Figure 3. The CTLA‐4 cytoplasmic domain promotes efficient transendocytosis of CD80 and CD86.

• A, B
  CTV‐labelled B cells (DG‐75) co‐expressing PD‐L1mCherry and CD80GFP (A) or PD‐L1mCherry and CD86GFP (B) were incubated with Jurkat cells expressing no CTLA‐4, CTLA‐4 WT or a mutant CTLA‐4 (Del36) lacking the cytoplasmic tail. Transendocytosis was carried out at a 1:1 ratio for the indicated times and analysed by flow cytometry. FACS plots show GFP ligand loss from donor B cells (upper left quadrants) and acquisition by Jurkat recipients (lower right quadrants).
• C
  Levels of CD80GFP and PD‐L1mCherry remaining on donor cells over a 24 h transendocytosis period with CTLA‐4 WT or Del36, plotted as a percentage relative to no CTLA‐4 control (mean ± SEM, six independent experiments). ****P ≤ 0.0001, RM one‐way ANOVA.
• D
  Levels of CD86GFP and PD‐L1mCherry remaining on donor cells over a 24 h transendocytosis period with CTLA‐4 WT or Del36 plotted relative to no CTLA‐4 control (mean ± SEM, six independent experiments). ****P ≤ 0.0001, RM one‐way ANOVA.
• E
  Comparison of CTLA‐4 surface expression levels in Jurkat cells expressing CTLA‐4 WT or CTLA‐4 Del36 as determined by anti‐CTLA‐4 (clone BNI3 at 1:100 dilution) stain on ice and analysed by flow cytometry. Graph shows mean ± SEM from four independent experiments, **P ≤ 0.01, Student's two‐tailed independent samples t‐test.