Enhancement of Th2 cell output in HDM-stimulated PBMC cultures is reciprocally regulated by RARα modulators. Cell trace violet (CTV)-labeled PBMCs from allergic asthmatic subjects were stimulated with HDM Ag extract (40 U/ml) for 10d in the presence of ATRA (RARα agonist), Ro41 (RARα antagonist), or DMSO vehicle control. After 10d cultures were restimulated with PMA and ionomycin and ICCS was performed. (A) Representative flow plots are shown. Combined results from 3 cultures showing (B) the frequency of CTVlow cells, after gating on viable HDM proliferated CD3+, CD4+, CD8- cells. Combined results from 3 cultures showing (C) the cell number and (D) percentage of Th2 cytokine producing HDM-expanded T cells. (E) Representative flow plots showing IL-13 vs. IL-5 expression after gating on viable HDM proliferated CD3+ CD4+ CTVlow cells. Combined results from 3 cultures showing (F) cell number and (G) percentage of of IL-5+ (IL-5+ IL-13+) and IL-5- (IL-5- IL-13+) Th2 cell populations. Data points represent independent HDM-stimulated PBMC cultures. P-values were generated with 2-way ANOVA. In B and C, only 2 experiments were performed using IL-4 as an analyte.