Little is known concerning the stimulatory receptors responsible for tumor cell lysis by NK cells. We generated a monoclonal antibody specific for murine NKG2D in order to investigate its function. Blocking of NKG2D inhibited natural cytotoxicity of all tumor cells tested that express ligands for the receptor. Staining analysis showed that NKG2D is also expressed by activated CD8(+) T cells and macrophages, and subsets of TCRgammadelta(+) and NK1.1(+) T cells. Contradicting reports that NKG2D is solely a costimulatory receptor, we observed that cross-linking of NKG2D directly stimulates NK cells and activated macrophages. In contrast, NKG2D costimulates activated CD8(+) T cells. Thus, NKG2D engagement directly stimulates NK cells and macrophages, costimulates CD8(+) T cells, and plays a substantial role in natural killing.

Natural killer cells express inhibitory receptors specific for MHC class I proteins and stimulatory receptors with diverse specificities. The MHC-specific receptors discriminate among different MHC class I alleles and are expressed in a variegated, overlapping fashion, such that each NK cell expresses several inhibitory and stimulatory receptors. Evidence suggests that individual developing NK cells initiate expression of inhibitory receptor genes in a sequential, cumulative, and stochastic fashion. Superimposed on the receptor acquisition process are multiple education mechanisms, which act to coordinate the stimulatory and inhibitory specificities of developing NK cells. One process influences the complement of receptors expressed by individual NK cells. Other mechanisms may prevent NK cell autoaggression even when the developing NK cell fails to express self-MHC-specific inhibitory receptors. Together, these mechanisms ensure a self-tolerant and maximally discriminating NK cell population. Like NK cells, a fraction of memory phenotype CD8(+) T cells, as well as other T cell subsets, express inhibitory class I--specific receptors in a variegated, overlapping fashion. The characteristics of these cells suggest that inhibitory receptor expression may be a response to prior antigenic stimulation as well as to poorly defined additional signals. A unifying hypothesis is that both NK cells and certain T cell subsets initiate expression of inhibitory receptors in response to stimulation.

Mouse NK cells express at least seven inhibitory Ly49 receptors. Here we employ a semiquantitative cell-cell adhesion assay as well as class I/peptide tetramers to provide a comprehensive analysis of specificities of Ly49 receptors for class I MHC molecules in eight MHC haplotypes. Different Ly49 receptors exhibited diverse binding properties. The degree of class I binding was related to the extent of functional inhibition. The tetramer studies demonstrated that neither glycosylation nor coreceptors were necessary for class I binding to Ly49 receptors and uncovered peptide-specific recognition by a Ly49 receptor. The results provide a foundation for interpreting and integrating many existing functional studies as well as for designing tests of NK cell development and self-tolerance.

NK cells express several families of receptors that play central roles in target cell recognition. These NK cell receptors are also expressed by certain memory phenotype CD8+ T cells, and in some cases are up-regulated in T cells responding to viral infection. To determine how the profile of NK receptor expression changes in murine CD8+ T cells as they respond to intracellular pathogens, we used class I tetramer reagents to directly examine Ag-specific T cells during lymphocytic choriomeningitis virus and Listeria monocytogenes infections. We found that the majority of pathogen-specific CD8+ T cells initiated expression of the inhibitory CD94/NKG2A heterodimer, the KLRG1 receptor, and a novel murine NK cell marker (10D7); conversely, very few Ag-specific T cells expressed Ly49 family members. The up-regulation of these receptors was independent of IL-15 and persisted long after clearance of the pathogen. The expression of CD94/NKG2A was rapidly initiated in naive CD8+ T cells responding to peptide Ags in vitro and on many of the naive T cells that proliferate when transferred into lymphopenic (Rag-1−/−) hosts. Thus, CD94/NKG2A expression is a common consequence of CD8+ T cell activation. Binding of the CD94/NKG2A receptor by its ligand (Qa-1b) did not significantly inhibit CD8+ T cell effector functions. However, expression of CD94 and NKG2A transgenes partially inhibited early events of T cell activation. These subtle effects suggest that CD94/NKG2A-mediated inhibition of T cells may be limited to particular circumstances or may synergize with other receptors that are similarly up-regulated.

In this article (Immunity 17, 19-29, July 2002), a monoclonal antibody was employed to examine the expression and function of NKG2D in various cell types, including NK cells, CD8 T cells, NK T cells, ␥␦ T cells, and activated macrophages. The paper showed that NKG2D is expressed in several immune cell types, that it functions as an important receptor for tumor cell recognition by NK cells, and that crosslinking the receptor provides a primary activation signal to NK cells and an enhancing or costimulatory signal to activated CD8 ϩ T cells. These major conclusions of the paper are unchanged. However, we wish to alert the community that in recent repetitions of the staining experiments we have failed to obtain clear-cut staining of activated bone marrow or peritoneal macrophages with antibodies to NKG2D. We observed either no staining or only weak staining of undemonstrated specificity. Similarly, efforts to elicit a functional response from activated macrophages by crosslinking with antibodies to NKG2D yielded either no response or a questionable response. We conclude that bone marrow and peritoneal macrophages activated in vitro under our conditions do not express easily detectable levels of NKG2D on the cell surface. Low-level expression is still possible, because we confirmed earlier studies that activated macrophages do contain detectable levels of NKG2D mRNA, and it is known that these cells express necessary adapter proteins for surface expression of the receptor.