K118I/K120P CD28 is polarized toward CD80-positive cells in the absence of TCR signaling.

<p>(<b>A</b> and <b>B</b>) CD28-deficient, DO11.10 CD4 T cells were retrovirally transduced with WT or K118I/K120P CD28 fused to YFP, stained with anti-CD28 and analyzed by flow cytometry. Two color display (<b>A</b>) shows total transduced protein expression as detected by YFP (FL1, x-axis) and cell surface expression of CD28 as detected by anti-CD28 staining (FL3, y-axis). Single color display (<b>B</b>) shows relative cell surface expression of WT CD28 (thick line) and K118I/K120P (KK/IP) CD28 (thin line). (<b>C</b> and <b>D</b>) Representative images of WT and K118I/K120P CD28-YFP localization in cell:cell conjugates with CD80-negative (<b>C</b>) and CD80-positive (<b>D</b>) APC in the presence (+Ag) and absence (-Ag) of TCR signaling. DIC (digital image correlation) and fluorescent images are shown. (<b>E</b>) Individual conjugates were visually scored for CD28 polarization toward the interacting APC and the percentage of conjugates displaying polarized CD28 is shown (n = 25–28). (<b>F</b>) The efficiency of CD28 recruitment to CD80-positive APC was calculated by determining the ratio of YFP fluorescence within the T cell:APC contact site to the YFP fluorescence in the T cell plasma membrane distal to the contact site. Values for individual cells, population medians and statistical analysis (non-parametric Kruskal-Wallis ANOVA with Dunn’s multiple comparison) are shown (ns, not significant; * p<0.05; ***p<0.001).</p>