Sorted TCM, TTM, TEM, and TTE cells from people without HIV were treated with the GSK3 inhibitor or vehicle control, followed by incubation under basal conditions or anti-CD3/anti-CD28 stimulation for 48 hours. (A) Representative histograms showing the expression of CCR7, CD27, CD28, and TCF-1 in sorted TTM cell populations (pop) under basal conditions. The median fluorescence intensity of each marker is indicated. Analysis of the expression of each marker in memory T cells after vehicle control or GSK3 inhibitor treatment, in the absence of stimulation, is shown. *P < 0.05, by Wilcoxon test. (B) Flow cytometric analysis of CD127⁺T-bet^– and CD127^–T-bet⁺ cells after anti-CD3/anti-CD28 stimulation, and fold change in the indicated subsets among memory T cells induced by anti-CD3/anti-CD28 stimulation, relative to the unstimulated condition. *P < 0.05, by Wilcoxon test. (C) Frequencies of granzyme B⁺, IFN-γ⁺, IL-2⁺, and TNF-α⁺ cells among memory cells after anti-CD3/anti-CD28 stimulation. At least 5 donors were included for each comparison; data shown are from 7 independent experiments. *P < 0.05 and **P < 0.01, by Wilcoxon test. (D) PCA of gene expression by TCM cells treated with vehicle control or the GSK3 inhibitor. (E) Heatmap of genes differentially expressed (P < 0.05 according to a mixed-effects model) in reprogrammed versus nonreprogrammed TCM cells. Heatmap scale indicates mRNA levels relative to the housekeeping gene GAPDH and are plotted as log 2^–ΔCt in unstimulated and polyclonally stimulated cells (at least 5 donors were included for each comparison; data are from 2 independent experiments).