Figure 1.

Effect of pregnenolone sulfate on cell viability and T cell proliferation in PBMCs and activated expanded T cells. (a) Experimental design of PBMC and expanded T-cell viability, proliferation and apoptosis assays. PBMCs and expanded T cells from 5 healthy donors were activated or non-activated with anti-CD3/anti-CD28 beads, and non-treated or treated with 50, 100, 200, or 400 μM of pregnenolone sulfate (PS) 24 h post-activation. Viability and proliferation were measured on day 6 for PBMCs and day 4 for expanded T cells post-activation. The apoptosis test was performed 24, 48, and 72 h after treatment using the same experimental design. All assays were measured using flow cytometry. Normalized bar plots showing for each of the PBMCs and expanded T cells respectively the (b,c) viability (7AAD^-), (d,e) apoptosis (AnnexinV⁺PI^-, AnnexinV⁺PI⁺, AnnexinV^-PI⁺) and (f,g) proliferation (CFSE dye dilution) of TCR-activated cells upon treatment with the assigned concentration of pregnenolone sulfate. All recorded values plotted per donor are relative to that of the corresponding non-treated condition (and to the 24-h non-treated condition for the apoptosis test). Statistical analyses were performed using one-way ANOVA for the proliferation and viability assays, and two-way ANOVA for the apoptosis assay (*p < 0.05, **p < 0.01; otherwise, nonsignificant). Figure 1a was created using biorender.com.