The Journal of Immunology Enhanced In Vivo Growth of Lymphoma Tumors in the Absence of the NK-Activating Receptor NKp46/NCR11 Gili G. Halfteck, Moran Elboim, Chamutal Gur, Hagit Achdout, Hormas Ghadially, and Ofer Mandelboim2 The in vitro elimination of virus-infected and tumor cells by NK cells is regulated by a balance between signals conveyed via specific inhibitory and activating receptors. Whether NK cells and specifically the NK-activating receptor NKp46 (NCR1 in mice) are directly involved in tumor eradication in vivo is still largely unknown. Since the NKp46/NCR1 tumor ligands have not been identified yet, we use a screening technique to identify functional ligands for NKp46/NCR1 which is based on a cell reporter assay and discover a NCR1 ligand in the PD1.6 lymphoma line. To study whether NKp46/NCR1 is important for the eradication of PD1.6 lymphoma in vivo, we used the Ncr1 knockout Ncr1gfp/gfp mice generated by our group. Strikingly, all Ncr1 knockout mice developed growing PD1.6 tumors, whereas initial tumor growth was observed in the wild-type mice and tumors were completely rejected as time progressed. The growth of other lymphoma cell lines such as B10 and EL4 was equivalent between the Ncr1 knockout and wild-type mice. Finally, we show that PD1.6 lymphoma cells are less killed both in vitro and in vivo in the absence of NKp46/NCR1. Our results therefore reveal a crucial role for NKp46/NCR1 in the in vivo eradication of some lymphoma cells. The Journal of Immunology, 2009, 182: 2221–2230. t was hypothesized that the immune system surveys the body reduced resistance to transplanted tumor cell lines (20–22). for nascent malignancies, eliminating some tumors and slow- Subsequent studies demonstrated that mice carrying the ho- I ing the growth of others (1). This hypothesis is supported by mozygous beige mutation, which are defective in granule exo- the findings that humans with congenital or acquired immunode- cytosis and have a profound defect in NK cell cytolytic func- ficiency have a significantly higher incidence of malignancies (2, tion, develop virus-induced and carcinogen-induced tumors at a 3). Consistent with such a role, various components of the immune higher frequency (23–25). However, these studies did not prove system, such as CTLs, NK cells, and Abs can exert potent activity a role for NK cells in tumor immunity, since the defects caused against different types of tumors in vitro (4–6). by the beige mutation are not limited to NK cells. Other studies NK cells are bone marrow-derived lymphocytes of the innate demonstrated an increased rate of carcinogen-induced sarcomas immune system comprising ϳ5–15% of PBLs. NK cells play an (and of tumors induced by oncogenic viruses) in mice lacking important role in the early elimination of virus-infected cells, bac- perforin, the pore-forming granule protein involved in cytotox- teria, intracellular parasites, and tumor cells (7–14). In addition, icity by NK cells and CTLs (26). In contrast to perforin-defi- NK cells have been shown to also perform several noncytotoxic cient mice, mice genetically deficient for CD8ϩ T cells did not functions, including: Ag presentation (15), activation of dendritic show significant defects in the control of carcinogen-induced cells (16), an important regulatory function in the maternal-fetal tumor growth, suggesting a possible role of NK cells in tumor interface (17), and secretion of immunomodulatory cytokines such surveillance (26). Importantly, perforin-deficient mice also de- ␥ ␣ as IFN- , TNF- , and chemokines causing T cells to shift to a TH1 veloped spontaneous disseminated lymphomas at a much higher phenotype (18). frequency than immunocompetent control mice (27). Whether The antitumor cell activity of NK cells was the hallmark of the perforin dependence of tumor resistance in normal mice their original discovery (19). However, only a limited number reflects the activity of NK cells or T cells, or both, was not of in vivo studies investigated whether NK cells in general and determined in this system. NK receptors in particular are involved in tumor eradication. NK cell’s activation is governed by numerous receptors Studies involving depletion of NK cells in mice often showed (some of which are activating while others are inhibitory) that recognize various classes of cell surface ligands (14, 28). The balance between these countervailing signals tightly regulates Lautenberg Center for General and Tumor Immunology, The Hebrew University, Hadassah Medical School, Jerusalem, Israel NK cell activation (14, 28). Many of the inhibitory receptors Received for publication June 11, 2008. Accepted for publication December 10, 2008. expressed by NK cells are specific for MHC class I molecules as predicted by the “missing self-recognition” hypothesis (29) 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 (although other, non-MHC-restricted receptors were also iden- with 18 U.S.C. Section 1734 solely to indicate this fact. tified (30–32)). In contrast, activating NK cell receptors recog- 1 This work was supported by the US–Israel Bi-national Science Foundation, the nize self, stress-inducible, pathogen-derived, and tumor ligands, Israeli Cancer Research Foundation, the Israeli Science Foundation, the European Consortium (Grants MRTN-CT-2005 and LSCH-CT-2005-518178), and the AICR. whose expression is induced or up-regulated in target cells (14, O.M. is an Edward Crown Professor of Molecular Immunology. 28). In that respect, a significant breakthrough in understanding 2 Address correspondence and reprint requests to Prof. Ofer Mandelboim, The He- NK cell activation was made following the identification of brew University, Hadassah Medical School, P.O.B. 12272 Jerusalem, Israel. E-mail three novel, NK-specific activating receptors, including NKp30, address: [email protected] NKp44, and NKp46 and its mouse ortholog NCR1 (33–35), Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 which are collectively known as natural cytotoxicity receptors www.jimmunol.org/cgi/doi/10.4049/jimmunol.0801878 2222 ENHANCED TUMOR GROWTH IN THE ABSENCE OF Ncr1 (NCRs).3 Among the NCRs, NKp46 and its mouse ortholog Fusion proteins, Abs, and flow cytometry NCR1 are constitutively expressed by NK cells and, in fact, are The NCR1-Ig, NKp46-Ig, NKp46 D1-Ig, and NKG2D-Ig fusion proteins the best marker which distinguishes NK cells from other im- were generated in COS-7 cells and purified by affinity chromatography mune cells in all tested mammals, including humans (35), mice using a protein G column, as previously described (44). The staining of cell (36, 37), monkeys (38), rats (39), and bovines (40). lines was visualized using a secondary PE-conjugated goat anti-human Ab Several in vitro studies have demonstrated that NKp46 in hu- (Jackson ImmunoResearch Laboratories). Abs used in this work included: a hybridoma-producing mAb 12CA5 (anti-HA tag), a polyclonal Ab mans and NCR1 in mice are major killer receptors involved in the against mouse NKp46 AF2225 (R&D Systems), staining of both Abs was recognition and killing of tumor cells (5, 41). In keeping, a low visualized using fluorescein-conjugated goat anti-mouse Ab and fluores- concentration of NKp46 has been observed in several types of cein-conjugated donkey anti-goat Ab (Jackson ImmunoResearch Labora- malignancies, including acute myeloid leukemia (42). We have tories), respectively, PE-conjugated anti-CD3 Ab, and negative control mouse IgG1/R-PE (DakoCytomation). Additionally, a hybridoma-produc- previously demonstrated that viral hemagglutinins (HA) of various ing mAb PK136 (anti-NK1.1) was used for the depletion of NK cells influenza viruses are recognized by NKp46 and NCR1 (14, 37, 43, in vivo. 44); however, a cellular tumor ligand for NKp46 or NCR1 still BW assay remains to be identified. To study the in vivo function of NKp46, we have previously For measurements of IL-2 production resulting from the interaction be- generated a knockout mouse in which a GFP cassette was tween NCR1 and ligands expressed by target tumor cell lines, 50,000 BW ␨ “knocked in” into the Ncr1 locus, thereby rendering Ncr1 non- or BW/NCR1- cells were coincubated with 50,000 irradiated (3000 rad) gfp/gfp cells of various mouse cell lines for 48 h at 37°C and 5% CO2. Superna- functional (Ncr1 ) (37). As we have previously reported, tants were collected and the level of IL-2 was quantified by using com- influenza infection was lethal in Ncr1gfp/gfp mice in both 129/Sv mercially available anti-mouse IL-2 mAbs (BD Pharmingen) and standard and C57BL/6 backgrounds, demonstrating that the recognition ELISA. of HA by NKp46/NCR1 is essential for influenza virus eradi- NK cell depletion cation in vivo (37). That being said however, the direct role of Fifty micrograms of anti-NK1.1 Ab in a volume of 300 ␮l of sterile PBS NKp46/NCR1 in the elimination of tumor cells in vivo has not ϩ was injected i.v. into the tail vein of C57BL/6 Ncr1gfp/ mice. Sterile PBS been clarified. The results obtained in these mice regarding tu- was injected as a control. Depletion was verified 2 days after injection: mor growth were much less conclusive, as we have observed mice were bled; RBCs were lysed (using ACK buffer), and GFP-positive reduced clearance of the RMAS tumor cells in vivo in Ncr1gfp/gfp NK cells were detected in peripheral blood cells by flow cytometry. NK mice in the 129/Sv background but not in the C57BL/6 back- depletion was further verified every 2 days during the experiments. ground (37). In vitro cytotoxicity assays In this study, we identified an unknown ligand for NKp46/ Heterozygous and Ncr1-knockout C57BL/6 mice were injected i.p.