Optimal CD4 ⁺ T cell stimulation conditions. (A and B) (A) Activation markers and (B) number of cell divisions of memory CD4⁺ T cells from a PWH stimulated at high cell density on day 0 for 24 h prior to distribution into 96-well culture plates on day 1. A 1:1 anti-CD3/CD28 bead-to-cell ratio was used. (C) Fold increase in total healthy donor CD4⁺ T cells from a starting number of 800 cells per well after 7 days of stimulation with the indicated anti-CD3/CD28 bead:cell ratio. Each data point is a replicate microculture well. Median fold increase for each condition is represented by a black dot.