B7 ligands acquired by trogocytosis initially colocalize with CD28 prior to subsequent cis capture and internalization by CTLA4. (A) Experimental scheme for examining the localization of trogocytosed CD80 in Jurkat cells. After coculture with CD80^−/−CD86^−/−CD80-GFP⁺Raji cells, Jurkat (JF646-SPY-CD28⁺CTLA4-mCherry⁺) cells were isolated, incubated alone or in the presence of ipilimumab for indicated durations, fixed, and imaged for CD80-GFP, CD28, and CTLA4. (B) Left: Representative maximum intensity z-projection confocal micrographs showing the CD28/CD80/CTLA4 localizations in CD80-GFP acquired Jurkat (JF646-SPY-CD28⁺CTLA4-mCherry⁺) cells at indicated time points of post-sorting incubation. Right: A violin plot showing the relative FI values of CD80-GFP colocalized with CTLA4. (C and D) Same as A and B except replacing CD80-GFP with CD86-GFP and showing single images instead of maximum intensity z-projection images. (E) Cartoon depicting dual EM labeling strategy using CD4⁺ T cells expressing CTLA4-HaloTag conjugated with CD80^−/−CD86^−/−CD80-GFP⁺ Raji cells. Laser excitation at 549 nm induces oxidative polymerization of electron-dense DAB proximal to CTLA4-HaloTag (JFX-549), CD80 was labeled by 10-nm nanogold conjugated secondary antibody. (F) Electron micrographs showing CTLA4-HaloTag (DAB staining, white arrowheads) and CD80 (nanogold, red arrowheads). Scale bar: 5 µm. Error bars are SD from 40 cells. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; unpaired two-tailed Student’s t test.