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. 2016 Oct 5;113(42):E6437–E6446. doi: 10.1073/pnas.1603321113

Fig. 7.

Fig. 7.

The superantigen strongly enhances B7-2/CD28-dependent synapse formation. (A) Effect of SEB on binding of B7-2 to cell-surface CD28. HEK 293T cells were transfected to express cell-surface CD28 (12) or with empty vector (EV). Cells were incubated with soluble B7-2 in the absence or presence of SEB at concentrations shown. Western blots show binding of B7-2 and expression of CD28 by the cells. Bound B7-2 is quantitated in the bar graphs (error bars, SEM; n = 3). (B) Effect of SEB on binding of CD28 to cell-surface B7-2. HEK 293T cells were transfected to express cell-surface B7-2 or with empty vector. Cells were incubated with soluble CD28 in the absence or presence of SEB at concentrations shown. Western blots show binding of CD28 and expression of B7-2 by the cells. Bound CD28 is quantitated in the bar graphs (error bars, SEM; n = 3). (C and D) SEB specifically enhances the B7-2/CD28 interaction. HEK 293T cells were transfected to express cell-surface CD28 (C) or B7-2 (D) or with empty vector. Cells were incubated with soluble B7-2 (C) or CD28 (D) in the absence or presence of SEB (μg/mL). Western blots show binding of B7-2 (C) and CD28 (D) and expression of CD28 and B7-2, respectively, by the cells. Representative experiments of three are shown. (E) SEB enhances B7-2/CD28-mediated intercellular synapse formation. HEK 293T cells transfected to express CD28/GFP fusion protein (green label) were incubated with HEK 293T cells transfected to express B7-2/Cherry fusion protein (red label), in the absence or presence of SEB at concentrations shown. As negative control served mutant B7-2C/Cherry, which lost the ability to bind CD28. Intercellular B7-2/CD28-dependent synapse formation was scored using flow cytometry to quantitate percent doubly labeled cells (error bars, SEM; n = 4). (F–J) Contour plots are shown for a representative experiment in E, upon incubation of cells expressing CD28/GFP with cells expressing B7-2C/Cherry (F) or B7-2/Cherry (G–J). Incubation was done in the absence of SEB (F and G) or presence of SEB at 0.1 (H), 0.3 (I), or 1 (J) μg/mL. Percent doubly labeled cells in the upper right quadrant denote B7-2/CD28 synapse formation. (K) tk2 mutant SEB fails to enhance B7-2/CD28-mediated intercellular synapse formation. Effect of tk2 SEB on intercellular B7-2/CD28 engagement was analyzed by flow cytometry as in E (error bars, SEM; n = 3).

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