Freshly isolated CD4+ T cells were plated with IL-2 (25 U/ml) in 24-well plates precoated with 2C-11 (1 μg/ml) and PV1. After 48 hours, cells were harvested, centrifuged, and resuspended in viral supernatants. Cells were then spinoculated by centrifugation for 2 hours at 1200g at 37°C. Cells were then cultured with IL-2 (25 U/ml) and used on day 5 after initial harvest. For imaging, Lab-Tek 8 chamber slides (Thermo Fisher Scientific) were coated with ICAM-1 (2 μg/ml) overnight at 4°C. CD4+ T cells were washed and resuspended in L15 medium (Gibco) supplemented with glucose (2 mg/ml) and added to the chambers. After 20 min, chambers were gently washed to remove unbound cells and then imaged using the confocal system described above. Image analysis and preparation was done using Velocity v6.3 and ImageJ software. Briefly, T cells were divided into thirds, and the mean fluorescence intensity of the membrane edge of the front third and the rear third was determined. Data are displayed as the ratio of the front third to the rear third, which corresponds to relative enrichment of PIP3.

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