PMP inhibition of Treg differentiation is P-selectin dependent. 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) Intracellular IL-17 expression in Tregs that were cocultured with PMPs as a standard mixed culture (control) or separated by a porous membrane (transwell). (B) The presence of the platelet-specific marker CD41 was assessed on the CD4⁺ Tregs after coculture with increasing PMP concentrations. Each donor’s Tregs are shown as a dot representing the mean response of those Tregs to the 3 different donors’ PMPs. The mean is presented as bars. *P < .05. (C) Representative contour plots of PMPs stained with anti–CD41-PE and anti–P-selectin-FITC or isotype mAbs. The contour plots are representative of 3 donors. (D) Treg expression of PSGL-1 and CD25 at day 0 of culture. (E) Surface CD41, CD27, and intracellular expression of IL-17 on Treg cocultured with untreated, isotype, or anti–P-selectin mAb-neutralized PMPs. The graphs are representative of 2 Treg donors. Mean and SD are shown. *P < .05. (F) Confocal laser scanning microscopy of Tregs cocultured with 1.5 × 10⁶ PMPs for 16 hours in the presence of anti-CD3/anti-CD28 mAb–coated beads and recombinant human IL-2 and stained for CD41 (FITC, green) and CD25 (APC, red) or (G) P-selectin (FITC, green) and PSGL-1 (APC, red). Scale bar, 5 µm. Representative images are shown. Images were acquired with a TCS SP5 confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with an HCX-PL-APO 63× 1.2 water-immersion lens while cells were maintained at 37°C in HBS buffer. Leica LAS AF acquisition software was used.