Fig 2: 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

Fig 3: 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 4: T-dependent GC responses and Tfh cells arise spontaneously in spleens of CKO mice.a Spleen cells from indicated mice were analyzed by FACS. Live myeloidneg CD3neg CD19+ splenic B cells were gated to show IgMneg IgDneg B cells, which were further gated to reveal IgG1+ cells. b Data summarizing the numbers of GC B cells (CD19+ CD38neg CD95+) in spleens of control, heterozygous and CKO mice treated with indicated antibodies is shown. Each dot represents data for an individual mouse, n = 4–6 mice per mouse group. *p < 0.01, ns, not significant (p < 0.1), nonparametric Wilcoxon one-way test. c Live myeloidneg CD19neg CD3+ CD4+ splenic T cells from indicated mice were gated to show PD-1, CXCR5, ICOS, and intracellular Foxp3 expression. Fluorescent minus one (FMO) staining, in which the fluorescent anti-CXCR5 antibody was omitted from the staining cocktail, is used to define CXCR5-expressing cells. FACS histogram on the right shows the intracellular Bcl6 expression by Tfh (PD-1hi CXCR5+, red line) and non-Tfh cells (PD-1neg CXCR5neg, blue line) in CKO mice. d Data summarizing numbers of Tfh cells (CD3+ CD4+ PD-1hi CXCR5+) in spleens of each mouse group is shown, n = 5–9 mice per mouse group, *p < 0.001, **p < 0.003, ns, not significant (p < 0.1), All mice are 2–3 months old. Box plots in c, b: box draws 75% (upper), 50% (center line), and 25% (down) quartile, the maxima and minima outliers are shown as top and bottom line, respectively.

Fig 5: CTLA-4 is selectively expressed by B-1 cells within the resting B-cell compartment.a Ctla4 expression by mature B-cell subsets in adult C57BL/6J mice (2–3 months old) was measured by qRT-PCR. B-cell subsets were phenotypically defined as: B-1a, CD19+ IgMhi IgDlo/- CD21lo/- CD43+ CD5+; B-1b, CD19+ IgMhi IgDlo/- CD43+ CD5neg; MZB, CD19+ IgMhi IgDlo/- CD21hi CD43neg CD5neg; FOB and peritoneal B-2, CD19+ IgMlo IgDhi CD43neg CD5neg; PCs, CD19+ IgDneg CD138+ CD267+. Ctla4 expression levels are shown as the data relative to the Ctla4 level expressed by splenic CD3+ CD4+ CD25+ Treg cells (>90% Foxp3+) using comparative CT method 2–??CT. Each dot represents data for an individual mouse, n = 5–9 mice per subset. *p < 0.003, **p < 0.007, ***p < 0.001, ****p < 0.002. b Ctla4 expression by splenic B-1a (sB-1a) and non B-1a cells in neonatal, young or adult mice was measured by qRT-PCR. D, day, W, week, M, month. FACS gating is shown in Supplementary Fig. 1B. Ctla4 expression levels are shown as the data relative to the Ctla4 level expressed by adult sB-1a. n = 5–7 mice per group, *p < 0.001, **p < 0.003, ***p < 0.006, ns, not significant (p < 0.5). c CTLA-4 expression by sB-1a, peritoneal B-1a (pB-1a), FOB, MZB, peritoneal B-2 (pB-2), and splenic Treg (sTeg) cells from indicated mice were measured by intracellular CTLA-4 FACS analysis. FACS gating is shown in Supplementary Fig. 1C. y axis shows surface CD5 expression for B-cell subsets and intracellular Foxp3 expression for sTreg cells, x axis shows data for cells stained with phycoerythrin (PE)-conjugated anti-CTLA-4 or isotype control antibodies. d Data summarizing independent FACS analyses (n = 6) is shown, each dot represents data for an individual mouse. y axis shows ratio of medium fluorescent intensity (MFI) values of the indicated B-cell subsets stained with PE-conjugated anti-CTLA-4 vs. isotype control antibody. *p < 0.0001, **p < 0.03, ***p < 0.01, ****p < 0.05, *****p < 0.007, ns, not significant (p < 0.6). Box plots in a, b, d: box draws 75% (upper), 50% (center line), and 25% (down) quartile, the maxima and minima outliers are shown as top and bottom line, respectively. Statistical significance was tested using nonparametric Wilcoxon one-way test.