Fig 2: Liquid platelet rich fibrin (iPRF) polarizes M0-macrophages in a more “M0/M2-like” phenotype through a GARP independent mechanism. Human monocyte derived macrophages were polarized, treated with 400 µL iPRF/well, 10 μg/mL anti-GARP antibody (Ab) or were left untreated for 2 days as indicated. Surface marker expression was analyzed via flow cytometry. Bar diagram shows the mean fluorescence intensity (MFI) of each surface marker normalized to the respective M0 control. Representative histograms of 3 independent experiments are shown (n = 5 donors, bar means ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 determined by two-way ANOVA).

Fig 3: Isolated platelets from liquid platelet rich fibrin (iPRF) induce a regulatory phenotype in CD4+ T cells. CD4+ T cells and platelets (Plt.) were cocultured at a ratio of 1 to 50 and were treated with 10 µg/mL anti-GARP antibody (Ab). CD4+ T cells were prelabled with carboxyfluorescein succinimidyl ester (CFSE) to track their proliferation and stimulated with 0.5 µg/mL anti-CD3 and 1.0 µg/mL anti-CD28 Abs in the presence or absence of platelets. (A–C) The expression of Foxp3 (A), GARP (B), and proliferation (C) of CD4+ T cells was measured on day 3 of coincubation via flow cytometry. (D,E) After 6 days of coincubation, cells were stimulated with phorbol 12-myristate 13-acetate (PMA), ionomycin, and monesin for 5 h. IL-2 and IFN-γ production was quantified via intracellular flow cytometry. Representative dot plots of three independent experiments are shown. Graphs show CD4+ T cells cocultured with platelets normalized to untreated CD4+ T cells alone control (n = 3, means ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, and ns determined by one-way ANOVA).