apyrase and Head-and-Neck-Neoplasms

Tumor antigen-specific CD4 T cells accumulate at tumor sites, evoking their involvement in antitumor effector functions in situ. Contrary to CD8 cytotoxic T lymphocyte exhaustion, that of CD4 T cells remains poorly appreciated. Here, using phenotypic, transcriptomic, and functional approaches, we characterized CD4 T cell exhaustion in patients with head and neck, cervical, and ovarian cancer. We identified a CD4 tumor-infiltrating lymphocyte (TIL) population, defined by high PD-1 and CD39 expression, which contained high proportions of cytokine-producing cells, although the quantity of cytokines produced by these cells was low, evoking an exhausted state. Terminal exhaustion of CD4 TILs was instated regardless of TIM-3 expression, suggesting divergence with CD8 T cell exhaustion. scRNA-Seq and further phenotypic analyses uncovered similarities with the CD8 T cell exhaustion program. In particular, PD-1hiCD39+ CD4 TILs expressed the exhaustion transcription factor TOX and the chemokine CXCL13 and were tumor antigen specific. In vitro, PD-1 blockade enhanced CD4 TIL activation, as evidenced by increased CD154 expression and cytokine secretion, leading to improved dendritic cell maturation and consequently higher tumor-specific CD8 T cell proliferation. Our data identify exhausted CD4 TILs as players in responsiveness to immune checkpoint blockade.

Topics: Antigens, Neoplasm; Apyrase; CD8-Positive T-Lymphocytes; Female; Gene Expression; Head and Neck Neoplasms; Humans; Immune Tolerance; Immunity, Cellular; In Vitro Techniques; Lymphocyte Activation; Lymphocyte Cooperation; Lymphocytes, Tumor-Infiltrating; Male; Ovarian Neoplasms; Programmed Cell Death 1 Receptor; RNA, Messenger; T-Lymphocytes, Helper-Inducer; Tumor Escape; Uterine Cervical Neoplasms

CD39 is the rate-limiting enzyme in the generation of immunosuppressive adenosine and its expression and activity are significant in tumor progression. Squamous cell carcinoma of the head and neck (HNSCC) shows an overall poor prognosis due to high local recurrence rates and early metastatic spread.. Primary tumor specimens and lymph node specimens harvested during neck dissection of 65 patients with a diagnosis of HNSCC were subjected to immunohistochemical and H-score analysis of CD39 expression. Demographics, histopathology and subsequent outcome were analyzed.. The primary cancer was squamous cell carcinoma in all patients (male/female 55:10). H-score for CD39 expression in the primary lesion and metastatic lymph nodes was significantly higher in advanced compared to early stages with no significant differences among different tumor locations. High intratumoral and intrametastatic CD39 expression was associated with an inferior patients' overall survival at a mean follow-up of 83.4 months (6-204 months).. CD39 expression in HNSCC correlated positively with tumor stage and appears to predict poor prognosis. Therefore, CD39 expression in primary lesions and metastatic lymph nodes seems to identify patients at high risk in HNSCC of all tumor sites. Immunotherapeutic approaches targeting CD39 might be promising for this patient population.

Topics: Aged; Apyrase; Carcinoma, Squamous Cell; Female; Germany; Head and Neck Neoplasms; Humans; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Retrospective Studies

The EGFR-targeted antibody cetuximab is effective against head and neck cancer (HNSCC), but in only 15% to 20% of patients, and the variability and extent of cetuximab-mediated cellular immunity is not fully understood. We hypothesized that regulatory T cells (Treg) may exert a functional and clinical impact on antitumor immunity in cetuximab-treated individuals. The frequency, immunosuppressive phenotype, and activation status of Treg and natural killer (NK) cells were analyzed in the circulation and tumor microenvironment of cetuximab-treated patients with HNSCC enrolled in a novel neoadjuvant, single-agent cetuximab clinical trial. Notably, cetuximab treatment increased the frequency of CD4(+)FOXP3(+) intratumoral Treg expressing CTLA-4, CD39, and TGFβ. These Treg suppressed cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and their presence correlated with poor clinical outcome in two prospective clinical trial cohorts. Cetuximab expanded CTLA-4(+)FOXP3(+) Treg in vitro, in part, by inducing dendritic cell maturation, in combination with TGFβ and T-cell receptor triggering. Importantly, cetuximab-activated NK cells selectively eliminated intratumoral Treg but preserved effector T cells. In ex vivo assays, ipilimumab targeted CTLA-4(+) Treg and restored cytolytic functions of NK cells mediating ADCC. Taken together, our results argue that differences in Treg-mediated suppression contribute to the clinical response to cetuximab treatment, suggesting its improvement by adding ipilimumab or other strategies of Treg ablation to promote antitumor immunity.

Topics: Antibodies, Monoclonal, Humanized; Antigens, CD; Apyrase; Cetuximab; Clinical Trials, Phase II as Topic; CTLA-4 Antigen; Cytotoxicity, Immunologic; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Killer Cells, Natural; Male; Middle Aged; Prognosis; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Although regulatory T cells (Treg) are highly enriched in human tumours compared with peripheral blood, expression of the immune-checkpoint receptors, immunosuppressive molecules and function of Treg in these two sites remains undefined.. Tumour-infiltrating lymphocytes and peripheral blood lymphocytes were isolated from a cohort of head and neck squamous cell carcinoma (HNSCC) patients. The immunosuppressive phenotypes and function of intratumoral Treg were compared with those of peripheral blood Treg.. The frequency of immune-checkpoint receptor-positive cells was higher on intratumoral FOXP3(+)CD25(hi) Treg compared with circulating Treg (CTLA-4, P=0.002; TIM-3, P=0.002 and PD-1, P=0.002). Immunosuppressive effector molecules, LAP and ectonucleotidase CD39 were also upregulated on intratumoral FOXP3(+) Treg (P=0.002 and P=0.004, respectively). CTLA-4 and CD39 were co-expressed on the majority of intratumoral FOXP3(+)CD4(+) Treg, suggesting that these molecules have a key role in regulatory functions of these cells in situ. Notably, intratumoral Treg exhibited more potently immunosuppressive activity than circulating Treg.. These results indicate that intratumoral Treg are more immunosuppressive than circulating Treg and CTLA-4 and CD39 expressed can be potential target molecules to inhibit suppressive activities of intratumoral Treg in situ.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Apyrase; Carcinoma, Squamous Cell; CTLA-4 Antigen; Cytokines; Female; Head and Neck Neoplasms; Humans; Immune Tolerance; Immunosuppressive Agents; Lymphocytes, Tumor-Infiltrating; Male; Middle Aged; Squamous Cell Carcinoma of Head and Neck; T-Lymphocytes, Regulatory

Adaptive regulatory T cells (Tr1) are induced in the periphery upon encountering cognate antigens. In cancer, their frequency is increased; however, Tr1-mediated suppression mechanisms are not yet defined. Here, we evaluate the simultaneous involvement of ectonucleotidases (CD39/CD73) and cyclooxygenase 2 (COX-2) in Tr1-mediated suppression. Human Tr1 cells were generated from peripheral blood mononuclear cell-derived, sorted CD4(+)CD25(-) T cells and incubated with autologous immature dendritic cells, irradiated COX-2(+) or COX-2(-) tumor cells, and IL-2, IL-10, and IL-15 (each at 10-15 IU/ml) for 10 days as described (Bergmann, C., Strauss, L., Zeidler, R., Lang, S., and Whiteside, T. L. (2007) Cancer Immunol. Immunother. 56, 1429-1442). Tr1 were phenotyped by multicolor flow cytometry, and suppression of proliferating responder cells was assessed in carboxyfluorescein diacetate succinimidyl ester-based assays. ATP hydrolysis was measured using a luciferase detection assay, and levels of adenosine or prostaglandin E(2) (PGE(2)) in cell supernatants were analyzed by mass spectrometry or ELISA, respectively. Intracellular cAMP levels were measured by enzyme immunoassay. The COX-2(+) tumor induced a greater number of Tr1 than COX-2(-) tumor (p < 0.05). Tr1 induced by COX-2(+) tumor were more suppressive, hydrolyzed more exogenous ATP (p < 0.05), and produced higher levels of adenosine and PGE(2) (p < 0.05) than Tr1 induced by COX-2(-) tumor. Inhibitors of ectonucleotidase activity, A(2A) and EP(2) receptor antagonists, or an inhibitor of the PKA type I decreased Tr1-mediated suppression (p < 0.05), whereas rolipram, a PDE(4) inhibitor, increased the intracellular cAMP level in responder cells and their susceptibility to Tr1-mediated suppression. Tr1 present in tumors or the peripheral blood of head and neck squamous cell carcinoma patients co-expressed COX-2, CD39, and CD73. A concomitant inhibition of PGE(2) and adenosine via the common intracellular cAMP pathway might be a novel approach for improving results of immune therapies for cancer.

Topics: 5'-Nucleotidase; Adaptive Immunity; Adenosine; Amino Acids, Cyclic; Antigens, CD; Apyrase; Carcinoma, Squamous Cell; Cell Line, Tumor; Cyclooxygenase 2; Dinoprostone; Down-Regulation; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Immune Tolerance; Intracellular Space; Signal Transduction; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory

Human regulatory T cells (Treg) have been variously defined as CD4(+)CD25(+), CD4(+)CD25(high) or CD4(+)CD25(high)FOXP3(+) cells which are responsible for maintaining peripheral tolerance. Their isolation from human peripheral blood or tissues depends on the expression level of CD25(IL-2Ralpha) - a surface marker which is also expressed on activated effector helper T cells. CD39, a cell surface associated ectonucleotidase, can be used to purify Treg with strong suppressor functions. The CD4(+)CD39(+) T cells catalyze cleavage of adenosine triphosphate (ATP) to adenosine monophosphate (AMP), which is then further cleaved to adenosine. CD4(+)CD39(+) T cells largely overlap with CD4(+)CD25(high)FOXP3(+) but not CD4(+)CD25(+) T cell subset, and mediate equally potent immune suppression. Thus, CD39 surface marker can be successfully used for routine isolation of functionally-active human Treg from the peripheral blood of healthy donors or patients with cancer for studies of their role in health and disease.

Topics: Adult; Antigens, CD; Apyrase; Carcinoma, Squamous Cell; Case-Control Studies; Cells, Cultured; Female; Flow Cytometry; Forkhead Transcription Factors; Head and Neck Neoplasms; Humans; Immunomagnetic Separation; Immunophenotyping; Interleukin-2 Receptor alpha Subunit; Male; Middle Aged; Reproducibility of Results; T-Lymphocytes, Regulatory; Young Adult

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.. 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(+)CD25(high) 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.. 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.. 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.

Topics: 5'-Nucleotidase; Adenosine Triphosphate; Adult; Aged; Antigens, CD; Apyrase; Carcinoma, Squamous Cell; CD4-Positive T-Lymphocytes; Female; Head and Neck Neoplasms; Humans; Immunosuppression Therapy; Male; Middle Aged; N-Glycosyl Hydrolases; T-Lymphocytes, Regulatory