All data representative of at least 2 independent experiments. (A) Overview of our approach, including key methods applied. (B) Dose-response curves showing fractional enhancement (Fr enhance) of compounds (LD50/EC50 > 2) for Treg (blue), Th1 (orange) and Th17 (red) lineages. (C) Plot of LD50/EC50 vs maximal fractional Treg cell enhancement showing all 21 Treg-specific enhancers (9RA, 9-cis retinoic acid; 13RA, 13-cis retinoic acid; ADQ, amodiaquine; AMIO, amiodarone; AMR, amrinone; ATRA, all-trans retinoic acid; ART, artemisinin; CIS, cisapride; CLO, clotrimazole; HAR, harmine; LOV, lovastatin; MBCQ, 4-((3,4-methylenedioxybenzyl)amino)-6-chloroquinazoline;4-quinazolinamine); PEN, pentamidine; PG, proguanil; PYR, pyrvinium pamoate; RAPA, rapamycin; RIB, ribavirin; ROT, rotenone; SER, sertaconazole; SIM, simvastatin; WM, wortmannin; Supplementary file 2). Retinoic acids are in green; compounds with LD50/EC50 < 2 are in gray and LD50/EC50 > 2 are in blue. The orange cluster is described in the text. (D) Ability of selected compounds (LD50/EC50 > 2 and controls) to inhibit mTOR activity, as measured by S6 phosphorylation (***p < 0.001, 1-way ANOVA with Dunnett correction). (E) Fractional (Fr) inhibitory activity of all 21 Treg-specific enhancers on Th1 and Th17 cell differentiation. (F) SEA-predicted relationships between all 21 Treg enhancers. Black lines predict binding of compounds (red circles) to proteins (blue diamonds) with likelihood proportional to line width. Green lines denote connection via curated KEGG pathways. See also Figure 1—figure supplements 1–8.