Cytotoxic Markers and Frequency Predict Functional Capacity of Natural Killer Cells Infiltrating Renal Cell Carcinoma Julia S. Schleypen, 1,2 Nicole Baur, 1 Robert Kammerer, 1,2 Peter J. Nelson, 3 Karl Rohrmann, 4 Elisabeth F. Gro ¨ ne, 5 Markus Hohenfellner, 6 Axel Haferkamp, 6 Heike Pohla, 1,2 Dolores J. Schendel, 1 Christine S. Falk, 1 and Elfriede Noessner 1 Abstract Purpose: Renal cell carcinoma harbors high numbers of infiltrating lymphocytes with apparent limited efficacy in tumor control. This study focused on the natural killer (NK) cells infiltrating renal cell carcinoma. Experimental Design: Tumor-infiltrating lymphocytes (TIL) were isolated from renal cell carcinoma and analyzed for NK cell frequency and phenotype ( n = 34). NK cells were enriched and tested for effector function. Results: Two renal cell carcinoma subtypes were identified, one containing high (>20% of the lymphocyte population, n = 14), the other low (<20%, n = 20), NK cell numbers. NK cells of both groups were noncytolytic ex vivo but differed in CD16 and cytotoxic effector molecule expression as well as in their capacity to acquire cytotoxic activity: The majority of NK cells from tumors with high NK cell content (high NK-TIL) were CD16 bright , whereas few CD16 bright NK cells were found in tumors with low NK cell frequencies (low NK-TIL). The CD16 dichotomy correlated with different capacities to develop cytotoxicity after short-term activation with interleukin-2 ex vivo : Low NK- TIL remained noncytolytic against K562 and unresponsive to signals via the activating receptor NKp46 despite expression of receptor and adaptor molecules. In contrast, high NK-TIL acquired cytotoxic function. As described for peripheral CD16 bright NK cells, NK cells from high-NK tumors showed high per cell expression of granzyme A, granzyme B, and perforin. NK cells from low NK-TIL resembled CD16 neg/dim peripheral NK cells with few cytotoxin + cells and lower expression of perforin. Conclusion: The extent of NK cell infiltration and the expression of markers (CD16 and cyto- toxins) predict the functional capacity of NK cells infiltrating renal cell carcinoma and can be used to characterize subgroups of renal cell carcinoma. Renal cell carcinoma (RCC) is a progressive tumor that accounts for 80% to 85% of malignant kidney tumors and 3% of all adult malignancies in the Western world (1, 2). About 30% of patients exhibit metastases at the time of diagnosis (2). Few therapeutic options exist for renal cell carcinoma because it does not respond to chemotherapy or irradiation. Renal cell carcinoma seems to be immunogenic and some patients respond to systemic immunotherapeutic agents, including IFN-a and/or interleukin-2 (IL-2; refs. 3, 4). However, it is unclear why only some patients show remarkable regression of metastatic lesions whereas others exhibit rapid tumor progression under identical cytokine therapies. Because systemic cytokine therapies often inflict serious adverse effects, it is desirable to spare non- responding patients from treatment. Multiple studies have attempted to elucidate variables that distinguish responders from nonresponders, thereby showing prognostic significance for successful immunotherapies (5 – 10). Conflicting results have been reported regarding the importance of peripheral lympho- cyte subpopulations (6 – 8) and the presence of higher numbers of T cells in renal cell carcinoma tissues seems to correlate with a poor prognosis (9, 11, 12). For some solid tumors, such as squamous cell lung cancer (13), gastric (14), colorectal cancer (15), and head and neck squamous cell carcinoma (16), the presence of intratumoral natural killer (NK) cells was correlated with improved survival rates. NK cells are part of the first defense against neoplastic growth (17), because, unlike T cells, they are not tolerized against autologous cells and can attack cells with aberrant Human Cancer Biology Authors’ Affiliations: 1 Institute of Molecular Immunology, GSF National Research Center for Environment and Health; 2 Laboratory for Tumor Immunology, 3 Medizinische Poliklinik, and 4 Department of Urology, Ludwig-Maximilians- University, Munich, Germany; 5 Department of Cellular and Molecular Pathology, German Cancer Research Center (Deutsches Krebsforschungszentrum); and 6 Department of Urology, University Clinic Heidelberg, Heidelberg, Germany Received 4/19/05; revised 11/10/05; accepted 11/16/05. Grant support: Deutsche Forschungsgemeinschaft grants SFB455 (E. Noessner and D.J. Schendel), SFB571 (C.S. Falk and P.J. Nelson), and NE 648/2-1 (P.J. Nelson); Deutsche Krebshilfe, Project 70-3344 (IIID; D.J. Schendel/C.S. Falk); and Deutsche Krebshilfe, Project 70-2729 (R. Kammerer). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: J.S. Schleypen and N. Baur contributed equally to this work. C.S. Falk and E. Noessner are senior authors who contributed equally to this work. Requests for reprints: Elfriede Noessner, Institute of Molecular Immunology, GSF Research Center for the Environment and Health, Marchioninistrasse 25, 81377 Munich, Germany. Phone: 49-89-7099303; Fax: 49-89-7099300; E-mail: noessner@gsf.de. F 2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-0857 www.aacrjournals.org Clin Cancer Res 2006;12(3) February 1, 2006 718 Research. on June 17, 2016. © 2006 American Association for Cancer clincancerres.aacrjournals.org Downloaded from