Characterization of tumor-associated T-lymphocyte subsets and immune checkpoint molecules in head and neck squamous cell carcinoma

// Axel Lechner 1, 2, * , Hans Schloser 1, 3, * , Sacha I. Rothschild 1, 4, * , Martin Thelen 1 , Sabrina Reuter 1 , Peter Zentis 5 , Alexander Shimabukuro-Vornhagen 1, 6 , Sebastian Theurich 1, 6, 7 , Kerstin Wennhold 1 , Maria Garcia-Marquez 1 , Lars Tharun 8 , Alexander Quaas 8 , Astrid Schauss 5 , Jorg Isensee 9 , Tim Hucho 9 , Christian Huebbers 10 , Michael von Bergwelt-Baildon 1, 6, * and Dirk Beutner 2, * 1 Cologne Interventional Immunology, Department I of Internal Medicine, University Hospital of Cologne, Cologne, Germany 2 Department of Otorhinolaryngology, Head and Neck Surgery, University of Cologne, Cologne, Germany 3 Department of General, Visceral and Cancer Surgery, University of Cologne, Cologne, Germany 4 University Hospital Basel, Department of Internal Medicine, Medical Oncology, Basel, Switzerland 5 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany 6 Department I of Internal Medicine, Center for Integrated Oncology (CIO), University Hospital of Cologne, Cologne, Germany 7 Max-Planck-Institute for Metabolism Research, Cologne, Germany 8 Institute of Pathology, University of Cologne, Cologne, Germany 9 Department of Anesthesiology and Intensive Care Medicine, Experimental Anesthesiology and Pain Research, University Hospital of Cologne, University of Cologne, Germany 10 Jean-Uhrmacher Institute for Clinical ENT Research, University of Cologne, Cologne, Germany * These authors contributed equally to this work Correspondence to: Michael von Bergwelt-Baildon, email: michael.bergwelt@uk-koeln.de Keywords: squamous cell carcinoma, head and neck, microenvironment, T cells, immune checkpoint Received: January 10, 2017 Accepted: April 25, 2017 Published: May 16, 2017 ABSTRACT The composition of tumor-infiltrating lymphocytes (TIL) reflects biology and immunogenicity of cancer. Here, we characterize T-cell subsets and expression of immune checkpoint molecules in head and neck squamous cell carcinoma (HNSCC). We analyzed TIL subsets in primary tumors ( n = 34), blood (peripheral blood mononuclear cells (PBMC); n = 34) and non-cancerous mucosa ( n = 7) of 34 treatment-naive HNSCC patients and PBMC of 15 healthy controls. Flow cytometry analyses revealed a highly variable T-cell infiltration mainly of an effector memory phenotype (CD45RA − /CCR7 − ). Naive T cells (CD45RA + /CCR7 + ) were decreased in the microenvironment compared to PBMC of patients, while regulatory T cells (CD4 + /CD25 + /CD127 low and CD4 + /CD39 + ) were elevated. Furthermore, we performed digital image analyses of entire cross sections of HNSCC to define the ‘Immunoscore’ (CD3 + and CD8 + cell infiltration in tumor core and invasive margin) and quantified MHC class I expression on tumor cells by immunohistochemistry. Immune checkpoint molecules cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) were increased in TILs compared to peripheral T cells in flow-cytometric analysis. Human papillomavirus (HPV) positive tumors showed higher numbers of TILs, but a similar composition of T-cell subsets and checkpoint molecule expression compared to HPV negative tumors. Taken together, the tumor microenvironment of HNSCC is characterized by a strong infiltration of regulatory T cells and high checkpoint molecule expression on T-cell subsets. In view of increasingly used immunotherapies, a detailed knowledge of TILs and checkpoint molecule expression on TILs is of high translational relevance.