Two Novel NKG2D Ligands of the Mouse H60 Family with Differential Expression Patterns and Binding Affinities to NKG2D 1 Akio Takada, 2 * † Shigeru Yoshida, 2 * ‡ Mizuho Kajikawa, § Yukiko Miyatake,* Utano Tomaru,* Masaharu Sakai, ‡ Hitoshi Chiba, ‡ Katsumi Maenaka, § Daisuke Kohda, § Kazunori Fugo,* and Masanori Kasahara 3 * H60, originally described as a dominant minor histocompatibility Ag, is an MHC class I-like molecule that serves as a ligand for the NKG2D receptor. In the present study, we identified two novel mouse chromosome 10-encoded NKG2D ligands structurally resembling H60. These ligands, which we named H60b and H60c, encode MHC class I-like molecules with two extracellular domains. Whereas H60b has a transmembrane region, H60c is a GPI-anchored protein. Recombinant soluble H60b and H60c proteins bound to NKG2D with affinities typical of cell–cell recognition receptors (K d  310 nM for H60b and K d  8.7 M for H60c). Furthermore, expression of H60b or H60c rendered Ba/F3 cells susceptible to lysis by NK cells, thereby estab- lishing H60b and H60c as functional ligands for NKG2D. H60b and H60c transcripts were detected only at low levels in tissues of healthy adult mice. Whereas H60b transcripts were detectable in various tissues, H60c transcripts were detected mainly in the skin. Infection of mouse embryonic fibroblasts with murine cytomegalovirus induced expression of H60b, but not H60c or the previously known H60 gene, indicating that transcriptional activation of the three types of H60 genes is differentially regulated. The present study adds two new members to the current list of NKG2D ligands. The Journal of Immunology, 2008, 180: 1678 –1685. C ytolytic activities of NK cells are controlled by the interplay of a multitude of inhibitory and activating receptors expressed on their surfaces (1, 2). Inhibitory receptors of the members of the mouse Ly49 and human killer Ig-related receptor families recognize self-MHC class I mole- cules on target cells (3, 4). Abnormal cells, whether trans- formed or infected, frequently lose expression of class I mole- cules. Because these cells are unable to engage inhibitory receptors, they become susceptible to lysis by NK cells. This mode of recognition by NK cells forms the basis of the “miss- ing-self” phenomenon (5). In comparison to inhibitory receptors, less is known about the ligands and biologic functions of activating receptors (6). Among the known activating receptors, the best characterized is NKG2D, a homodimeric C-type, lectin-like receptor encoded by the NK gene complex (7–10). In mice, NKG2D is expressed on NK cells, activated CD8   T cells, subsets of  T cells, subsets of NK T cells, macrophages, and IFN-producing killer dendritic cells (11–14). The ligands for NKG2D are usually expressed only poorly or not at all by normal cells, but their expression is up-regulated in response to cellular distress such as transformation (15), infection (16, 17), heat shock (18), and DNA damage (19). This up-regulation alerts the immune sys- tem to the presence of damaged and potentially dangerous cells via NKG2D receptors. Whereas NKG2D is a direct activating receptor in NK cells and macrophages, it acts as a costimulatory receptor in T cells (20). A unique property of the NKG2D receptor-ligand system is the presence of multiple ligands for a single receptor. In hu- mans, known ligands include MHC class I-related chains A and B (MICA and MICB) 4 molecules encoded in the MHC (20), as well as a total of five retinoic acid early inducible-1 (RAE1)/ UL16-binding protein (ULBP) molecules encoded outside the MHC (21, 22). Similarly, seven ligand molecules for NKG2D have been identified in mice: RAE1, RAE1, RAE1, RAE1, RAE1, H60, and MULT1 (murine ULBP-like transcript 1) (11, 23–25). All of these ligands are encoded outside the MHC and are more closely related to human retinoic acid early transcript (RAET)/ULBP than to MICA and MICB. Thus far, no ligands corresponding to human MICA/MICB have been identified in mice. *Department of Pathology, Hokkaido University Graduate School of Medicine, Sap- poro, Japan; † Department of Pathology, Sapporo City General Hospital, Sapporo, Japan; ‡ Department of Health Sciences, Hokkaido University School of Medicine, Sapporo, Japan; and § Division of Structural Biology, Medical Institute of Bioregu- lation, Kyushu University, Fukuoka, Japan Received for publication April 30, 2007. Accepted for publication November 27, 2007. 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. 1 This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by grants from the Japan Science and Technology Agency, the Northtec Foundation, the Takeda Science Foundation, and the Yasuda Medical Foundation. 2 These authors contributed equally to this work. The order of the first two authors is arbitrary. 3 Address correspondence and reprint requests to Dr. Masanori Kasahara, Department of Pathology, Hokkaido University Graduate School of Medicine, North-15, West-7, Sapporo 060-8638, Japan. E-mail: mkasaha@med.hokudai.ac.jp 4 Abbreviations used in this paper: MICA, MICB, MHC class I-related chains A and B; EST, expressed sequence tag; GSP, gene-specific primer; MCMV, murine CMV; MEF, mouse embryonic fibroblast; PI-PLC, phosphatidylinositol-specific phospholipase C; RAE1, retinoic acid early inducible-1; RAET, retinoic acid early transcript; SPR, surface plasmon resonance; ULBP, UL16-binding protein; UT, untranslated. Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 The Journal of Immunology www.jimmunol.org