Fig. 3

CD28 dimer interface mimetic peptide p2TA inhibits intercellular engagement of costimulatory receptor CD28 with B7-2 or B7-1. A p2TA inhibits binding of B7-2 to cell-surface CD28. HEK293T cells were transfected to express cell-surface CD28 or with empty vector (EV). Cells were incubated with soluble B7-2 in the absence or presence of p2TA at concentrations shown. Western blots show binding of B7-2 and equal expression of CD28 by the cells. Bound B7-2 is quantitated in the bar graphs; data are mean and SEM of three independent experiments. B p2TA inhibits binding of CD28 to cell-surface B7-2. HEK293T 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 p2TA at concentrations shown. Western blots show binding of CD28 and equal expression of B7-2 by the cells. Bound CD28 is quantitated in the bar graphs; data are mean and SEM of three independent experiments. C HEK293T cells were transfected to express cell-surface B7-2. Cells were incubated with soluble CD28 in the absence or presence of p2TA or its randomly scrambled form, p2TAsc [13], at concentrations shown. Western blots show binding of CD28 and expression of B7-2 by the cells. D p2TA attenuates intercellular B7-2/CD28 receptor engagement. HEK293T cells transfected to express CD28/GFP fusion protein (green label) were incubated with HEK293T cells transfected to express B7-2/mCherry fusion protein (red label), in absence or presence of p2TA at concentrations shown. As negative control served B7-2C/mCherry, which lacks the ability to bind CD28. Intercellular B7-2/CD28 receptor engagement was scored using flow cytometry to quantitate per cent doubly labeled cells. Data are mean and SEM of three independent experiments. E–I Contour plots for a representative experiment in D, upon incubation of cells expressing CD28/GFP with cells expressing B7-2/mCherry (E–H) or B7-2C/mCherry (I) in the absence or presence of p2TA (µg/mL). Per cent doubly labeled cells is denoted in upper right quadrant. J p2TAsc fails to attenuate intercellular B7-2/CD28 engagement, assayed as in D. Data are mean and SEM of three independent experiments (contour plots: Additional file 1: Fig. S2). K p2TA attenuates intercellular B7-1/CD28 engagement. Synapse formation was assayed as in D, using B7-1/mCherry fusion protein instead of B7-2/mCherry (contour plots: Additional file 1: Fig. S3). Intercellular receptor engagement was compared using the one-tailed unpaired student’s t-test; *p < 0.05, **p < 0.005