Abstract
Background: Programmed death ligand 1 (PD-L1) is expressed on various cell types, including platelets. The PD-L1/PD-1 pathway plays a central role in maintenance of immunologic homeostasis. Platelets express PD-L1 at low levels in healthy individuals, and upregulate PD-L1 in cancer. Platelet PD-L1 has been suggested to reflect intratumoral PD-L1 and predict response to immune checkpoint inhibitor (ICI) therapy. We hypothesized that platelet-derived EVs contain PD-L1 and may be involved in immunomodulatory and/or adverse effects of ICI. Methods: Whole blood was collected in 3.2% sodium citrate from healthy subjects. PRP was isolated by centrifugation, diluted 1:1 in Tyrode's Solution, and platelets isolated by centrifugation at 1400 x g for 5 minutes. The platelet pellet was resuspended in Tyrode's and stimulated by incubation with 10 uM ADP or 1U/mL thrombin. Platelets were removed by centrifugation, and supernatants serially centrifuged at 16,000 x g for 40 min, and at 100,000 x G for two hours to obtain larger EV (e.g. microparticles) and smaller EV (e.g. exosomes). Large and small EV fractions, and pellets from platelets before and after stimulation were solubilized and PD-L1 content determined by immunoblotting using anti-human PD-L1 antibodies. The concentration of platelet-derived EV was also measured using a ZetaView NTA PMX-120. Results: PD-L1 was detected in lysates of unstimulated platelets but reduced after stimulation. PD-L1 was present primarily in the small EV fraction (exosomes), with greater amounts detected after platelet stimulation (Figure). Platelet EV counts increased substantially after platelet stimulation with ADP (120 x 108/ml), or thrombin (74 x 108/ml) compared to unstimulated platelets (31 x 108/ml). Conclusion: These pilot studies suggest that platelet activation results in release of EV containing PD-L1. Studies are in progress to determine whether these EV may regulate responses or toxicities of ICI through transfer of PD-L1 to other cell types, or other mechanisms. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal
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