Fig 2: ROR?tS182A mice have exacerbated diseases in DSS-induced colitis(A) Weight changes of ROR?tWT and ROR?tS182A 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 ROR?tWT (n = 5) and ROR?tS182A (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 ROR?tWT (n = 4) and ROR?tS182A (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]ß+ROR?t+Foxp3-), ROR?t+ Treg (TCRß+ROR?t+Foxp3+), and conventional Tregs (TCRß+ROR?t-Foxp3+) in colons from DSS-challenged ROR?tWT and ROR?tS182A 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 ROR?tWT and ROR?tS182A 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: Normal T cell development and differentiation in ROR?tS182A mice(A) Proportion of modified and unmodified peptides identified by tandem mass spectrometry (MS/MS) mapping to murine ROR?t from whole-cell lysates (WCL) of HEK293 cells transfected with a 2xFLAG-mROR?t expression construct for 48 h. Phosphorylation (p), methylation (m), and ubiquitination (u).(B) Model diagram of ROR?t protein domains. Black vertical line indicates the position of the evolutionarily conserved serine 182. AF-1, activation function domain 1; DBD, DNA-binding domain; LBD, ligand-binding domain.(C) Weights of 2- to 4-month-old wild-type (ROR?tWT, n = 49) and ROR?tS182A (n = 57) adult mice obtained from heterozygous crosses. Each dot represents the result from 1 mouse.(D) Representative flow cytometry analysis of cell surface CD4 and CD8a on splenocytes from ROR?tWT and ROR?tS182A cohoused littermates. This experiment was repeated 3 times on independent biological samples with similar results.(E) UMAP plots depicting the transcriptomes of immune cell clusters (top) and cluster 2 subsets (bottom) obtained from colonic lamina propria (cLP) CD4+ T cells (10X Genomics droplet-based 3' scRNA-seq).(F) Violin plots of selected gene expressions in cells from the indicated clusters.(G) Proportions of colonic Cd4+Cd3e+ (left) and Cd4+Cd3e+Foxp3+ (right, cluster 2) T cells in each cluster from 2 pairs of ROR?tWT and ROR?tS182A littermates. Each bar represents the sample mean. * p < 0.05 (ratio paired t test).(H) Heatmap of mean scaled average expression of selected genes from the indicated clusters.(I) Violin plots (left) and UMAP (right) showing expression of Il17a and Il1r1 in colonic Th17 cell subsets (clusters 0 and 3) in steady-state ROR?tWT and ROR?tS182A mice.