Fig. 6: CTLA-4 Arg70 mutants are defective in CD86 TE.
a, CTLA-4 WT or mutant proteins (Arg70Gln and Arg70Trp) expressed in Jurkat T cells tested for TE of CD80–mCherry and CD86–mCherry from ligand-expressing DG-75 B cells. CTV-labeled, ligand-expressing cells were incubated with CTLA-4-expressing cells (CTV−) and assessed for TE overnight. Detection of CD80 and CD86 acquisition is highlighted in the gray- and blue-shaded quadrants, respectively. b, Quantification of ligand remaining on the donor cell relative to no CTLA-4 controls. c, The amount of CD80–mCherry ligand detected inside CTLA-4+ recipient cells shown for the CTLA-4 mutants. BafA was added to evaluate the impact of lysosome blockade. d, The impact of Arg70Gln mutation on TE by human Treg cells. Arg70Gln or WT CTLA-4 was knocked into the endogenous CTLA-4 locus by HDR using a CRISPR–Cas9/AAV6 system. Knock-in cells were detected using a GFP reporter. GFP+ Treg cells expressing Arg70Gln mutant cDNA were compared with endogenous CTLA-4 (GFP−) or GFP+-expressing Treg cells containing WT cDNA for their ability to capture CD80 (gray quadrant) or CD86 (blue quadrant) from DG-75 B cells. e, Quantification of the experiment shown in d, using data from three independent samples. The statistical significance was determined by two-way ANOVA with Sidak’s multiple comparison correction (b and c) or two-tailed, unpaired Student’s t-test (e): **P < 0.01, ***P < 0.001, ****P < 0.0001. All data are presented as mean ± s.d. from three independent experiments (b and c) or three biologically independent samples (e).
