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.