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.

Purpose: Head and neck cancers (HNCs) often induce profound immunosuppression, which contributes to disease progression and interferes with immune-based therapies. Body fluids of patients with HNC are enriched in exosomes potentially engaged in negative regulation of antitumor immune responses. The presence and content of exosomes derived from plasma of patients with HNC are evaluated for the ability to induce immune dysfunction and influence disease activity.Experimental Design: Exosomes were isolated by size-exclusion chromatography from plasma of 38 patients with HNC and 14 healthy donors. Morphology, size, numbers, and protein and molecular contents of the recovered exosomes were determined. Coculture assays were performed to measure exosome-mediated effects on functions of normal human lymphocyte subsets and natural killer (NK) cells. The results were correlated with disease stage and activity.Results: The presence, quantity, and molecular content of isolated, plasma-derived exosomes discriminated patients with HNC with active disease (AD) from those with no evident disease (NED) after oncologic therapies. Exosomes of patients with AD were significantly more effective than exosomes of patients with NED in inducing apoptosis of CD8+ T cells, suppression of CD4+ T-cell proliferation, and upregulation of regulatory T-cell (Treg) suppressor functions (all at P < 0.05). Exosomes of patients with AD also downregulated NKG2D expression levels in NK cells.Conclusions: Exosomes in plasma of patients with HNC carry immunosuppressive molecules and interfere with functions of immune cells. Exosome-induced immune suppression correlates with disease activity in HNC, suggesting that plasma exosomes could be useful as biomarkers of HNC progression. Clin Cancer Res; 23(16); 4843–54. ©2017 AACR.

Nuclear factor-κB (NF-κB) has been described as one of the most important molecules linking inflammation to cancer. More recently, it has become clear that NF-κB is also involved in the regulation of immune checkpoint expression. Therapeutic approaches targeting immune checkpoint molecules, enabling the immune system to initiate immune responses against tumor cells, constitute a key breakthrough in cancer treatment. This review discusses recent evidence for an association of NF-κB and immune checkpoint expression and examines the therapeutic potential of inhibitors targeting either NF-κB directly or molecules involved in NF-κB regulation in combination with immune checkpoint blockade.

Head and neck squamous cell carcinoma (HNSCC) is a highly immunosuppressive malignancy. Exosomes in HNSCC patients' plasma are enriched in inhibitory cargo and mediate immunosuppression. As these exosomes are products of various cells, the cellular origin of immunoregulatory proteins they carry is unknown. To test whether tumour- or T cell-derived exosomes in patients' plasma are immunosuppressive and impact upon disease activity, we separated CD3^(–) from CD3⁽⁺⁾ exosomes by immunocapture using anti-CD3 antibodies. The exosome protein cargo was evaluated for immunoregulatory proteins using on-bead flow cytometry. Tumour protein-enriched CD3^(–) exosomes were CD44v3⁽⁺⁾. Surprisingly, mean levels of programmed death ligand 1 (PD-L1), cytotoxic T lymphocyte antigen 4 (CTLA-4) and cyclooxygenase-2 (COX-2) were similar in CD3⁽⁺⁾ and CD3^(–) exosomes, although the latter induced higher (P < 0·0025) ex-vivo apoptosis of CD8⁽⁺⁾ T cells and greater (P < 0·005) conversion of CD4⁺ T cells to CD4⁽⁺⁾CD39⁽⁺⁾ regulatory T cells (T_reg). CD3⁽⁺⁾ and CD3^(–) exosomes carrying high levels of immunosuppressive proteins were highly effective in mediating these functions. Exosomes of patients with Union for International Cancer Control (UICC) stages III/IV disease had higher levels of PD-L1 and COX-2 than stages I/II patients (P < 0·005). Patients with nodal involvement had exosomes with the higher inhibitory protein content than N0 patients (P < 0·03). CD3⁽⁺⁾ and CD3^(–) exosomes of HNSCC patients had higher PD-L1, COX-2 and CD15s levels than healthy donors' exosomes (P < 0·009), although levels of immunostimulatory OX40 or OX40L were not different. By isolating CD3^(–)/CD44v3-enriched and CD3⁽⁺⁾ exosomes from plasma, the cellular origins of immunoregulatory proteins they carry were identified. Association of exosome molecular profiles with disease progression supports the exosome potential as future cancer biomarkers.

Exosomes, the smallest group of extracellular vesicles, carry proteins, miRNA, mRNA, DNA, and lipids, which they efficiently deliver to recipient cells, generating a communication network. Exosomes strongly contribute to the immune suppressive tumor microenvironment of head and neck squamous cell carcinomas (HNSCC). Isolation of exosomes from HNSCC cell culture or patient’s plasma allows for analyzing their molecular cargo and functional role in immune suppression and tumor progression. Immune affinity-based separation of different exosome subsets, such as tumor-derived or T cell-derived exosomes, from patient’s plasma simultaneously informs about tumor status and immune dysfunction. In this review, we discuss the recent understanding of how exosomes behave in the HNSCC tumor microenvironment and why they are promising liquid biomarkers for diagnosis, prognosis, and therapy in HNSCC.

MAGE expression in head and neck squamous cell carcinoma primary tumors, lymph node metastases and respective recurrences-implications for immunotherapy