Fig. 3: CD80 and CD86 direct CTLA-4 trafficking via different intracellular compartments.
a, PLAs in CHO cells showing the association between CTLA-4 and ubiquitin (red) and their co-localization (yellow) with CD80 or CD86 (green) after overnight TE with CTLA-4+ CHO cells labeled with CTFR and CD80/CD86–GFP CHO cells or CHO cells expressing NL at a 1:1 ratio in the presence of NH4Cl. Images were acquired by confocal microscopy and cells quantified for the number of PLA puncta (CTLA-4+Ubq+). The significance was calculated using the Mann–Whitney U-test: **P < 0.01 All data are presented as mean ± s.d. and show individual data points (n = 30 cells from one experiment representing three independent experiments). b, Co-localization of CTLA-4, ligand–GFP and markers of intracellular compartments in CTLA-4-expressing HeLa cells (human cells with appropriate morphology) and quantified using CellProfiler. The statistical significance was determined by two-way ANOVA with Sidak’s multiple comparison correction: *P < 0.05, **P < 0.01. All data are presented as mean ± s.d. and show individual data points (n = 8 fields of view examined over two independent experiments). c, CHO cells expressing CD80 or CD86 surface stained using CTLA-4–Ig and then washed at the pH indicated. CTLA-4–Ig remaining bound was detected by Immunoblot using anti-IgG. Lysates were immunoblotted for tubulin as a sample-processing control. Data represent four individual experiments. d, Impact of NH4Cl on CD80 and CD86 TE over time. CHO cells expressing GFP–ligands were labeled with CTV and co-incubated with CTLA-4-expressing CHO cells for 8 h (d) or the times shown (e) in the presence of 25 mM NH4Cl, and analyzed by flow cytometry. Detection of CD80 and CD86 acquisition is highlighted in the gray- and blue-shaded quadrants, respectively. All data are presented as mean ± s.d. (n = 4 independent experiments). f, Detection of available CTLA-4 in Jurkat T cells after overnight TE of DG-75 B cells expressing CD80, CD86 or without ligand (‘no TE’). Histograms show available CTLA-4 measured using anti-CTLA-4 antibody at 37 °C for 60 min and MFI of CTLA-4 staining quantified. The statistical significance was determined by two-way ANOVA with Sidak’s multiple comparison correction: ****P < 0.0001. All data are presented as mean ± s.d. and show individual data points. g, The TE experiment in f was repeated using CTLA-4+CD4+CD25+ human Treg cells. The statistical significance was determined by two-way ANOVA with Sidak’s multiple comparison correction: ****P < 0.0001. All data are presented as mean ± s.d. and show individual data points from four independent experiments.
