag1−/− recipient. Each bar represents the mean from 5 Rag1−/− recipients. *p < 0.05; n.s., not significant (multiple t test).(D) Representative flow cytometry analysis of IL-17A and IL-10 in colonic Th17 and RORγt+ Treg cells from (C).

Fig 4: GPR183 Enhances IL-22 Production by Colonic ILC3s(A) IL-22 production by ILC3s from the colon or small intestine of Gpr183+/+ and Gpr183-/- mice. Numbers indicate cell frequencies.(B) Frequency of intestinal ILC3s producing IL-17A, IL-17F, and IL-22 and frequency of ILC2s producing IL-5 from Gpr183+/+ and Gpr183-/- mice (n = 3–4).(C) Frequency of IL-22-producing ILC3s from mesenteric lymph nodes (MLN), small intestine (SI), and colon of B6 mice after stimulation with IL-1ß and IL-23 in the presence of solvent control dimethyl sulfoxide (DMSO) or 10 nM 7a,25-OHC (n = 10).(D) Csf2 mRNA expression in the colon (top; n = 8–9) or in sorted colonic ILC3s (bottom; n = 2) from Gpr183+/+ and Gpr183-/- mice on a Rag1-deficient background. mRNA expression was normalized to Hprt.Data are represented as means ± SEM. **p < 0.01, ***p < 0.001 by Student’s t test. Data are representative of or combined from two experiments. See also Figure S6.

Fig 5: Cultured RORγtS182A Th17 cells harbor augmented cytokine production potential in response to IL-1β signaling(A) Left: Workflow of the co-culture experiment using the indicated transduced vectors and marked RORγtWT and RORγtS182A CD4+ T cells polarized in the presence of IL-6 (20 ng/mL), IL-1β (20 ng/mL), IL-23 (25 ng/mL), and TGF-β (0.3 ng/mL). Right: Summarized proportion of IL-17A+ in cultured cells 3 days postpolarization. Each line represents results from 1 experimental well. ****p < 0.0001 (paired t test).(B) Summarized proportion of IL-17A+ in Th17 cells cultured in the indicated conditions. Each dot represents the result from 1 mouse. Each bar represents the sample mean. *p < 0.05, **p < 0.01; n.s., not significant (paired multiple t test).(C) Representative flow cytometry analysis of IL-17A and IL-17F expression in cultured Th17 cells from (B).(D) RORγt gMFI in cultured Th17 cells from (B). Each dot represents the result from an independent experiment. Each bar represents the sample mean. n.s., not significant.(E) Immunoblot (IB) analysis of total RORγt or RORγt phosphorylated at S182 (pS182) in cultured Th17 WCL from 2 pairs of RORγtWT and RORγtS182A littermates.(F) Proportion of IL-17A+ among cultured RORγtS182A Th17 cells transduced with retroviruses carrying RORγtWT (circle) (Huh et al., 2011), RORγtS182A (triangle), or RORγtS182D (square) expression constructs. Each dot represents the result from an independent experiment. Each bar represents the sample mean. *p < 0.05 (t test).(G) HEK293 cells transfected with empty or 2xFLAG-RORγt expression constructs for 16 h were stimulated with the indicated cytokines. RORγt was captured by anti-FLAG beads and pS182 status was detected by immunoblot.