p110δ is essential for PIP3 accumulation to the plasma membrane. (A) Images showing fluorescence and transmitted light from a movie of AktPH-GFP⁺ WT, p110δ^D910A/D910A, p110γ^−/−, CD28^−/−, and CD28^Y170F CD4⁺ T-cell blasts interacting with LPS-activated APCs presenting OVA_323-339 peptide. In some experiments, WT cells were pretreated with PI3K inhibitor (LY294002) for 15 minutes before stimulation. (B) Quantification of AktPH-GFP redistribution in WT (n = 11), LY294002 pretreated WT (n = 8), p110δ^D910A/D910A (n = 9), p110γ^−/− (n = 12), CD28^−/− (n = 9), and CD28^Y170F (n = 7) CD4⁺ T cells. A few LY294002-treated T cells showed apparently normal AktPH-GFP accumulation (Figure S3). It is possible that a subset of T cells express sufficient levels of multidrug resistance protein transporter and could potentially thus evade inhibition.^81,82  Therefore, to establish a realistic baseline for minimal PI3K activity, these outliers were excluded from the analysis, but they are shown in Figure S3. The recruitment of AktPH-GFP at the plasma membrane is expressed as a ratio between the fluorescence measured at the contact area and the average fluorescence within the cell. Fluorescence was measured on at least 2 independent days. (C) Average ratio of PIP3 at membrane during the plateau phase of the response (approximately 40-400 seconds). The mean fluorescence values were determined as described in “Methods” (**P < .01, ***P < .001). (D) Acute inhibition of PI3K activity with the p110δ-selective inhibitor IC87114. Two T cell–B cell conjugates are shown after the addition of IC87114 as indicated by arrows. PIP3 depletion from the membrane occurred rapidly in both cases.