Microenvironment and Immunology Metalloprotease-Mediated Tumor Cell Shedding of B7-H6, the Ligand of the Natural Killer Cell–Activating Receptor NKp30 Eva Schlecker 1 , Nathalie Fiegler 1 , Annette Arnold 1 , Peter Altevogt 2 , Stefan Rose-John 3 , Gerhard Moldenhauer 2 , Antje Sucker 4 , Annette Paschen 4 , Elke Pogge von Strandmann 5 , Sonja Textor 1 , and Adelheid Cerwenka 1 Abstract Natural killer (NK) cells are potent immune effector cells capable of mediating antitumor responses. Thus, during immunoediting, tumor cell populations evolve strategies to escape NK-cell–mediated recognition. In this study, we report a novel mechanism of immune escape involving tumor cell shedding of B7-H6, a ligand for the activating receptor NKp30 that mediates NK-cell binding and NK-cell–mediated killing. Tumor cells from different cancer entities released B7-H6 by ectodomain shedding mediated by the cell surface proteases "a disintegrin and metalloproteases" (ADAM)-10 and ADAM-17, as demonstrated through the use of pharmacologic inhibitors or siRNA-mediated gene attenuation. Inhibiting this proteolytic shedding process increased the levels of B7-H6 expressed on the surface of tumor cells, enhancing NKp30-mediated activation of NK cells. Notably, we documented elevated levels of soluble B7-H6 levels in blood sera obtained from a subset of patients with malignant melanoma, compared with healthy control individuals, along with evidence of elevated B7-H6 expression in melanoma specimens in situ. Taken together, our results illustrated a novel mechanism of immune escape in which tumor cells impede NK-mediated recognition by metalloprotease-mediated shedding of B7-H6. One implication of our findings is that therapeutic inhibition of specific metalloproteases may help support NK-cell–based cancer therapy. Cancer Res; 74(13); 3429–40. Ó2014 AACR. Introduction Natural killer (NK)-cell activation is determined by a delicate balance of signals received via inhibitory and activating recep- tors (1–3). Inhibitory receptors mainly recognize self-MHC class I molecules. Activating receptors, such as natural cyto- toxicity receptors (NCR), or NKG2D, interact with virus-, stress-, transformation-, or senescence-inducible ligands (1, 4, 5). These ligands are normally not expressed on the cell surface of healthy cells. Tumor cells frequently express high levels of activating NK-cell receptor ligands and low levels of MHC class I rendering these cells highly susceptible to NK-cell– mediated lysis (6). The family of NCRs comprises three members, NKp30 (7), NKp44, and NKp46, all of which are involved in the killing of tumor cells (7, 8). Recently, B7-H6 (natural cytotoxicity trig- gering receptor 3 ligand 1, NR3LG1) was identified as a tumor cell surface–expressed ligand for NKp30 (natural cytotoxicity triggering receptor 3, NCR3; ref. 9). Neither NKp30 nor B7-H6 orthologues exist in mice. B7-H6 is a 454-aa-long type I transmembrane protein with a predicted molecular weight of 51 kDa belonging to the B7 family (9). The extracellular domain of B7-H6 comprises two immunoglobulin-like domains and harbors several potential N-glycosylation sites (9, 10). The interaction of B7-H6 on tumor cells with NKp30 on NK cells leads to efficient NK-cell activation and target cell killing (9). To date, B7-H6 expression was found on tumor cell lines or monocytes and neutrophils after inflammatory stimulation and has not been detected on healthy cells (9, 11, 12). So far, mechanisms regulating B7-H6 expression on tumor cells are still poorly explored. To evaluate B7-H6 as a potential target for tumor therapy, it is crucial to obtain an in-depth understand- ing about its expression and regulation. Release of soluble ligands for the activating NK-cell receptor NKG2D has been implied in immune escape mechanisms of tumor cells from NK- or T-cell–mediated killing (13, 14). The release of soluble NKG2D ligands was described to be either mediated by proteases and/or by exosomal secretion (15–21). Authors' Affiliations: 1 Innate Immunity and 2 Translational Immunology, Department of Tumor Immunology, German Cancer Research Center, Heidelberg; 3 Department of Biochemistry, Christian-Albrechts-Universit€ at zu Kiel, Kiel; 4 Department of Dermatology, University Hospital, University Duisburg-Essen, Essen; and 5 Department of Internal Medicine I, Centre for Integrated Oncology Koeln Bonn, University of Cologne, Cologne, Germany Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). S. Textor and A. Cerwenka contributed equally to this work. Corresponding Author: Sonja Textor, German Cancer Research Center (DKFZ; D080), D-69120 Heidelberg, Germany. Phone: 49-6221-424484; Fax: 49-6221-423755; E-mail: s.textor@dkfz.de doi: 10.1158/0008-5472.CAN-13-3017 Ó2014 American Association for Cancer Research. Cancer Research www.aacrjournals.org 3429