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

(A and 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 (A) 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) shows relative cell surface expression of WT CD28 (thick line) and K118I/K120P (KK/IP) CD28 (thin line). (C and D) Representative images of WT and K118I/K120P CD28-YFP localization in cell:cell conjugates with CD80-negative (C) and CD80-positive (D) APC in the presence (+Ag) and absence (-Ag) of TCR signaling. DIC (digital image correlation) and fluorescent images are shown. (E) 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). (F) 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).