Therefore, we used TRAP-6, an additional canonical platelet activator, in our coculture. target the immunosuppressive TME. Abstract Platelets have been recently described as an important component of the innate and adaptive immunity through their interaction with immune cells. However, information on the plateletCT cell interaction in immune-mediated diseases remains limited. Glycoprotein A repetitions predominant (GARP) expressed on platelets and on activated regulatory T cells (Treg) is involved in the regulation of peripheral immune responses by modulating the bioavailability of transforming growth factor (TGF-). Soluble GARP (sGARP) exhibits strong regulatory and anti-inflammatory capacities both in vitro and in vivo, leading to the induction of peripheral Treg. Herein, we investigated the effect of platelet-derived GARP on the differentiation, phenotype, and function of T effector cells. CD4+CD25? T cells cocultured with platelets upregulated FoxP3, the master transcription factor for Treg, were anergic, and were strongly suppressive. These effects were reversed by using a blocking anti-GARP antibody, indicating a dependency on GARP. Importantly, melanoma patients in different stages of disease showed a significant upregulation TAS4464 of GARP on the platelet surface, correlating to a reduced responsiveness to immunotherapy. In conclusion, our data indicate that platelets induce peripheral Treg via GARP. These findings might contribute to diseases such as cancer-associated thrombocytosis, wherein poor prognosis and metastasis are associated with high counts of circulating platelets. = 3, means SD * 0.05, and n.s. determined by Students = 12, means SD, * 0.05, ** 0.01, *** 0.001, and n.s. determined by KruskalCWallis test). Having thus far used a non-canonical activation method, we next wanted to investigate a canonical, agonist-induced platelet activation, e.g., by thrombin being clinically more relevant. Therefore, we added 10 U/mL thrombin to the coculture. In comparison to the coculture without thrombin, this led to similar results, with increased Foxp3 and GARP expression and decreased cytokine production. Nevertheless, as described by Metelli et al. , thrombin leads to the cleavage of GARP on thrombocytes, which might partially explain the slightly reduced significances in the thrombin treated versus the 1:50 coculture control group (Figure A4). Therefore, we used TRAP-6, an additional canonical platelet activator, in our coculture. Here, we could again see similar results without any significant difference between TRAP-6-activated platelets (canonical activation) and the 1:50 coculture control group (non-canonical activation, Figure A5). In sum, platelet-cocultured Teff displayed typical characteristics of induced peripheral Treg (iTreg), namely, reduced proliferation and effector cytokine production and increased FoxP3 expression. To analyze whether these phenotypically altered anergic T cells resembling iTreg also had a suppressive function, we used them in a conventional suppressor assay. In detail, CD4+FoxP3+ iTreg (T cells pre-cultured with platelets for 6 days at the ratio 1:50) were harvested, washed, and then cultured together with untreated Teff (Figure A3) to investigate their suppressive function. Herein, platelet-induced Treg showed a significant TAS4464 TAS4464 suppressive capacity (Figure 3), as demonstrated by the reduced proliferation of T cells by Ki-67 staining in the suppression assay. Herein, decreasing numbers of platelet-induced Treg in the culture led to an increased proliferation of T cells, showing a dose-dependent suppression by the iTreg. Open in a separate window Figure 3 Platelet-induced iTreg suppressed T effector cells (Teff) cells. To analyze iTreg induction by platelets, we expanded CD4+CD25? T cells for 6 days in the presence of platelets at the ratio of 1 1:50, as described previously, and were subsequently incubated at various ratios with 0.5 105 CD4+CD25? T cells and restimulated with 1 105 irradiated peripheral blood mononuclear cells (PBMC) and 0.5 g/mL anti-CD3 mAb. Proliferation was determined on day 3 of culture by Ki-67 staining (= 3, means SD, * 0.05, and n.s. determined by one-way ANOVA). In order to determine whether the induction of iTreg is GARP-dependent, we added 10 g/mL of a blocking anti-GARP Ab to the Rabbit Polyclonal to FAKD1 cocultures and again analyzed Foxp3 and GARP expression, proliferation, (Figure.