Increased Ectonucleotidase Expression and Activity in Regulatory T Cells of Patients with Head and Neck Cancer

Abstract

Purpose: Regulatory T cell (Treg) frequency and activity are increased in cancer patients and play a major role in tumor escape. Although disease progression is favored by the presence of Treg, mechanisms used by Treg to suppress antitumor immunity are unknown. The ectonucleotidases CD39 and CD73 are expressed in Treg and convert ATP into immunosuppressive adenosine. In this study, the involvement of the adenosinergic pathway in Treg-mediated suppression in head and neck squamous cell carcinoma (HNSCC) patients was evaluated. Experimental Design: HNSCC patients with an active disease (n = 19) and patients with no evident disease after therapy (n = 14) were studied. Ectonucleotidase expression on CD4 + T cells and CD4 + CD25high Treg was evaluated by flow cytometry and compared with normal controls. Ectonucleotidase activity was also compared within these three groups. The data were analyzed for associations of ectonucleotidase expression/function with disease stage. Results: The percentages and expression levels of CD39 and CD73 in CD4 + T cells and Treg were greater in HNSCC than in normal controls and highest in patients with no evident disease. Patients' Treg hydrolyzed ATP at higher rates and produced higher levels of adenosine than normal controls' Treg. The increased frequency and enzymatic activity of CD4 + CD39 + cells corresponded to increased adenosine-mediated suppression of effector T cells, which was partly inhibited by ARL67156, an ectonucleotidase inhibitor, and by ZM241385, a selective A 2a /A 2b receptor antagonist. Conclusions: CD39+ Treg frequency and adenosine-mediated suppression are significantly increased in HNSCC patients. The adenosinergic pathway is involved in Treg-mediated immunosuppression in cancer and its attenuation could be a promising immunotherapeutic strategy for patients with HNSCC. ( Head and neck squamous cell carcinoma (HNSCC), arising from the mucosal epithelium, is the fifth most common type of cancer worldwide and the sixth most common cause of cancerrelated mortality (1, 2). Although advances in surgical methods, chemotherapy, and radiation have improved patients' treatment and quality of life, the overall outcome and survival rate among patients with this disease have not notably improved (3). Therefore, novel therapeutic strategies are necessary for the treatment of HNSCC. Recent studies have focused on the dynamic interplay and coevolution of antitumor immune responses and tumor progression in HNSCC (4, 5). These studies have shown that T lymphocytes play a major albeit contradictory role in local tumor expansion, metastasis, and neoangiogenesis. In most human cancers, regulatory T cells (Treg), a small subset of CD4 + T cells, are significantly increased in the peripheral blood as well as in the tumor microenvironment (6-8). Treg are phenotypically defined as CD4 + CD25high FOXP3 + and modulate immune responses by suppressing functions of other T cells. To date, at least two types of Treg are known to exist in man: (a) naturally occurring Treg, which devel...

Systemic and local human papillomavirus 16‐specific T‐cell immunity in patients with head and neck cancer

“…It was reported for CD4þ T cells isolated from oropharyngeal tumors that they exert suppressive function via ectonucleotidases CD39 and CD73. 31 Interestingly, all isolated CD4þ T cells tested expressed the ectonucleotidases CD39 and CD73 on the surface whereas this is not the case for peripheral CD4þ T cells isolated from a healthy control (Fig. 5b).…”

“…The expression of CD39 and CD73 has been found on CD4þ T cells as well as on the great majority of regulatory T cells in HNSCC, and has been implicated as a mechanism for T cell suppression. 31 Analyses of these markers on the isolated T cell clones revealed that all CD4þ T-cell clones highly expressed CD39 and CD73 at the cell surface in resting state, but these included CD4 helper T cells that were not suppressive in a classical suppression assay. Notably, also influenza virus-specific CD4þ helper T cells and regulatory T cells that were isolated from PBMC cultures 40 express similar high levels of CD39 and CD73 (unpublished observations).…”

“…The presence of HPV in the diluted samples was determined by genotyping the samples that demonstrated 65 basepair PCR amplimers on a 3% agarose gel using an INNOLiPA Genotyping Extra test (Innogenetics, Ghent, Belgium), according to the manufacturer's instruction. This assay allows the detection of the following HPV types: HPV 6,11,16,18,26,31,33,35,39,40,43,44,45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69/71, 73 and 74. Hybridization patterns were visually inspected and interpreted using a grid.…”

Phenotypic and functional characteristics of CD39highhuman regulatory B cells (Breg)

“…10 In cancer, ADO is viewed as a pro-tumor factor which suppresses antitumor functions of Teff and promotes functions of suppressor cells (Treg and MDSC). 11,12 Generally, B cells are viewed as promoters of T-cell activation and proliferation. However, recent studies identified a small subset of CD19 C CD25 C circulating B cells that can suppress functions of other lymphocytes.…”

Immune responses to malignancies

“…43,44 These cells suppress functions of other immune cells in the microenvironment by mechanisms that might be cell contact dependent or might involve the production of inhibitory cytokines or adenosine. [43][44][45][46] Recently, a potent proinflammatory T-cell subset, IL-17-producing T H 17 cells, were observed among CD4 + cells in patients with ovarian carcinoma. The presence of these cells was significantly correlated to enhanced survival in these patients and was found to inversely correlate with the number of FOXP3 + Treg cells.…”