Functional stability of MDSCs isolated from cancer patients requires continuous PGE₂-COX2 feedback involving EP2 and EP4 signaling. (A) Expression of immunosuppressive factors in cancer-isolated CD11b⁺ cells pretreated (24 hours) or not with celecoxib, the EP4 antagonist AH23848, the EP2/1 antagonist AH6809, and the EP3 antagonist L798106. (B) PGE₂ production and COX2 expression in primary cells (white bars) and CD11b⁺ cells (black bars) isolated from cancer, compared with control CD11b⁺ cells isolated from blood treated or not with celecoxib. Measurements were performed in triplicates. (C) Celecoxib pretreatment of MDSCs isolated from cancer abolishes their suppressive impact on CD3/CD28–activated naive CD8⁺ T cells. (D) Inhibition of COX2, arginase-1, IL-10, or IDO1 counteracts the suppressive functions of MDSCs isolated from cancer (n = 3). (E) Celecoxib, the EP4 antagonist AH23848, and the EP2/1 antagonist AH6809, but not the EP3 antagonist L798106, similarly reverse the suppressive functions of MDSCs. The addition of synthetic PGE₂ to celecoxib-pretreated MDSCs isolated from cancer restores immunosuppressive functions (n = 3). Neither celecoxib nor the EP antagonists showed any cytotoxic effects at the concentrations used. (F) CD3/CD28 activation of CD8⁺ T cells (absence of MDSCs) ± 1μM PGE₂ or different concentrations of the commercially available agonists butaprost (EP2) or sulprostone (EP3/1 agonist, negative control). All data (panels A-F) were confirmed in at least 3 independent experiments. Histograms present data from a single representative experiment with different donors as means ± SD.