Fig. 4: Altering CTLA-4 recycling affects TE of CD86 but not CD80.
a, TE assays carried out for 16 h using Jurkat T cells at a ratio of three donors (DG-75 mCherry ligand):one recipient to provide excess ligand. Recipient Jurkat T cells either lacked CTLA-4 (‘no CTLA-4’) or expressed CTLA-4 WT or CTLA-4 K-less variants. CTLA-4 WT was also expressed in Jurkat cells lacking LRBA after CRISPR–Cas9 targeting (LRBA KD). Representative FACS plots are shown in a and quantified in b for the amount of ligand acquired by the CTLA-4-expressing cell (shaded quadrant: gray CD80 and blue CD86). The far right-hand graph shows the uptake of CD86 relative to CD80 for each condition. The statistical significance was determined by the paired Student’s t-test: **P < 0.01. All data are presented as mean ± s.d. and show individual data points from three independent experiments. c, Kinetic analysis of the TE assay used in b. All data are presented as mean ± s.d. from four independent experiments. d, The impact of the K-less mutation on TE by human T cells. K-less or WT CTLA-4 was knocked into the endogenous CTLA-4 locus by homology-directed repair (HDR) using a CRISPR–Cas9/AAV6 system. Knock-in cells were detected using a GFP reporter. GFP+ T cells expressing WT or K-less cDNA, were analyzed for their ability to capture CD80–mCherry (gray quadrants) or CD86–mCherry (blue quadrants) from DG-75 B cells. The statistical significance was calculated using two-way ANOVA with Sidak’s multiple comparison correction: **P < 0.01. All data are presented as mean ± s.d. and show individual data points from three biologically independent samples.
