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Expansion of Human T Regulatory Type 1 Cells in the Microenvironment of Cyclooxygenase 2 Overexpressing Head and Neck Squamous Cell Carcinoma

¹ University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania; ² Department of Otorhinolaryngology, Ludwig-Maximilians-University of Munich, Munich, Germany; and ³ Department of Otorhinolaryngology, University of Duisburg-Essen, Essen, Germany

Requests for reprints: Theresa L. Whiteside, University of Pittsburgh Cancer Institute, Research Pavilion at the Hillman Cancer Center, 5117 Centre Avenue, Suite 1.27, Pittsburgh, PA 15213-1863. Phone: 412-624-0096; Fax: 412-624-0264; E-mail: whitesidetl{at}upmc.edu.

Cyclooxygenase 2 (COX-2) overexpression and production of prostaglandin E₂ (PGE₂) by head and neck squamous cell carcinomas (HNSCC) induce type 1 regulatory T (Tr1) cells and contribute to carcinogenesis by creating a tolerogenic milieu. To test this hypothesis, CD4⁺CD25^– T cells obtained from the peripheral blood of 10 normal donors were cocultured with autologous dendritic cells, irradiated HNSCC cells and cytokines, interleukin 2 (IL-2), IL-10, and IL-15. HNSCC cells were either COX-2 negative, constitutively expressed COX-2, were transfected with COX-2, or had COX-2 expression knocked down by small interfering RNA. Other modifications included coculture plus or minus the COX-inhibitor, Diclofenac, or synthetic PGE₂ in the absence of HNSCC. Lymphocytes proliferating in 10-day cocultures were phenotyped by flow cytometry, studied for cytokine production by ELISA and for suppressor function in CFSE inhibition assays plus or minus anti–IL-10 or anti–transforming growth factor-ß₁ (TGF-ß₁) monoclonal antibodies (mAb). COX-2⁺ HNSCC or exogenous PGE₂ induced outgrowth of Tr1 cells with the CD3⁺CD4⁺CD25^–IL2Rß⁺IL2R⁺FoxP3⁺CTLA-4⁺IL-10⁺TGF-ß₁⁺IL-4^– phenotype and high suppressor functions (range, 46–68%). Small interfering RNA knockout of COX-2 gene in HNSCC led to outgrowth of lymphocytes with decreased IL2R (P = 0.0001), FoxP3 (P = 0.05), and IL-10 (P = 0.035) expression and low suppressor activity (range, 26–34%). Whereas COX-2⁺ cocultures contained IL-10 and TGF-ß₁ (medians, 615 and 824 pg/mL), cytokine levels were decreased (P < 0.0001) in COX-2^– cocultures. Inhibition of COX-2 enzymatic activity in HNSCC abrogated outgrowth of Tr1 cells. Neutralizing mAbs to IL-10 and/or TGF-ß₁ abolished Tr1-mediated suppression. COX-2 overexpression in HNSCC plays a major role in the induction of Tr1 cells in the tumor microenvironment. [Cancer Res 2007;67(18):8865–73]

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				C. Bergmann, L. Strauss, Y. Wang, M. J. Szczepanski, S. Lang, J. T. Johnson, and T. L. Whiteside T Regulatory Type 1 Cells in Squamous Cell Carcinoma of the Head and Neck: Mechanisms of Suppression and Expansion in Advanced Disease Clin. Cancer Res., June 15, 2008; 14(12): 3706 - 3715. [Abstract] [Full Text] [PDF]