Fig. 3. CD28 and CTLA4 target trogocytosed CD80 to different cellular compartments.

a Images of trogocytosed CD80-TagRFP acquired by CD28- or CTLA4-TagGFP-expressing 58αβ A2 T cells, whose cell-surface and endosomal membranes were stained with wheat germ agglutinin lectin (WGA-L). Scale bar 5 µm. b–d Trogocytosed CD80 experiences different pH-levels upon acquisition via CD28 versus CTLA4. b Cartoon depicting design of pHluorin-CD80-mScarlet reporter. c, d CD28- or CTLA4-expressing 58αβ A2 T cells were co-cultured with CHO cells expressing pHluorin-CD80-TagRFP. Representative dot plots depicting pHluorin and TagRFP signals of T cells (c), quantification of pHluorin/TagRFP ratio (d, left) and extrapolation of pH-levels experienced by trogocytosed CD80 (d, right) based on standard curve shown in Figure S3. e Images and quantification of co-localization of late lysobisphosphatidic acid (LBPA) (late endosome) staining with CD80 trogocytosed by CD28- or CTLA4-TagGFP expressing 58αβ T cells. Scale bar 2 µm. f CTLA4 is more efficient in targeting trogocytosed CD80 for degradation than CD28. CD28 or CTLA4 expressing 58αβ A2 T cells were co-cultured with CHO cells expressing CD80-TagRFP-pHluorin with or without Bafilomycin. Plot depicts increase of TagRFP gMFI upon Bafilomycin treatment. See also Figure S3. Statistics: d: pHluorin/TagRFP: pooled data from 6 independent experiments. Two-sided paired t test (n = 6 biologically independent samples). pH: pooled data from 3 independent experiments. Two-sided paired t test (n = 3 biologically independent samples). e: Two-sided unpaired t test with Welch’s correction; p-value: 2 × 10⁻¹⁰ (n = 16 cells per group examined over 2 independent experiments). f: Pooled data from 8 independent experiments. Two-way repeated-measure ANOVA with Šídák’s correction, treated vs untreated (n = 8 biologically independent samples per group). Source data are provided as a Source data file.