CTLA4-mediated CD80 cis depletion is promoted by TCR stimulation. (A) FACS histograms showing surface levels of CTLA4 and CD28 on human Tregs after 12 h coculture with Raji (CD80^−/−CD86^−/−CD80-GFP⁺) in the presence or absence of SEB. (B) Upper: Experimental scheme. Human Tregs were precultured with CD80-GFP-expressing Raji (CD80-GFP⁺CD80^−/−CD86^−/−) in the presence or absence of SEB, isolated by FACS, and incubated with 30-fold excess of CD28^−/− Jurkat filler cells for 9 h, in the presence or absence of ipilimumab and/or 28.3scFv before FACS measurement of CD80 and HLA-DR amounts. Lower: Representative FACS 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 Raji (CD80^−/−CD86^−/−CD80-GFP⁺) as shown in A. Normalized MFIs of anti-CD80 and anti–HLA-DR were calculated by setting the MFI of “Treg, isotype” as 0 and the MFI of “0 h, +, −, −” as 100. Error bars are SD from three independent coculture experiments, data were generated using PBMCs from three independent age-matched donors. *, P < 0.05; ***, P < 0.001; unpaired two-tailed Student’s t test.