Fig 2: CD28 and CTLA4 trogocytose CD80, but direct it towards different fates.a–d Trogocytosis of CD80 (a, c) or CD86 (b, d) by naive WT, Cd28−/− or Ctla4−/− CD8+ T cells (a, b) or WT CD8+ T cells upon CD28- or CTLA4-blockade (c, d). e Quantification of CD80 trogocytosed by GFP-, Cd28- or Ctla4-transduced Cd80−/−Cd86−/− CD8+ T cells. f, g Expression of CTLA4 (f) and its correlation with trogocytosed CD80 on T cell surface (g) of titrated numbers of Cd80−/−Cd86−/− P14 CD8+ T cells activated in flat-, u-, or v-bottom plates for 28 h. To correct for different trogocytosis rates, surface CD80 levels were normalized to total acquired CD80-TagRFP (αCD80-PE-Cy7/TagRFP). h Quantification of trogocytosed CD80 on surface of GFP-, Cd28- or Ctla4-transduced Cd80−/−Cd86−/− CD8+ T cells. Note that only CD28-, but not CTLA4-overexpression in T cells increases CD80-surface expression. i Images and quantification of co-localization of wheat germ agglutinin lectin (WGA-L) surface staining with CD80 trogocytosed by CD28- or CTLA4-expressing 58αβ T cells. Scale bar 2 µm. j Trogocytosis of CD80 via CD28 but not via CTLA4 enables co-stimulation of neighboring cells. Cd28- or Ctla4-transduced Cd80−/−Cd86−/− B cells were co-cultured with CD80-mScarlet expressing MEFs, FACS-sorted for mScarlet-expression, gp33-antigen-loaded and co-cultured with Cd80−/−Cd86−/− P14 CD8+ T cells with or without addition of CTLA4-Fc (“specificity control”) or agonistic αCD28 antibodies (“positive control”). Plots depict day 3 T cell counts (left) and CD25 expression (right). See also Figure S2. Statistics: a: Pooled data from 3 independent experiments. Brown-Forsythe/Welch’s ANOVA with Dunnett’s T3 correction. WT and Cd28−/− (n = 6 biologically independent samples), Ctla4−/− (n = 4 biologically independent samples). b: Pooled data from 3 independent experiments. Brown-Forsythe/Welch’s ANOVA with Dunnett’s T3 correction. p-values: WT vs. Cd28−/−: 0.00001; WT vs. Ctla4−/−: 0.27 WT (n = 9 biologically independent samples), CD28−/− (n = 4 biologically independent samples), Ctla4−/− (n = 7 biologically independent samples). c: Pooled data from 3 independent experiments. Brown-Forsythe/Welch’s ANOVA with Dunnett’s T3 correction. p-values: medium vs. α-CD28-Fab: 1 × 10−15; medium vs. α-CTLA4-Fab: 0.84 (medium and α-CD28-Fab: n = 10 biologically independent samples, α-CTLA4-Fab: n = 8 biologically independent samples). d: Pooled data from 3 independent experiments. Brown-Forsythe/Welch’s ANOVA with Dunnett’s T3 correction. p-values: medium vs. α-CD28-Fab: 4 × 10−8; medium vs. α-CTLA4-Fab: 0.61. (medium and α-CD28-Fab: n = 11 biologically independent samples, α-CTLA4-Fab: n = 8 biologically independent samples). e: Pooled data from 3 independent experiments. Repeated-measures one-way ANOVA with Dunnett’s T3 correction. p-values: GFP vs. Cd28: 4 × 10−7; GFP vs. Ctla4: 0.000017 (n = 10 biologically independent samples per group). f: Representative data from 1 of 2 independent experiments. (n = 3 biologically independent samples per group). g: Representative data from 1 of 2 independent experiments (n = 3 biologically independent samples per group). h: Pooled data from 3 independent experiments. Friedmann test with Dunn’s correction. p-values: GFP vs. Cd28: 0.015; GFP vs. Ctla4: p value: 0.36 (n = 10 biologically independent samples per group). i: Two-sided Mann–Whitney test; p-value: 2 × 10−10 (CD28: n = 28 cells and CTLA4: n = 25 cells, examined over 2 independent experiments). j: Representative data from 1 of 3 independent experiments. Repeated-measures one-way ANOVA with Holm-Šídák’s correction (n = 3 biologically independent samples per group). Source data are provided as a Source data file.

Fig 3: Fc.Mut24 activates different Foxp3+ TR cell subsets(A) Number and proliferation (Ki-67) of CD25+ Foxp3+ TR and the expression of indicated TR activation markers. Gates were set on live, CD4+ cells.(B) Percentage, number, and proliferation of CD44lo CD62Lhi central TR (cTR), CD44hi CD62Lhi central-effector TR (ceTR), and CD44hi CD62Llo effector TR (eTR) in each treatment group. Gates are indicated on representative flow-cytometry plots.(C) Expression of indicated TR activation markers on TR subsets.All graphs show mean ± SD with individual data points (n = 6–11); ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗∗p ≤ 0.0001, two-tailed unpaired t tests, or multiple unpaired t tests. n.s., not significant. Data are representative of 2–3 independent experiments.

Fig 4: Ezh2Y641F Treg cells have a competitive advantage over WT Treg cells(A) Generation of Treg cell mosaic mice.(B) Representative flow plots and normalized ratios of YFP+RFP+/GFP+RFP− Treg cells from LNs of Treg cell mosaic mice.(C) Frequencies of Treg cells in LN.(D) Normalized H3K27me3/histone H3 levels in Treg cells from LN.(E) Normalized ratios of YFP+RFP+/GFP+RFP− Treg cells from lung, gut, and skin of Treg cell mosaic mice.(F) Representative flow plots and frequencies of YFP−RFP+ in LN.(G) Frequencies of IL-10+ Treg cells in LNs of Treg.Ezh2WT/Ezh2WT and Treg.Ezh2Y641/Ezh2WT mice.(H–K) Frequencies of CD44+ (H); TIGIT+ or PD1+ (I); CD103+, CD69+, or CD25+ (J); and CXCR3+, CCR8+, CCR6+, and IL33R+ (K) of YFP+RFP+ Treg cells in LNs of Treg.Ezh2WT/Ezh2WT and Treg.Ezh2Y641/Ezh2WT mice.Data are mean ± SEM and representative of at least two independent experiments (n = 4–11 mice total per genotype). Unpaired two-tailed Student’s t test was used; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.See also Figure S3.

Fig 5: Acute induction of Ezh2Y641F in Treg cells increases their activation and effector differentiation state(A) Generation of Treg.iEzh2Y641F model.(B) Normalized H3K27me3/histone H3 levels of CD4+Foxp3+ Treg cells in LN.(C) Frequencies (left) and numbers (right) of Treg cells in LN.(D–F) Frequencies of TIGIT+ or PD1+ (D); CD103+, CD69+, or CD25+ (E); and CXCR3+, CCR8+, and CCR6+ (F) Treg cells in LN.Data are mean ± SEM (n = 6–18 mice per group pooled from two independent experiments). Unpaired two-tailed Student’s t test was used; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.See also Figure S4.