Purpose: 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⁺ exosomes to immune suppression and disease activity are evaluated.

Experimental Design: 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⁺ and PD-1⁺ exosome/bead complexes were correlated with the patients’ clinicopathologic data. PD-L1^high or PD-L1^low exosomes were incubated with activated CD69⁺ human CD8⁺ 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.

Results: Levels of PD-L1 carried by exosomes correlated with patients’ disease activity, the UICC stage and the lymph node status (P = 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 (P < 0.03) by coincubation with PD-L1^high but not by PD-L1^low exosomes. Blocking of PD-L1⁺ exosome signaling to PD-1⁺ T cells attenuated immune suppression.

Conclusions: PD-L1 levels on exosomes, but not levels of sPD-L1, associated with disease progression in HNSCC patients. Circulating PD-L1⁺ exosomes emerge as useful metrics of disease and immune activity in HNSCC patients. Significance: Circulating PD-L1^high exosomes in HNC patients’ plasma but not soluble PD-L1 levels associate with disease progression. Clin Cancer Res; 24(4); 896–905. ©2017 AACR.