PMPs inhibit Treg differentiation into IL-17–producing cells. Tregs were isolated from healthy donors and cultured for 7 days with anti-CD3/anti-CD28 mAb–coated beads and recombinant human IL-2, IL-15, and IL-1β in the presence or absence of allogeneic PMPs isolated from platelet apheresis units of 3 healthy donors. Flow cytometry analyses of intracellular cytokines were performed after stimulation with PMA and ionomycin, in the presence of Brefeldin A. (A) CD41⁺PS⁺ PMPs <1 µm were purified from platelet apheresis units of healthy donors by differential centrifugation and analyzed by flow cytometry for phosphatidylserine (PS) and CD41 exposure as described in “Methods.” Size beads (500 nm) were used to set a size-exclusion gate, and fluorescent count beads (∼10 µm) were added for quantification. The contour plots are representative of 6 donors. (B) Representative experiment showing intracellular IL-17 and IFN-γ staining of the Treg at day 0. (C-D) Representative example of intracellular IFN-γ, IL-17, and FOXP3 expression by Tregs that were cultured for 7 days with and without 1.5 × 10⁶ PMPs. (E) A representative experiment performed with Tregs from a single donor and increasing PMP concentrations from 3 different healthy donors. Surface CCR6, CD27, CD62L, and intracellular IL-17, IFN-γ, and FOXP3 staining of the Treg at day 7 of culture is shown. Error bars represent standard deviation (SD). (F) Cumulative data of different Treg donors (n = 3-5) showing surface CCR6, CD27, CD62L, and intracellular IL-17, IFN-γ, and FOXP3 staining of Tregs cultured for 7 days with increasing PMP concentrations. Each donor’s Tregs are shown as a dot representing the mean response of those Tregs to the three different donor’s PMPs. The mean is presented as bars. *P < .05. FSC, forward scatter; SSC, side scatter.