Activation and Expansion of Memory CD4+ T Cells

A simplified VOA was performed as we previously described (55 (link)). Briefly, memory CD4⁺ T cells were cultured at 1 × 10⁶ cells/well in 1 mL media (RPMI, 10% FBS, 1% P/S) in a 48-well plate (Costar) coated with CD3 Abs (1 μg/mL; BD Biosciences, Clone UCHT1) and in the presence of soluble CD28 Abs (1 μg/mL; BD Biosciences, Clone CD28.2). At day 3 poststimulation, cells from each original replicate were individually washed with media, split into two new CD3 Abs-uncoated 48-well plates, and cultured in media containing IL-2 (5 ng/mL; R&D Systems) in the presence or in the absence of GGSK2691805A (5 μM). The cells from each well were further split into two new wells (without washing) at day 6 and 9 poststimulation, with 50% of the media being refreshed with IL-2 with/without GSK2691805A. The cells were kept in culture for a total of 12 d.