HPV-associated head and neck cancer is characterized by distinct profiles of CD8+ T cells and myeloid-derived suppressor cells.

Patients with HPV--localized head and neck cancer (HNC) show inferior outcomes after surgery and radiochemotherapy compared to HPV-associated cancers. The underlying mechanisms remain elusive, but differences in immune status and immune activity may be implicated. In this study, we analyzed immune profiles of CD8+ T cells and myeloid-derived suppressor cells (MDSC) in HPV+ versus HPV- disease.The overall frequency of CD8+ T cells was reduced in HNC versus healthy donors but substantially increased after curative therapy (surgery and/or radiochemotherapy). In HPV+ patients, this increase was associated with significant induction of peripheral blood CD8+/CD45RA-/CD62L- effector memory cells. The frequency of HPV-antigen-specific CD8+ cells was low even in patients with virally associated tumors and dropped to background levels after curative therapy. Pre-therapeutic counts of circulating monocytic MDSC, but not PMN-MDSC, were increased in patients with HPV- disease. This increase was accompanied by reduced fractions of terminally differentiated CD8+ effector cells. HPV- tumors showed reduced infiltrates of CD8+ and CD45RO+ immune cells compared with HPV+ tumors. Importantly, frequencies of tumor tissue-infiltrating PMN-MDSC were increased, while percentages of Granzyme B+ and Ki-67+ CD8 T cells were reduced in patients with HPV- disease.We report differences in frequencies and relative ratios of MDSC and effector T cells in HPV- HNC compared with more immunogenic HPV-associated disease. Our data provide new insight into the immunological profiles of these two tumor entities and may be utilized for more tailored immunotherapeutic approaches in the future.

HPV16 drives cancer immune escape via NLRX1-mediated degradation of STING.

The incidence of human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) has surpassed that of cervical cancer and is projected to increase rapidly until 2060. The coevolution of HPV with transforming epithelial cells leads to the shutdown of host immune detection. Targeting proximal viral nucleic acid-sensing machinery is an evolutionarily conserved strategy among viruses to enable immune evasion. However, E7 from the dominant HPV subtype 16 in HNSCC shares low homology with HPV18 E7, which was shown to inhibit the STING DNA-sensing pathway. The mechanisms by which HPV16 suppresses STING remain unknown. Recently, we characterized the role of the STING/type I interferon (IFN-I) pathway in maintaining immunogenicity of HNSCC in mouse models. Here we extended those findings into the clinical domain using tissue microarrays and machine learning-enhanced profiling of STING signatures with immune subsets. We additionally showed that HPV16 E7 uses mechanisms distinct from those used by HPV18 E7 to antagonize the STING pathway. We identified NLRX1 as a critical intermediary partner to facilitate HPV16 E7-potentiated STING turnover. The depletion of NLRX1 resulted in significantly improved IFN-I-dependent T cell infiltration profiles and tumor control. Overall, we discovered a unique HPV16 viral strategy to thwart host innate immune detection that can be further exploited to restore cancer immunogenicity.

T cell receptor richness in peripheral blood increases after cetuximab therapy and correlates with therapeutic response.

The role of T cell receptor (TCR) signaling for adaptive immune responses is essential. The ability to respond to a broad spectrum of tumor antigens requires an adaptive selection of various TCR. So far, little is known about the role of TCR richness and clonality in the cellular immune response to head and neck cancer (HNC), though the Endothelial Growth Factor Receptor (EGFR)-specific CD8+ T cell response can be enhanced by cetuximab therapy. Therefore, we investigated differences in TCR sequences between human papillomavirus (HPV)+ and HPV- HNC patients, as well as differences in TCR sequence characteristics between T cells of peripheral blood mononuclear cells (PBMC) and tumor infiltrating lymphocytes (TIL). Additionally, we were able to investigate the TCR richness and clonality in samples pre- and post- treatment in a prospective clinical trial of neoadjuvant cetuximab. Interestingly, HPV+ and HPV- HNSCC did not significantly differ in the extent of TCR clonality and richness in PBMC or TIL. However, neoadjuvant cetuximab treatment increased the number of unique TCR sequences in PBMC (p = 0.0003), which was more prominent in the clinical responder patients compared to non-responders (p = 0.04). A trend toward TCR gene focusing was observed in TIL (p = 0.1) post-treatment. Thus, an increase in richness of TCR sequences in the periphery with a focusing at the tumor site is associated with an improved treatment response, suggesting an influence of peripheral quantity and intratumoral quality on adaptive immunity in cetuximab treated patients.

Phase Ib Study of Immune Biomarker Modulation with Neoadjuvant Cetuximab and TLR8 Stimulation in Head and Neck Cancer to Overcome Suppressive Myeloid Signals.

Purpose: The response rate of patients with head and neck squamous cell carcinoma (HNSCC) to cetuximab therapy is only 15% to 20%, despite frequent EGFR overexpression. Because immunosuppression is common in HNSCC, we hypothesized that adding a proinflammatory TLR8 agonist to cetuximab therapy might result in enhanced T-lymphocyte stimulation and anti-EGFR-specific priming.Experimental Design: Fourteen patients with previously untreated HNSCC were enrolled in this neoadjuvant trial and treated preoperatively with 3 to 4 weekly doses of motolimod (2.5 mg/m2) and cetuximab. Correlative tumor and peripheral blood specimens were obtained at baseline and at the time of surgical resection and analyzed for immune biomarker changes. Preclinical in vitro studies were also performed to assess the effect of cetuximab plus motolimod on myeloid cells.Results: TLR8 stimulation skewed monocytes toward an M1 phenotype and reversed myeloid-derived suppressor cell (MDSC) suppression of T-cell proliferation in vitro These data were validated in a prospective phase Ib neoadjuvant trial, in which fewer MDSC and increased M1 monocyte infiltration were found in tumor-infiltrating lymphocytes. Motolimod plus cetuximab also decreased induction of Treg and reduced markers of suppression, including CTLA-4, CD73, and membrane-bound TGFß. Significantly increased circulating EGFR-specific T cells were observed, concomitant with enhanced CD8+ T-cell infiltration into tumors. These T cells manifested increased T-cell receptor (TCR) clonality, upregulation of the costimulatory receptor CD27, and downregulation of inhibitory receptor TIGIT.Conclusions: Enhanced inflammatory stimulation in the tumor microenvironment using a TLR agonist overcomes suppressive myeloid and regulatory cells, enhancing the cellular antitumor immune response by therapeutic mAb in HNSCC. Clin Cancer Res; 24(1); 62-72. ©2017 AACR.

PD-1 Status in CD8+ T Cells Associates with Survival and Anti-PD-1 Therapeutic Outcomes in Head and Neck Cancer.

Improved understanding of expression of immune checkpoint receptors (ICR) on tumor-infiltrating lymphocytes (TIL) may facilitate more effective immunotherapy in head and neck cancer (HNC) patients. A higher frequency of PD-1+ TIL has been reported in human papillomavirus (HPV)+ HNC patients, despite the role of PD-1 in T-cell exhaustion. This discordance led us to hypothesize that the extent of PD-1 expression more accurately defines T-cell function and prognostic impact, because PD-1high T cells may be more exhausted than PD-1low T cells and may influence clinical outcome and response to anti-PD-1 immunotherapy. In this study, PD-1 expression was indeed upregulated on HNC patient TIL, and the frequency of these PD-1+ TIL was higher in HPV+ patients (P = 0.006), who nonetheless experienced significantly better clinical outcome. However, PD-1high CD8+ TILs were more frequent in HPV- patients and represented a more dysfunctional subset with compromised IFN-γ secretion. Moreover, HNC patients with higher frequencies of PD-1high CD8+ TIL showed significantly worse disease-free survival and higher hazard ratio for recurrence (P < 0.001), while higher fractions of PD-1low T cells associated with HPV positivity and better outcome. In a murine HPV+ HNC model, anti-PD-1 mAb therapy differentially modulated PD-1high/low populations, and tumor rejection associated with loss of dysfunctional PD-1high CD8+ T cells and a significant increase in PD-1low TIL. Thus, the extent of PD-1 expression on CD8+ TIL provides a potential biomarker for anti-PD-1-based immunotherapy. Cancer Res; 77(22); 6353-64. ©2017 AACR.

Adenosine metabolism of human mesenchymal stromal cells isolated from patients with head and neck squamous cell carcinoma.

BACKGROUND: Mesenchymal stromal cells (MSC) are a major component of the tumor microenvironment in patients with head and neck squamous cell carcinoma (HNSCC). MSC display innate and regulatory immunologic functions, very similar to many hematopoietic 'classical' immune cells. Conversion of ATP to immunosuppressive adenosine is an immunosuppressive mechanism utilized by other hematopoietic immune cells. The present study explores the adenosine metabolism of tumor derived MSC in comparison to autologous MSC from non-malignant tissue. METHODS: From HNSCC patients (n=10), paired MSC were generated from tumor tissue (tMSC) and autologous healthy control tissue (cMSC). Differentiation properties and phenotype (CD105, CD73, CD39, CD90, CD26, CD29) were compared by flow cytometry. Production of immunosuppressive adenosine (ADO) by functionally active ectonucleotidases, CD39 and CD73, was determined by luminescence and mass spectrometry. Suppressive activity of ADO was tested in CFSE proliferation assays of isolated T-cells. Plasticity of cMSC was explored after incubation with tumor-cell conditioned media. RESULTS: Differentiation into osteogenic, chondrogenic and adipogenic directions was comparable in tMSC and cMSC. Expression of ectonucleotidases, CD39 and CD73, was decreased in tMSC as compared to corresponding cMSC, which correlated with decreased ATP metabolism in mass spectrometry. Proliferation of CD4+ T-cells was significantly suppressed by exogenous ADO. Tumor-conditioned medium was unable to down-regulate ADO production in cMSC. CONCLUSION: We identified MSC of the oropharyngeal mucosa as an important producer of exogenous ADO. In patients with HNSCC, reduced expression of ADO may contribute to excessive inflammation and tumor growth.

The bidirectional tumor--mesenchymal stromal cell interaction promotes the progression of head and neck cancer.

INTRODUCTION: Mesenchymal stromal cells (MSC) are an integral cellular component of the tumor microenvironment. Nevertheless, very little is known about MSC originating from human malignant tissue and modulation of these cells by tumor-derived factors. The aim of this study was to isolate and characterize MSC from head and neck squamous cell carcinoma (HNSCC) and to investigate their interaction with tumor cells. METHODS: MSC were isolated from tumor tissues of HNSCC patients during routine oncological surgery. Immunophenotyping, immunofluorescence and in vitro differentiation were performed to determine whether the isolated cells met the consensus criteria for MSC. The cytokine profile of tumor-derived MSC was determined by enzyme-linked immunosorbent assay (ELISA). Activation of MSC by tumor-conditioned media was assessed by measuring cytokine release and expression of CD54. The impact of MSC on tumor growth in vivo was analyzed in a HNSCC xenograft model. RESULTS: Cells isolated from HNSCC tissue met the consensus criteria for MSC. Tumor-derived MSC constitutively produced high amounts of interleukin (IL)-6, IL-8 and stromal cell-derived factor (SDF)-1α. HNSCC-derived factors activated MSC and enhanced secretion of IL-8 and expression of CD54. Furthermore, MSC provided stromal support for human HNSCC cell lines in vivo and enhanced their growth in a murine xenograft model. CONCLUSIONS: This is the first study to isolate and characterize MSC from malignant tissues of patients with HNSCC. We observed cross-talk of stromal cells and tumor cells resulting in enhanced growth of HNSCC in vivo.