Abstract
<div>Abstract<p><b>Purpose:</b> The microenvironment of head and neck squamous cell carcinomas (HNSCC) is highly immunosuppressive. HNSCCs expressing elevated levels of PD-L1 have especially poor outcome. Exosomes that carry PD-L1 and suppress T-cell functions have been isolated from plasma of patients with HNSCC. The potential contributions of PD-L1<sup>+</sup> exosomes to immune suppression and disease activity are evaluated.</p><p><b>Experimental Design:</b> Exosomes isolated from plasma of 40 HNSCC patients by size exclusion chromatography were captured on beads using anti-CD63 Abs, stained for PD-1 and PD-L1 and analyzed by flow cytometry. The percentages and mean fluorescence intensities (MFI) of PD-L1<sup>+</sup> and PD-1<sup>+</sup> exosome/bead complexes were correlated with the patients’ clinicopathologic data. PD-L1<sup>high</sup> or PD-L1<sup>low</sup> exosomes were incubated with activated CD69<sup>+</sup> human CD8<sup>+</sup> T cells ± PD-1 inhibitor. Changes in CD69 expression levels on T cells were measured. Patients’ plasma was tested for soluble PD-L1 (sPD-L1) by ELISA.</p><p><b>Results:</b> Levels of PD-L1 carried by exosomes correlated with patients’ disease activity, the UICC stage and the lymph node status (<i>P</i> = 0.0008–0.013). In contrast, plasma levels of sPD-L1 or exosome PD-1 levels did not correlate with any clinicopathologic parameters. CD69 expression levels were inhibited (<i>P</i> < 0.03) by coincubation with PD-L1<sup>high</sup> but not by PD-L1<sup>low</sup> exosomes. Blocking of PD-L1<sup>+</sup> exosome signaling to PD-1<sup>+</sup> T cells attenuated immune suppression.</p><p><b>Conclusions:</b> PD-L1 levels on exosomes, but not levels of sPD-L1, associated with disease progression in HNSCC patients. Circulating PD-L1<sup>+</sup> exosomes emerge as useful metrics of disease and immune activity in HNSCC patients. Significance: Circulating PD-L1<sup>high</sup> exosomes in HNC patients’ plasma but not soluble PD-L1 levels associate with disease progression. <i>Clin Cancer Res; 24(4); 896–905. ©2017 AACR</i>.</p></div>
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