CTLA4 mediates T cell–intrinsic cis depletion of CD80 but not MHCII. (A) Experimental scheme. Human Tregs were precultured with CD80^−/−CD86^−/−CD80-GFP⁺ Raji cells in the presence of SEB, isolated by FACS, and incubated alone or with 30-fold excess of CD28^−/− Jurkat filler cells for 9 h, in the presence or absence of ipilimumab and/or 28.3scFv before flow cytometry measurement of CD80 and HLA-DR amounts. (B) Representative flow cytometry histograms and quantification graph of CD80 and HLA-DR amounts on Tregs before (0 h) and after 9 h incubation under the indicated conditions. Tregs at 0 h were preconditioned by CD80^−/−CD86^−/−CD80-GFP⁺ Raji cells as shown in A. Normalized MFIs of anti-CD80 and anti–HLA-DR were calculated by setting the MFI of “Treg untreated, isotype” condition as 0 and the MFI of “0 h, −, −, −” condition as 100. (C and D) Same as A and B except replacing human Tregs with human Tconvs. Error bars are SD from three independent coculture experiments using PBMCs from three independent age-matched donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired two-tailed Student’s t test.