HLA-E is a major ligand for the natural killer inhibitory receptor CD 94 y NKG 2 A

We previously showed that the availability of a nonamer peptide derived from certain HLA class I signal sequences is a necessary requirement for the stabilization of endogenous HLA-E expression on the surface of 721.221 cells. This led us to examine the ability of HLA-E to protect HLA class I transfectants from natural killer (NK) cell-mediated lysis. It was possible to implicate the CD94yNKG2A complex as an inhibitory receptor recognizing this class Ib molecule by using as target a . 221 transfectant selectively expressing surface HLA-E. HLA-E had no apparent inhibitory effect mediated through the identified Ig superfamily (Ig-SF) human killer cell inhibitory receptors or ILT2yLIR1. Further studies of CD94yNKG21 NK cell-mediated recognition of . 221 cells transfected with different HLA class I allotypes (i.e., -Cw4, -Cw3, -B7) confirmed that the inhibitory interaction was mediated by CD94yNKG2A recognizing the surface HLA-E molecule, because only antibodies directed against either HLA-E, CD94, or CD94yNKG2A specifically restored lysis. Surface stabilization of HLA-E in cold-treated . 221 cells loaded with appropriate peptides was sufficient to confer protection, resulting from recognition of the HLA class Ib molecule by the CD94yNKG2A inhibitory receptor. Consistent with the prediction that the ligand for CD94yNKG2A is expressed ubiquitously, our examination of HLA-E antigen distribution indicated that it is detectable on the surface of a wide variety of cell types. Natural killer (NK) cells play an important role in the nonadaptive immune response, which is related to their ability to kill target cells lacking major histocompatibility (MHC) class I (1). NK cells express receptors that interact with MHC class I serving to inhibit cell-mediated cytotoxicity. In the mouse, these molecules are encoded by the Ly49 gene family, a group of type II membrane glycoproteins that are members of the C-type lectin superfamily (2, 3). The human killer cell inhibitory receptors (KIRs), which interact with HLA class I molecules, are type I glycoproteins, members of the Ig superfamily (Ig-SF) (4–6). In addition, the human CD94yNKG2 C-type lectin receptor complex also plays a role as NK cell receptor for HLA class I (6, 7). The invariant CD94 molecule is expressed as a heterodimer covalently associated with members of the NKG2 family on most NK cells and a subset of T lymphocytes (8, 9). The CD94yNKG2A heterodimer constitutes an inhibitory receptor, whereas the association of CD94 with other NKG2 proteins lacking immunoreceptor tyrosine-based inhibitory motifs (i.e., NKG2C) may form receptors with triggering function (10, 11). CD94yNKG2A has been proposed to be involved in the recognition of a wide variety of HLA-A, -B, and -C allotypes (12, 13), although these studies did not provide a clear rationale for the subdivision among the HLA class I apparently recognized by CD94yNKG2A. Additional inhibitory receptors of the Ig-SF have recently been identified (14); among them, ILT2yLIR1 is broadly distributed in different leukocyte lineages and has also been proven to interact with several HLA class I molecules as well as with the UL18 glycoprotein, an HLA homologue of human cytomegalovirus (15, 16). Recently, some studies have focused on NK recognition of nonclassical class I molecules, specifically HLA-G (17–20). Although conflicting results have been obtained on the role of Ig-SF KIRs and the CD94yNKG2 receptor recognition of cells expressing HLA-G, the general conclusion that this class Ib molecule can interact with at least some NK receptors to inhibit lysis in a manner similar to HLA class Ia antigens appears sound. Thus far, a formal proof for interaction with HLA-G has been reported only for the ILT2yLIR1 receptor (15). In addition to HLA-G, the human nonclassical or class Ib genes that are expressed include HLA-E and F (21). We previously obtained results indicating that the availability of a nonamer peptide derived from certain HLA class I signal sequences is a necessary requirement for HLA-E expression on the surface of 721.221 cells (22). These data showed that a number of HLA class I transfectants of . 221 were displaying not only the transfected class I allele on the surface, but also the endogenous HLA-E protein. This led us to form the hypothesis that HLA-E might play a role in the NK–HLA class I interactions in studies that used . 221 transfectants. In addition, because HLA-E has been highly conserved and exhibits low allelic polymorphism (23), we extended this hypothesis to propose that an interaction with HLA-E and the invariant CD94yNKG2A receptor was likely. In this study, we present strong evidence in support of this hypothesis. MATERIALS AND METHODS Antibodies and Antisera. mAb 3D12 (IgG1, ref. 22) reacts with the HLA-E free heavy chain as well as the heavy chain associated with b2-microglobulin and peptide. Anti-CD94 mAb HP-3B1 (IgG2a) and anti-HLA class I HP-1F7 (IgG1) have been described (18, 24). mAb Z199 (IgG2b) specific for the CD94yNKG2A heterodimer (9), anti-CD56 (C218), anti The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y955199-6$2.00y0 PNAS is available online at http:yywww.pnas. org. Abbreviations: Ig-SF, Ig superfamily; NK cells, natural killer cells; FITC, fluorescein isothiocyanate; KIR, human killer cell inhibitory receptor. A commentary on this article begins on page 4791. ‡N.L. and M.L. contributed equally to this work. ¶Present address: Department of Legal Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634, Japan. \M.L. -B. and D.E.G. share equal credit for senior authorship. **To whom reprint requests should be addressed. e-mail: geraghty@ fhcrc.org or mlbiotet/princesa@hup.es.