Fig 2: Sharpin deficiency in Treg causes cpdm-like skin inflammation. a Western blot analysis of LUBAC components from sorted CD4+CD25+Foxp3+ Treg. Actin is indicated as a loading control. Asterisks indicate non-specific bands. b Percentage of Foxp3+CD25+ Treg within the CD4+ T cell population, absolute cell number of Treg, and the mean fluorescent intensity (MFI) of Foxp3 in Treg in the spleen and peripheral LNs (pLNs) from 10-week-old mice. n = 5 biologically independent animals. c MFI of signal mediators in Treg upon CD3/28 antibody-based activation. Intracellular staining was performed. Data for p-p65 or I?Ba/p-ERK were acquired 5 and 10 min after activation, respectively. n = 6 biologically independent animals. d Expression of selected genes associated with Treg function and TCR-inducible Treg markers is presented in heat maps. Data are derived from three independent experiments. e Representative plots and percentage of effector (eTreg; CD44hiCD62Llo) and central (cTreg; CD44loCD62Lhi) Treg subsets in the spleen and pLNs. n = 4 biologically independent animals. f Percentage of CCR6+ effector Treg subsets. n = 9 biologically independent animals. g Percentage of Ki67+ Treg subsets. n = 5 biologically independent animals. h Appearance of skin inflammation at 10 weeks old. i Representative photos of H/E-stained skin of Sharpinfl/flFoxp3Cre, control, and cpdm mice. Scale bar: 200 µm. Small circles in the graphs indicate data from an individual mouse. Small horizontal lines indicate the mean (±s.e.m.). ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Two-tailed Mann–Whitney U-test was used for b, c, and e–g. Data are pooled from at least three independent experiments (b, c, e–g). Also, see Supplementary Fig. 1. Source data are provided in a Source Data file

Fig 3: PRMT5 inhibition suppresses Cyclin E1/CDK2 expression and Rb hyperphosphorylation in inflammatory T cells. (A) Diagram of the expression of cyclins and CDKs at the different cell cycle stages. Created with BioRender.com, modified from a template. (B–G) mTh1 cells were activated on anti-CD3/CD28 coated plates, treated with 5 µM HLCL65 or vehicle control (DMSO) and harvested at various timepoints for protein analyses. (B) Immunoblots of mTh1 cells probed with cell cycle proteins at resting, 24h and 48h post-plating. (C–G) Protein quantification of (C) Cyclin D1, (D) Cdk4, (E) Cyclin E1, (F) Cdk2, (G) ratio of phosphorylated Rb at S608 to total Rb. DMSO values are relative to resting. Data pooled from 4-6 independent experiments, n=6-10, student’s paired t-test. *p < 0.05, **p < 0.01.

Fig 4: PRMT5 inhibition arrests TCR-induced cell cycle progression at the G1/S checkpoint. (A, B) mTh1 cells were activated on anti-CD3/CD28 coated plates, treated with 5 µM HLCL65 or vehicle control (DMSO) and harvested at the time of plating (resting) or 24, 48 or 72h post-plating. They were analyzed by immunoblot with PRMT5 (A) or H4R3 (B). Data plotted represent relative protein expression at indicated timepoints. Plots are combined 4-6 experiments, n=8-13/timepoint/condition. (C) mTh1 cells were plated on anti-CD3/CD28 coated plates, treated with vehicle (DMSO), 5 µMHLCL65 or 5 µM EPZ, and pulsed with tritiated 3H-thymidine at 24, 48 or 72h post-plating with vehicle or HLCL65. Cells were harvested 16-18h later. This is a representative experiment with 6 technical replicates/group, analyzed by two-way ANOVA with Tukey’s multiple comparisons test. (D) Supernatants from mTh1 cells plated on anti-CD3/CD28 coated plates were collected at 4h, 8h, 18h, 24h and 48h ± HLCL65, EPZ or vehicle and analyzed for IL-2 cytokine expression by ELISA. Data pooled from 3 independent experiments. Data analyzed by two-way ANOVA with Tukey’s multiple comparisons test. (E) Diagram of the cell cycle stages including Gap 1 (G1), Synthesis (S), Gap 2 (G2) and Mitosis (M). Cells represent the stage the cell is in at each phase of the cell cycle. Red lines on the cell cycle represent cell cycle checkpoints. Created with BioRender.com, modified from a template. (F) Flow cytometry plots of activated mTh1 cells demonstrating the gating strategy and representative results in the negative control (no BrdU), vehicle (DMSO) or PRMT5 inhibitor (2.5 µM HLCL65). (G) Quantification of the results from analyses in (F), corresponding to four independent experiments. Graphs for Gap 0/Gap 1 (G0/G1), Synthesis (S) and Gap 2/mitosis (G2/M). Results analyzed by paired t-test, n=7 pairs. Graphs are line or bar graphs representing mean ± SD with individual data points plotted (A–D) or before-after (plot individual points) (G). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: Sharpin predisposes mice to spontaneous T cell-dependent dermatitis. a Representative appearance of 12-week-old control, healthy, and sick Sharpinfl/flLckCre (left) mice and photos of H/E-stained skin (right). Scale bar: 200 µm. b Incidence of spontaneous dermatitis. All mice were monitored over a period of 6 months. c Percentage of effector CD44hiCD62Llo or memory CD44hiCD62Lhi subsets within the CD4+ or CD8+ T cell populations in the spleen and pLNs from 12-week-old healthy Sharpinfl/flLckCre mice. n = 6 biological independent animals. d Percentage of recently activated CD69+ cells within the CD8 T cell population in pLNs. n = 4 biological independent animals. e MFI of I?Ba in T cells at 10 min after CD3/28 antibody-based TCR stimulation. Intracellular staining was performed. n = 6 biological independent animals. f MFI of Nur77 in T cells at 3 h after CD3 or CD3/CD28 antibody-based TCR stimulation. n = 3 biological independent animals. g CD69 and CD25 expression by CD4+ or CD8+ T cells at the indicated times after TCR stimulation. h Experimental induction of colitis by naïve CD4+ T cells. Rag2-/- mice were intravenously injected with HBSS alone or with 2.5 × 105 sorted naïve CD4+ T cells (CD45RBhiCD25-CD4+CD3e+), which were obtained from pooled cells isolated from the spleen and pLNs of 4-week-old mice. Bodyweight of injected Rag2-/- mice was monitored twice or thrice weekly. Data indicate average values. i Representative photos of H/E and immunofluorescent staining against CD3+ T cells within the inflamed colon of mice in h. j Percentage of CD25+CD4 T cells in the mesenteric LN (mLN) of mice in h. n = 4 biological independent animals. Small horizontal lines indicate the mean (±s.e.m.). ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001. Kruskal–Wallis test with Bonferroni correction (a value = 0.05) was used for h, while two-tailed Mann–Whitney U-test was used for c–f and j. Data are pooled from at least three independent experiments (c–g, j). Also, see Supplementary Fig. 2. Source data are provided in a Source Data file