Figure 8. Mutation at Y188 diminishes the efficiency of NF-κB nuclear translocation.

CD28-deficient T cells were retrovirally transduced with WT CD28, Y188F CD28, or empty vector (MIG). CD28+/+ and CD28-deficient (CD28KO) T cells were included as controls. Conjugates with these T cells and Ag-pulsed ProAd-ICAM-B7 were stained for CD28 (green), NF-κB p65 (red), and the nucleus (blue) 45 minutes after conjugate formation. A, Representative images are shown. The T cell is oriented toward the top in each panel. B, Percentage of conjugates that translocated visually detectable levels of NF-κB into the nucleus (n = 40 conjugates). CD28+/+ vs CD28KO (p<0.0001), WT vs Y188F (p = 0.121), and Y188F vs MIG (p<0.0001) by two population proportion Z-student statistical test. C, The intensity of NF-κB staining in the nucleus of individual T cells is shown and the thick line represents the median of each population sample. The thin line across all the samples represents the limit of detection for visual identification of NF-κB translocation into the nucleus. Although Y188F CD28 did not impact on the frequency of T cells that activated NF-κB, the magnitude of NF-κB nuclear translocation was significantly reduced compared to T cells expressing WT CD28. One experiment representative of three is shown. The population distribution was compared using the nonparametric Mann-Whitney test; statistical significance between relevant samples is shown.