Figure 3. Cis-B7:CD28 interactions occur head-to-head and are promoted by negative membrane curvatures.

(A) Dot plots summarizing cis-CD80:CD28 FRET and cis-CD86:CD28 FRET when indicated CD28 and B7 were co-expressed in HEK293T cells. Fluorophores coupled to ECDs as in Figure 1C. n > 20 cells from three independent experiments.

(B) Ribbon diagrams of predicted CD80:CD28 and CD86:CD28 structures. Dash circles: indicate B7:CD28 binding interfaces. Red residues crucial for interactions.

(C) FRET efficiency images and dot plots summarizing cis-CD28:B7 FRET, cis-CD28:PDL1 FRET, and cis-PDL1:CD80 FRET, when the indicated protein pairs were co-expressed in HEK293T cells, with the fluorophores coupled to their ICDs, indicated in the cartoons. n > 36 cells from ≥ 3 independent experiments.

(D) A BiLC assay probing the CD28-ICD:B7-ICD proximity with LgBiT and SmBiT fused to the C-terminus of ICDs. Cartoon depicts the theoretical outcomes of head-to-head versus side-by-side binding. Low transfection efficiency allowed untransfected cells to serve as filler cells, limiting trans-contact of double positive cells. Positive control: PDL1:CD80 pair. Bar graphs show luminescence intensity ± blockade drug (Abatacept for cis-B7:CD28, atezolizumab for cis-CD80:PDL1).

(E) Same as D except that LgBiT and SmBiT were coupled to the N-terminus of ECDs, to probe ECD proximity within a cis-complex. Cartoon depicts the theoretical outcomes. Bar graph summarizes the experimental data.

(F) Control experiments showing limited trans-B7:CD28 interactions due to excess filler cells. LgBiT-CD28 singly-transfected cells incubated with SmBiT-B7 singly-transfected cells in the presence of excess untransfected filler cells. Luminescence data summarized in the bar graph as mean ± SEM from four independent experiments.

(E) Effects of FBP17-induced membrane curvatures on cis-B7:CD28 FRET. HEK293T cells expressed CLIP-CD28, SNAP-B7, FKBP-TagBFP-CAAX, and mGFP-FRB-FBP17. Cartoon depicts a likely series of molecular events: Rapa induces membrane recruitment mGFP-FRB-FBP17, which invaginates the membranes to promote cis-CD28:B7 interactions. Confocal images show Rapa effects on mGFP-FRB-FBP17 localizations. Dot plot summarizes cis-B7:CD28 and cis-CD80:PDL1 FRET of HEK293T cells treated with DMSO or Rapa, ± Abata from n > 21 cells from ≥ 3 independent experiments.

Unpaired two-tailed Student’s t test (D, E) and One-way ANOVA (B-F): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S3.