CTLA4 binds and depletes T cell–intrinsic CD80 in cis. (A) Left: Experimental scheme. Right: Representative confocal images and quantification violin plot showing how coexpression of CD80 or CD86 affected the B7-Ig staining of CTLA4^ΔICD. (B) Flow cytometry histograms showing how coexpression of CD80 or CD86 affected the B7-Ig staining of CTLA4^ΔICD. (C) CTLA4 regulation of endogenous CD80 expression on human Tregs. Human Tregs were precultured with CD80^−/−CD86^−/−CD86-GFP⁺ Raji, isolated by FACS, and incubated with 30-fold excess of CD28^−/− Jurkat filler cells for 6 h, in the presence or absence of ipilimumab and/or 28.3scFv before flow cytometry measurement of anti-CD80 staining. Data presented as three technical replicates for Tregs from a single human donor (male, age 30). Normalized MFIs were calculated by setting the absolute 0 as 0 and the MFI of “Treg untreated” condition as 100 in each replicate. (D) CTLA4 regulation of Myc-CD80 expression in Jurkat cells. CD28^−/− or CD28^+/+ Jurkat cells cotransduced with CTLA4 and Myc-CD80 were incubated with a 30-fold excess of CD28^−/− Jurkat filler cells for 1 h, in the presence or absence of ipilimumab before flow cytometry measurement of anti-Myc staining. Normalized MFIs were calculated by setting the absolute 0 as 0 and the highest MFI value among all the conditions as 100 in each replicate. Error bars: SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired two-tailed Student’s t test.