CD28, but not p110δ, regulates PKCθ localization at the synapse. (A) Conjugates between naive WT, WT + LY294002, p110δ^D910A/D910A, p110γ^−/−, CD28^−/−, or CD28^Y170F CD4⁺ T cells and OVA_323-339–pulsed APCs were stained with antibodies against PKCθ and the TcRβ. Representative images of the distribution of AktPH-GFP, TcR, and PKCθ during conjugate formation are shown. (B) Quantification of AktPH-GFP recruitment at the plasma membrane (WT, n = 72; WT + LY294002, n = 31; p110δ^D910A/D910A, n = 53; p110γ^−/−, n = 23; CD28^−/−, n = 51; CD28^Y170F, n = 62), determined as described in “Methods” (**P < .01, ***P < .001). (C) Recruitment of PKCθ to the contact area (WT, n = 76; WT + LY294002, n = 24; p110δ^D910A/D910A, n = 28; p110γ^−/−, n = 28; CD28^−/−, n = 33; CD28^Y170F, n = 67), expressed as the ratio between the fluorescence at the contact area and the fluorescence within the cell. Only the means of the values for the CD28^−/− and CD28^Y170F values were significantly different from the WT values (***P < .001). (D) A 3-dimensional reconstruction of the contact area was done and representative pictures of the interface projection and the mean area (± SEM, *P < .05, **P < .001, Student 2-tailed t test; WT, n = 38; p110δ^D910A/D910A, n = 18; CD28^−/−, n = 23; CD28^Y170F, n = 21) occupied by PKCθ at the immune synapse are shown. Data are representative of 2 independent experiments. (E) The YMNM motif of CD28 is not required for NF-κB nuclear translocation. Conjugates between WT, CD28^−/−, or CD28^Y170F CD4⁺ T cells and OVA_323-339–pulsed APCs were stained for p65 NF-κ, and nuclei were stained with 7-AAD. The conjugates were scored for the frequency of nuclear localization of NF-κB. The p110δ inhibitor IC87114 blocked NF-κB nuclear translocation in CD28^−/− and CD28^Y170F T cells. Data represent mean (± SEM) with more than 30 cells analyzed in each of 3 experiments.