Figure 1. CD28 and B7 are co-expressed on CD8⁺ T cells, colocalize on plasma membranes, and interact in cis.

A) Flow cytometry histograms showing expression of CD80 and CD86 in OT-1 T cells during in vitro priming using OVA_257–264. The line plot shows the percentages of CD28⁺CD80⁺, CD28⁺CD86⁺, and CD28⁺CD80⁺CD86⁺ OT-1 T cells during priming. Mean ± SEM from three OT-1 mice.

(B) STORM of CD28 and Spy-CD80, CD28 and Spy-CD86, or CD28 and Spy-PDL1 on the plasma membranes of HEK293T cells. Leftmost cartoons depict the experimental scheme: CD28 stained with anti-CD28 1^st antibody (Ab) and CF568-labeled 2^nd Ab; SpyTagged proteins stained with JF646-labeled SpyCatcher. 2^nd column: raw two-color STORM images of indicated proteins. Bars = 1 μm. 3^rd column: point rendering for two-color localizations computed by Coloc-Tesseler. Blue: CD28 colocalized with B7 or PDL1. Cyan: CD28 not colocalized with B7 or PDL1. Red: B7 or PDL1 colocalized with CD28. Yellow: B7 or PDL1 not colocalized with CD28. Upper dot plot: Manders coefficients calculated using CD28 as a reference (orange) or using CD80, CD86, or PDL1 as a reference (cyan), based on 2–3 random 3 × 3 μm² areas from each cell, n ≥ 6 areas from three independent experiments. Lower dot plot: number of molecules counted from each area, grey lines connect data points within the same areas.

(C) FRET data of cis-B7:CD28 interactions. Leftmost cartoons show cells co-expressing CLIP-CD28 (Dy547 labeled) and SNAP-tagged CD80, CD86, or PDL1 (AF647 labeled), ± abatacept (Abata). Columns 2–5: confocal images of Dy547 and AF647 channels before and after AF647 photobleaching. Bar = 5 μm. Column 6: calculated pseudo-color FRET efficiency image (yellow to violet denotes strong to weak FRET). Column 7: DIC image. Dot plot: FRET efficiencies (N_FRET) from n > 33 cells in ≥ three independent experiments. Mean ± SEM. One-way ANOVA: ****p < 0.0001. See also Figure S1.