NK cell activation: distinct stimulatory pathways counterbalancing inhibitory signals

NK Cell Activation: Distinct Stimulatory Pathways Counterbalancing Inhibitory Signals Alexander B. H. Bakker, Jun Wu, Joseph H. Phillips, and Lewis L. Lanier ABSTRACT: A delicate balance between positive and negative signals regulates NK cell effector function. Activation of NK cells may be initiated by the triggering of multiple adhesion or costimulatory molecules, and can be counterbalanced by inhibitory signals induced by receptors for MHC class I. A common pathway of inhibitory signaling is provided by immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic domains of these receptors which mediate the recruitment of SH2 domain-bearing tyrosine phosphate-1 (SHP-1). In contrast to the extensive progress that has been made regarding the negative regulation of NK cell function, our

ABBREVIATIONS ADCC antibody-dependent cellular cytotoxicity cDNA complementary deoxyribonucleic acid HLA human leukocyte antigen IFN interferon Ig immunoglobulin Ig-SF Ig superfamily ILT Ig-like transcript ITAM immunoreceptor tyrosine-based activation motif ITIM immunoreceptor tyrosine-based inhibition motif

INTRODUCTION Natural killer (NK) cells are involved in the innate immune response against certain viruses, intracellular bacteria, parasites, and transformed cells [1– 4]. Like T cells, NK cells exert cell-mediated cytotoxicity, and produce an array of cytokines and chemokines. NK cell

From the Department of Immunobiology, DNAX Research Institute of Cellular and Molecular Biology, Palo Alto, California (all authors). Address reprint requests to: Dr. A. B. H. Bakker, Department of Immunobiology, DNAX Research Institute of Cellular and Molecular Biology, 901 California Ave., Palo Alto, California 94304-1104, USA. Received September 21, 1999; accepted September 27, 1999. Human Immunology 61, 18 –27 (2000) © American Society for Histocompatibility and Immunogenetics, 2000 Published by Elsevier Science Inc.

knowledge of the signals that activate NK cells is still poor. Recent studies of the activating receptor complexes have shed new light on the induction of NK cell effector function. Several NK receptors using novel adaptors with immunoreceptor tyrosine-based activation motifs (ITAMs) and with PI 3-kinase recruiting motifs have been implicated in NK cell stimulation. Human Immunology 61, 18 –27 (2000). © American Society for Histocompatibility and Immunogenetics, 2000. Published by Elsevier Science Inc. KEYWORDS: NK cells; DAP12; DAP10; 2B4

KIR LIR mAb MDL-1 MHC MIR NK PIR SH2 SHP

killer cell Ig-like receptor leukocyte Ig-like receptor monoclonal antibody myeloid DAP12-associating lectin-1 major histocompatibility complex monocyte Ig-like receptor natural killer paired Ig receptors src-homology 2 SH2 domain-bearing tyrosine phosphatase

activity is regulated by opposing signals from receptors that activate and inhibit effector function. Unlike B and T cells, NK cells do not possess a uniquely rearranged immunoglobulin or T-cell receptor that can trigger their activation [5–7]. Instead, activating signals for NK cells may be initiated by many different adhesion or costimulatory molecules. Once activated, NK cells are able to kill virus-infected and transformed cells, implying that they are able to discriminate between normal and abnormal host cells. Based on the ability of NK cells to kill preferentially tumor cells that lacked the expression of major histocompatibility complex (MHC) class I mol0198-8859/00/$–see front matter PII S0198-8859(99)00160-3

NK Cell Activation: Stimulatory Pathways and Inhibitory Signals

19

FIGURE 1 Activation of NK cells via distinct stimulatory pathways.

ecules, proposed a mechanism of immune surveillance has been proposed to eliminate cells that have down regulated MHC [8, 9]. As predicted, NK cells have been shown to express receptors for MHC class I that, upon ligation, inhibit NK cell-mediated cytotoxicity and cytokine secretion. The inhibitory receptors on NK cells have been extensively reviewed recently [10 –13]. MEMBRANE RECEPTORS IMPLICATED IN NK CELL ACTIVATION It has been proposed that the initiation of NK cell immune responses may result from signals originating from one or more adhesion or co-stimulatory molecules, rather than from a unique NK receptor [14]. As a consequence of inflammation, myeloid cells, endothelial cells, and other tissues have been shown to up regulate CD54 (ICAM-1), CD58 (LFA-3), VCAM-1, CD80, and CD86. Expression of an appropriate array of these costimulatory ligands at adequate levels may be sufficient to activate NK cells bearing the corresponding receptors. Resting or activated NK cells express CD2, CD11a/ CD18 (LFA-1), CD69, CD49d/CD29 (VLA-4), and DNAM-1 [15–19]. In addition, human fetal NK cells and mouse NK cells express CD28 [20, 21]. Experiments using Fc-receptor-bearing targets coated with monoclonal antibodies (mAbs) against some of these costimulatory molecules demonstrated that they are able to induce cytotoxicity. The most extensively characterized membrane receptor on NK cells is the low affinity receptor for immunoglobulin (Ig)G, CD16 [22]. Ligation of CD16 by immune complexes or antibody-coated target cells induces antibody-dependent cellular cytotoxicity (ADCC) and cytokine secretion [23–25]. CD16 is noncovalently associated with Fc⑀RI␥ or CD3␨, which mediates signal

transduction [26, 27]. Upon the engagement of CD16, src-family tyrosine kinases phosphorylate tyrosine residues contained within immunoreceptor tyrosine-based activation motifs (ITAM) in the cytoplasmic domains of Fc⑀RI␥ and/or CD3␨ [28, 29]. In turn, this triggers a signaling cascade similar to that activated by the TcR. While CD16 is responsible and required for ADCC, it has not been implicated in other modes of NK cellmediated cytotoxicity. Another receptor capable of triggering NK cells is NKR-P1, which in the mouse is also known as NK1.1 (for detailed reviews see [10, 30]). Crosslinking of NKR-P1 on NK cells induces cytotoxicity and IFN␥ secretion and, similar to CD16, this signaling pathway may require the Fc⑀RI␥ chain [31]. In rodents, three related genes have been identified (NKR-P1A, NKRP1B, and NKR-P1C). Interestingly, mouse and rat NKR-P1B contains an ITIM in its cytoplasmic domain and recently mouse NKR-P1B has been shown to function as an inhibitory receptor [32, 33]. Triggering of human NK cells with anti-NKR-P1 mAb can result in either activation or inhibition [34, 35], suggesting that functionally distinct isoforms of NKR-P1 may exist in humans as well. 2B4 2B4 is a cell surface receptor associated with NK and T cell-mediated non-MHC restricted cytotoxicity. Crosslinking of this receptor on mouse NK cells induces target cell lysis and IFN␥ secretion [36]. Cloning of the complementary deoxyribonucleic acid (cDNA) encoding mouse 2B4 revealed that the receptor belongs to the immunoglobulin (Ig) superfamily, sharing homology with Ly9, CD48, CD58 [37]. Recently, the human homologue of mouse 2B4 has been cloned [38, 39]. Human

20

2B4 is recognized by the C1.7 mAb, a mAb described as being expressed on all NK cells and a subset of CD8⫹ T cells mediating non-MHC-restricted lysis [40]. Human 2B4 is also expressed on monocytes and basophils [39, 40]. Similar to mouse 2B4, mAb-mediated ligation of human 2B4 on NK cells induced lysis of Fc-bearingtarget cells, demonstrating that human 2B4 is an activating receptor. Human 2B4 exhibits approximately 70% identity with mouse 2B4, and its cytoplasmic domain contains four previously unrecognized TxYxxV/I motifs which, aligned with identical motifs, present in mouse 2B4. Interestingly, the same motifs can be found in the intracellular domain of SLAM, another two Ig domain receptor sharing homology with 2B4 [41]. Upon pervanadate treatment, human 2B4 becomes phosphorylated and is able to recruit SHP-2, but not SHP-1 [38]. The cytoplasmic domain of SLAM constitutively associates with the SH2 domain-containing adapter protein SAP, and SAP competes with SHP-2 for binding to phosphorylated SLAM [42]. By analogy, human 2B4 also binds SAP, thereby, preventing its association with SHP-2 [38]. Unlike SLAM, SAP could only bind to phosphorylated 2B4. The X-linked lymphoproliferative disease is caused by a defect in the SAP gene [42]. Although SAP was identified by its ability to associate with SLAM, it is likely that NK and CD8⫹ T cells from XLP patients also display disregulated signaling through human 2B4 and possibly other receptors, thus, contributing to disease. Provided that the positive signaling through SLAM and 2B4 is mediated via SHP-2, the recruitment of SAP could negatively influence this response by displacing SHP-2 from SLAM and 2B4. Alternatively, in the case of 2B4, SAP might bring in adaptor molecules that are capable of transmitting the positive signal, thereby, alleviating a potential negative signal mediated via SHP-2. A recent report suggests that, indeed, a splice variant of mouse 2B4 can function as an inhibitory receptor in transfected RNK16 cells through an association with SHP-2 [43]. However, this study is incomplete because the transfectant expressing this isoform of 2B4 appears to have lost its cytotoxic function, and the investigators did not analyze the function of SAP in the RNK16 cells. Recently, CD48 has been identified as the ligand for 2B4 by protein binding assays and surface plasmon resonance [44, 45]. Previously, CD48 was shown to be a counterstructure for CD2 [46]. Interestingly, CD48⫺/⫺ mice display decreased immune responses [47]. Since mice with a targeted disruption in the CD2 gene have a normal immune system [48], this suggests that the lack of signaling through 2B4 receptor complexes may contribute to the immunocompromized phenotype observed in CD48⫺/⫺ mice.

A. B. H. Bakker et al.

MHC CLASS I INHIBITORY RECEPTORS Four distinct types of membrane receptors expressed on NK cells bind MHC class I molecules and inhibit the lysis of MHC class I-bearing target cells (reviewed in [10 –13]. Mouse NK cells express MHC class I receptors encoded by a family of genes designated Ly49A-I, which localize within the NK complex on mouse chromosome 6 [49]. Ly49 receptors are type II glycoproteins of the C-type lectin superfamily that are expressed as disulfidebonded homodimers on overlapping subsets of mouse NK cells. A single human Ly49-like gene has been cloned that appears to be a pseudogene and may represent a remnant of a common ancestral gene [50, 51]. Most Ly49 receptors contain an immunoreceptor tyrosine-based inhibition motif (ITIM) in their cytoplasmic domains. Upon tyrosine phosphorylation, the ITIM recruits the cytoplasmic tyrosine phosphatase SHP-1 or SHP-2 that, subsequently, inhibits NK cell effector function [52, 53]. Human NK cells express another set of C-type lectin receptors that are heterodimers composed of an invariant common subunit, CD94, which is disulfide-linked to a distinct glycoprotein encoded by a gene of the NKG2 family [54 –56]. The CD94 and NKG2 genes are present on human chromosome 12 at the syntenic region to the mouse NK complex on chromosome 6. CD94/NKG2A is an inhibitory receptor that mediates negative signaling via the ITIM-bearing NKG2A subunit [57]. The CD94/ NKG2A complex was initially believed to be involved in the recognition of a broad spectrum of human leukocyte antigen (HLA) molecules, including HLA-G. However, there were no obvious structural similarities in the extracellular domains of the HLA-A, -B, -C, or -G alleles that could explain the specificity of recognition by CD94/NKG2A⫹ NK cells. Recent investigations revealed that HLA-E, which binds leader peptides originating from HLA-A, -B, -C, or -G alleles, is recognized by the CD94/NKG2A complex [58 – 60]. As a result, HLA-E monitors the overall expression level of MHC class I on the cell surface, thereby, allowing NK cells to scrutinize a multitude of HLA class I molecules, yet using only one invariant receptor to bind the non-polymorphic HLA-E ligand. In the mouse the class Ib molecule Qa-1, like HLA-E, binds similar peptides derived from mouse H2 class I proteins [61]. Recently, it has been shown that mouse CD94/NKG2A recognizes Qa-1b [62]. Thus this elegant mechanism by which NK cells can monitor MHC class I expression on target cells is evolutionary conserved. A second set of MHC class I receptors expressed on human NK cells are the killer cell Ig-like receptors (KIRs) (reviewed in [12]). Most KIR molecules are monomeric type I glycoproteins that contain either 2 or 3

NK Cell Activation: Stimulatory Pathways and Inhibitory Signals

Ig-like domains in their extracellular region (KIR2D or KIR3D). Isoforms with long intracellular tails (KIR2DL and KIR3DL) contain ITIMs that bind SHP-1 and mediate NK cell inhibition, while other isoforms contain short cytoplasmic regions that lack signaling motifs (KIR2DS). The KIR2D molecules predominantly recognize HLA-C alleles, while KIR3D receptors have been implicated in the recognition of HLA-Bw4 alleles (for a detailed review see [10]). KIR2DL4, originally called KIR103, has been demonstrated to recognize HLA-G [63, 64]. Another family of glycoproteins designated immunoglobulin-like transcripts (ILTs), leukocyte immunoglobulin-like receptors (LIRs) or monocyte immunoglobulinlike receptor (MIRs) includes at least eight genes clustered with the KIR gene family on human chromosome 19p13.4 [65– 68]. ILT proteins contain either 2 or 4 Ig-like domains and most ILT receptors bear cytoplasmic ITIMs. ILT-2 and ILT-4 proteins interact with a broad spectrum of HLA class I molecules and upon ligand binding recruit SHP-1, which leads to the inhibition of several cellular effector functions [68 –71]. For most members of the ILT family, the ligands have not yet been identified. Interestingly, the expression of ILT family members is predominantly on myeloid cells. ILT-2 is expressed on subsets of NK cells, B cells and monocytes and macrophages, while ILT-4 is expressed exclusively on monocytes, macrophages, and dendritic cells [69, 70, 72]. Therefore, ILT-2 and -4 may inhibit cellular responses on myelomonocytic cells upon interaction with MHC class I molecules. Although ILT homologues have not been identified in rodents, a family of mouse receptors related to the ILT family are the paired immunoglobulin receptors (PIR) or p91 [73, 74]. PIR molecules contain six Ig-like domains, and are expressed on myeloid cells and B cells. PIR family members either carry a short intracytoplasmic tail lacking ITIMs (PIR-A) or they contain a long cytoplasmic region containing one ITIM (PIR-B). So far, no ligands for PIR have been identified. The PIR genes map to a region on mouse chromosome 7 that is syntenic with the KIR/ILT complex at human chromosome 19q13.4 [66, 73, 75]. Activating short isoforms of both the ILT (ILT-1) and PIR families (PIR-A1) has been demonstrated to associate with Fc⑀RI␥ to form functional activating receptor complexes [76 –78]. ACTIVATING MHC CLASS I RECEPTORS USE THE SIGNALING ADAPTOR MOLECULE DAP12 Certain isoforms of the MHC class I receptors on NK cells lack ITIM sequences in their cytoplasmic domains and it has been proposed that these isoforms activate,

21

rather than inhibit, NK cells [79 – 81]. These activating receptors have very short intracellular regions lacking any signaling motifs and they all share a positively charged residue within their transmembrane domain, which suggested the association with an adapter molecule capable of signaling. DAP12, a type I disulfidelinked homodimer containing an ITAM, non-covalently assembles with the human KIR2DS receptors [82]. DAP12 has a negatively charged aspartic acid residue in its transmembrane region and corresponds to the previously reported 12–13 kD phosphoprotein that was found to co-immunoprecipitate with KIR2DS [83, 84]. Upon receptor engagement, DAP12 becomes phosphorylated and recruits the Syk kinase, thus, inducing a signaling cascade similar to TcR [82, 85]. In addition to being associated with KIR2DS, a receptor for HLA-C, DAP12 is also expressed at the cell surface of NK cells associated with the activating mouse Ly49D and Ly49H receptors that recognize H-2 [86, 87] and with the human CD94/NKG2C heterodimer receptor complex that recognizes HLA-E [88]. The conservation of activating NK cell receptors for MHC class I during evolution suggests that their role is of importance to the immune system. However, the biological rationale for paired inhibitory and activating receptors for MHC class I is still unclear. The KIR2DS receptors bind HLA-C allotypes with lower affinities than their KIR2DL inhibitory counterparts [89, 90]. This difference may ensure that the inhibitory signal overrules the activating signal when both activating and inhibitory receptors recognizing HLA molecules are expressed on the same NK cell. A potential role for the activating MHC class I receptor isoforms might be that they serve to recruit the tyrosine kinase responsible for the phosphorylation of the ITIM sequences in the inhibitory isoforms. However, this is unlikely, since activating and inhibitory receptors are expressed differentially within the total NK cell population and only a minor subset of NK cells expresses both forms that can recognize identical HLA allotypes [91–93]. Alternatively, the engagement of activating MHC class I receptors might function during development potentially by positively selecting immature NK cells. However, there is no evidence for the appearance of activating receptors prior to inhibitory receptors during development. In addition, NK cell development is normal in DAP12⫺/⫺ mice (Bakker and Lanier, unpublished observation). A third model involves NK cells that express activating and inhibitory MHC class I receptors recognizing distinct MHC class I molecules. In this model, engagement of the inhibitory receptor dominates when ligands for both types of receptors are encountered. Now activating receptors may only signal when target cells have lost the expression of an HLA allele recognized by the

22

inhibitory receptor co-expressed on the NK cell, thus, allowing NK cells to attack cells selectively that lack a single MHC class I allele but leaving normal cells unaffected. Physiologic circumstances in which this latter scenario might apply are virus-infected or transformed cells, which may selectively down regulate HLA-A and -B allotypes while leaving HLA-C and -E unaffected [94, 95]. OTHER DAP12-ASSOCIATING RECEPTORS NKp44 represents a NK cell specific receptor involved in non-MHC-restricted cytotoxicity that is specifically expressed on IL-2-activated NK cells [96]. The induced expression of NKp44 may contribute to the high cytolytic activity displayed by IL-2 activated NK cells against tumor cells. NKp44, an Ig superfamily (Ig-SF) member containing one Ig-like domain, contains a charged lysine in its transmembrane region that is responsible for the association of NKp44 with DAP12 [97]. In contrast to most Ig-SF NK receptors that are encoded by genes on chromosome 19, the NKp44 gene is located on human chromosome 6. NKp44 may synergize with NKp46, another stimulatory receptor specifically expressed on NK cells [98]. NKp46 is expressed on both resting and activated NK cells, and crosslinking of this receptor results in cytokine production and cytolytic activity. Cloning of the cDNA encoding NKp46 revealed that this receptor belongs to the Ig-SF and bears resemblance with the ILT/MIR family [99]. The gene encoding NKp46 is localized on human chromosome 19. To signal, NKp46 may associate with CD3␨ [96]. Natural cytotoxicity of NK cells against MHC class I-negative targets has been shown to correlate with the level of NKp46 expression on the NK cells [100]. DAP12 is not only expressed in NK cells, but also in peripheral blood granulocytes, monocytes, and dendritic cells. Polymerase chain reaction (PCR) analysis revealed DAP12 transcripts in a mouse neuroblastoma cell line as well, suggesting that DAP12 might also be expressed in brain [101]. However, biochemical analyses of both mouse and human neuroblastoma cell lines, and immunohistochemistry of mouse brain tissue sections failed to detect any DAP12 protein other than on microglial cells (Bakker, Hoek, and Lanier, unpublished results). Therefore, DAP12 expression is confined to hematopoietic cells. The expression of DAP12 in myeloid cells suggested that there could be other novel receptors associating with this adaptor. Application of an indirect expression cloning strategy resulted in the identification of a novel DAP12-associated receptor myeloid cells [102]. When expressed in the absence of associating receptors, DAP12 largely remains located intracellularly. However, upon

A. B. H. Bakker et al.

the introduction of an associating partner, DAP12 complexes can be formed, resulting in the translocation of DAP12 to the cell surface. Using a 293T cell line stably expressing DAP12 intracellularly, a cDNA clone was obtained from a macrophage cDNA library that rescued DAP12 cell surface expression. This cDNA encoded a novel type II transmembrane protein belonging to the C-type lectin superfamily exclusively expressed in monocytes and macrophages, termed myeloid DAP12-associating lectin-1 (MDL-1). The gene encoding human MDL-1 is localized on chromosome 7q33. Although the physiologic role or ligand of this molecule is not known, the receptor complex is functional, since crosslinking of the MDL-1/DAP12 complex on J774 mouse macrophage cells resulted in calcium mobilization. Signaling via MDL-1/DAP12 complexes may, therefore, constitute a significant activation pathway in myeloid cells. MDL-1 transcripts were detected at very low levels in dendritic cells that had been generated by culturing human monocytes in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. However, these cells express a significant amount of DAP12, suggesting the presence of other receptors. Candidate dendritic cell receptors that associate with DAP12 are currently under investigation. These data suggest that MDL-1 receptors may be involved in the proinflammatory activation of macrophages, while DAP12 complexes involving other novel receptors may activate dendritic cells. Together, DAP12 may function in cellular activation mediated by a diverse array of receptors in both NK cells and in the myeloid cell lineage. DAP10: THE NEIGHBOR’S NEW TALE Recent efforts to identify potential membrane signaling proteins by searching the EST databases have led to the identification of DAP10, a novel 10-kD surface adaptor primarily expressed in hematopoietic cells [103]. Although DAP10 has only limited homology with DAP12, its transmembrane domain contains a negatively charged residue that is conserved in the transmembrane regions of DAP12 and all CD3 subunits of the TCR. In addition, the conserved cysteine residues within the extracellular domain of DAP12 and the CD3 chains are also present in DAP10. Interestingly, the human DAP10 and DAP12 genes lie adjacent on chromosome 19q13.1, in opposite transcriptional orientation and separated by only approximately 130 bp, presumably as a result of gene duplication. One unique feature of DAP10 is its short but conserved cytoplasmic tail, which contains a YxxM signaling motif, a potential src-homology 2 (SH2) domainbinding site for the p85 regulatory subunit of the phosphatidylinositol 3-kinase (PI 3-kinase) [104]. The presence of the acidic amino acid within its

NK Cell Activation: Stimulatory Pathways and Inhibitory Signals

transmembrane segment and the precedent for interaction between proteins of multisubunit immune receptor complexes through oppositely charged residues in the transmembrane regions (such as the KIR2DS2/DAP12 complex and the TCR/CD3 complex) predicted that DAP10 would associate with a membrane receptor with a basic residue in the transmembrane region. Indeed, a membrane protein was found to co-immunoprecipitate with DAP10 in NK and T cells [103]. Subsequent biochemical and cotransfection experiments have identified this DAP10-associated protein as NKG2D, an orphan C-type lectin receptor within the NK complex on human chromosome 12 and on mouse chromosome 6 [103, 105]. Despite its name, NKG2D is not a member of the NKG2 family and, thus, does not form heterodimers with CD94. Instead, NKG2D contains a basic residue within its transmembrane domain and requires an association with DAP10 for surface expression. The assembly of such receptor complexes appears to be highly specific, since in vivo DAP10 and DAP12 interact exclusively with their partners, despite similarities in their transmembrane regions. Preliminary mutagenesis experiments suggested that residues other than the charged amino acids within the transmembrane regions of DAP10 and DAP12 are critical for providing such specificity (Wu, unpublished results). The activating function of the NKG2D/DAP10 receptor complex has been demonstrated by the potent anti-NKG2D mAb-mediated redirected lysis of the FcR⫹ target cells by NK cells and CD8⫹ T cells [103, 106]. Since the cytoplasmic domain of NKG2D is not well conserved and lacks any positive signaling motif, one region likely to mediate such activating function is the cytoplasmic domain of DAP10, in which the conserved YxxM sequence resembles the predicted binding motif for the SH2 domain of the p85 subunit of PI 3-kinase. In fact, the DAP10 cytoplasmic domain, upon tyrosine phosphorylation, was shown to specifically recruit p85 both in vitro and in vivo [103]. Consistent with this, treatment of NK cells with PI 3-kinase specific inhibitors diminished the functions mediated by NKG2D/DAP10 receptor (Wu, unpublished results). Thus, DAP10 is likely to function as a membrane signaling adaptor leading to downstream PI 3-kinase activation, which subsequently results in NK cell effector functions. The functional significance of the NKG2D/DAP10 receptor is further underscored by the identification of the human non-classic MHC class I molecule MICA as the ligand for NKG2D [106]. MICA and its related gene were previously known to function as stress-inducible antigens in epithelium and are recognized by ␥␦ T cells [107, 108]. However, recent studies have revealed that MICA antigens are also frequently expressed in epithelial

23

tumors, including lung, breast, kidney, ovary, prostate, and colon carcinoma [109]. Interestingly, NKG2D on NK cells, CD8⫹ ␣␤ T cells, and ␥␦ T cells are capable of mediating killing of MICA transfectants and MICAbearing tumor cells [106]. Therefore, the activating NKG2D/DAP10 receptor complex may represent yet another weapon in the innate immune surveillance against tumors and various danger signals. CONCLUDING REMARKS Recent progress in the field of NK cell biology led to the identification of distinct receptor families with paired activation/inhibition isoforms. While the presence of inhibitory receptors on NK cells can be readily explained from the perspective of self-protection, a physiological role for the activating isoforms is still missing. The identification of stress-inducible ligands as targets for NK cells has further emphasized their role in the innate immune system. The characterization of new Ig-like receptors on macrophages and dendritic cells has widened the field and adds another layer of immune regulation. Experimental models, including gene knockouts, may clarify the in vivo function of these receptors in the immune system. REFERENCES 1. Scott P, Trinchieri G: The role of natural killer cells in host-parasite interactions. Curr Opin Immunol 7:34, 1995. 2. Unanue ER: Inter-relationship among macrophages, natural killer cells and neutrophils in early stages of Listeria resistance. Curr Opin Immunol 9:35, 1997. 3. Scharton-Kersten TM, Sher A: Role of natural killer cells in innate resistance to protozoan infections. Curr Opin Immunol 9:44, 1997. 4. Biron CA, Nguyen KB, Pien GC, Cousens LP, SalazarMather TP: Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol 17:189, 1999. 5. Lanier LL, Cwirla S, Phillips JH: Genomic organization of T cell ␥ genes in human peripheral blood natural killer cells. J Immunol 137:3375, 1986. 6. Lanier LL, Cwirla S, Federspiel N, Phillips JH: Human natural killer cells isolated from peripheral blood do not rearrange T cell antigen receptor ␤ chain genes. J Exp Med 163:209, 1986. 7. Loh EY, Cwirla S, Serafini AT, Phillips JH, Lanier LL: Human T-cell receptor ␦ chain: genomic organization, diversity, and expression in populations of cells. Proc Natl Acad Sci USA 85:9714, 1988. 8. Karre K, Ljunggren HG, Piontek G, Kiessling R: Selective rejection of H-2-deficient lymphoma variants

24

9.

10. 11. 12. 13.

14.

15.

16.

17.

18.

19.

20.

21.

22. 23.

24.

A. B. H. Bakker et al.

suggests alternative immune defense strategy. Nature 319:675, 1986. Ljunggren H-G, Karre K: In search of the ‘missing self’: MHC molecules and NK cell recognition. Immunol Today 11:237, 1990. Lanier LL: NK cell receptors. Annu Rev Immunol 16: 359, 1998. Yokoyama WM: Natural killer cell receptors. Curr Opin Immunol 10:298, 1998. Long EO: Regulation of immune responses through inhibitory receptors. Annu Rev Immunol 17:875, 1999. Lopez-Botet M, Bellon T: Natural killer cell activation and inhibition by receptors for MHC class I. Curr Opin Immunol 11:301, 1999. Hersey P, Bolhuis R: ‘Nonspecific’ MHC-unrestricted killer cells and their receptors. Immunol Today 8:233, 1987. Siliciano RF, Pratt JC, Schmidt RE, Ritz J, Reinherz EL: Activation of cytolytic T lymphocyte and natural killer cell function through the T11 sheep erythrocyte binding protein. Nature 317:428, 1985. Timonen T, Patarroyo M, Gahmberg CG: CD11a-c/ CD18 and GP84 (LB-2) adhesion molecules on human large granular lymphocytes and their participation in natural killing. J Immunol 141:1041, 1988. Lanier LL, Buck DW, Rhodes L, Ding A, Evans E, Barney C, Phillips JH: Interleukin 2 activation of natural killer cells rapidly induces the expression and phosphorylation of the Leu-23 activation antigen. J Exp Med 167:1572, 1988. Robertson MJ, Caligiuri MA, Manley TJ, Levine H, Ritz J: Human natural killer cell adhesion molecules: differential expression after activation and participation in cytolysis. J Immunol 145:3194, 1990. Shibuya A, Campbell D, Hannum C, Yssel H, FranzBacon K, McClanahan T, Kitamura T, Nicholl J, Sutherland GR, Lanier LL, Phillips JH: DNAM-1, a novel adhesion molecule involved in the cytolytic function of T lymphocytes. Immunity 4:573, 1996. Azuma M, Cayabyab M, Buck D, Phillips JH, Lanier LL: Involvement of CD28 in major histocompatibility complex-unrestricted cytotoxicity mediated by a human NK leukemia cell line. J Immunol 149:1115, 1992. Nandi D, Gross JA, Allison JP: CD28-mediated costimulation is necessary for optimal proliferation of murine NK cells. J Immunol 152:3361, 1994. Daeron M: Fc receptor biology. Annu Rev Immunol 15:203, 1997. Perussia B, Trinchieri G, Jackson A, Warner NL, Faust J, Rumpold H, Kraft D, Lanier LL: The Fc receptor for IgG on human natural killer cells: phenotypic, functional, and comparative studies with monoclonal antibodies. J Immunol 133:180, 1984. Cassatella MA, Angeon I, Cuturi MC, Griskey P,

25.

26.

27.

28.

29.

30. 31.

32.

33.

34.

35.

36.

37.

Trinchieri G, Perussia B: Fc␥R (CD16) interaction with ligand induces Ca2⫹ mobilization and phosphoinositide turnover in human natural killer cells. Role of Ca2⫹ in Fc␥R (CD16)-induced transcription and expression of lymphokine genes. J Exp Med 169:549, 1989. Anegon I, Cuturi MC, Trinchieri G, Perussia B: Interaction of Fc receptor (CD16) ligands induces transcription of interleukin 2 receptors (CD25) and lymphokine genes and expression of their products in human natural killer cells. J Exp Med 167:452, 1988. Ravetch JV, Perussia B: Alternative membrane forms of Fc␥RIII (CD16) on human natural killer cells and neutrophils: cell type-specific expression of two genes that differ in single nucleotide substitutions. J Exp Med 170:481, 1989. Lanier LL, Yu G, Phillips JH: Co-association of CD3␨ with a receptor (CD16) for IgG Fc on human natural killer cells. Nature 342:803, 1989. O’Shea J, Weissman AM, Kennedy ICS, Ortaldo JR: Engagement of the natural killer cell IgG Fc receptor results in tyrosine phosphorylation of the ␨ chain. Proc Natl Acad Sci USA 88:350, 1991. Vivier E, da Silva AJ, Ackerly M, Levine H, Rudd CE, Anderson P: Association of a 70-kDa tyrosine phosphoprotein with the CD16:␨;␥ complex expressed in human natural killer cells. Eur J Immunol 23:1872, 1993. Ryan JC, Seaman WE: Divergent functions of lectin-like receptors on NK cells. Immunol Rev 155:79, 1997. Arase N, Arase H, Park SY, Ohno H, Ra C, Saito T: Association with FcR␥ is essential for activation signal through NKR-P1 (CD161) in natural killer (NK) cells and NK1.1⫹ T cells. J Exp Med 186:1957, 1997. Kung SK, Su RC, Shannon J, Miller RG: The NKR-P1B gene product is an inhibitory receptor on SJL/J NK cells. J Immunol 162:5876, 1999. Carlyle JR, Martin A, Mehra A, Attisano L, Tsui FW, Zuniga-Pflucker JC: Mouse NKR-P1B, a novel NK1.1 antigen with inhibitory function. J Immunol 162:5917, 1999. Lanier LL, Chang C, Phillips JH: Human NKR P1A: a disulfide linked homodimer of the C-type lectin superfamily expressed by a subset of NK and T lymphocytes. J Immunol 153:2417, 1994. Poggi A, Costa P, Morelli L, Cantoni C, Pella N, Spada F, Biassoni R, Nanni L, Revello V, Tomasello E, Mingari MC, Moretta A, Moretta L: Expression of human NKRP1A by CD34⫹ immature thymocytes: NKRP1Amediated regulation of proliferation and cytolytic activity. Eur J Immunol 26:1266, 1996. Garni-Wagner BA, Purohit A, Mathew PA, Bennett M, Kumar K: A novel function-associated molecule related to non-MHC-restricted cytotoxicity mediated by activated natural killer cells and T cells. J Immunol 151:60, 1993. Mathew PA, Garni-Wagner BA, Land K, Takashima A,

NK Cell Activation: Stimulatory Pathways and Inhibitory Signals

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

Stoneman E, Bennett M, Kumar V: Cloning and characterization of the 2B4 gene encoding a molecule associated with non-MHC-restricted killing mediated by activated natural killer cells and T cells. J Immunol 151:5328, 1993. Tangye SG, Lazetic S, Woollatt E, Sutherland GR, Lanier LL, Phillips JH: Human 2B4, an activating NK cell receptor, recruits the protein tyrosine phosphatase SHP-2 and the adaptor signaling protein SAP. J Immunol 162:6981, 1999. Nakajima H, Cella M, Langen H, Friedlein A, Colonna M: Activating interactions in human NK cell recognition: the role of 2B4-CD48. Eur J Immunol 29:1676, 1999. Valiante NM, Trinchieri G: Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes. J Exp Med 178:1397, 1993. Cocks BG, Chang C-CJ, Carballido JM, Yssel H, de Vries JE, Aversa G: A novel receptor involved in T-cell activation. Nature 376:260, 1995. Sayos J, Wu C, Morra M, Wang N, Zhang X, Allen D, van Schaik S, Notarangelo L, Geha R, Roncarolo MG, Oettgen H, De Vries JE, Aversa G, Terhorst C: The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAM. Nature 395:462, 1998. Schatzle JD, Sheu S, Stepp SE, Mathew PA, Bennett M, Kumar V: Characterization of inhibitory and stimulatory forms of the murine natural killer cell receptor 2B4. Proc Natl Acad Sci USA 96:3870, 1999. Brown MH, Boles K, Anton van der Merwe P, Kumar V, Mathew PA, Barclay NA: 2B4, the natural killer and T cell immunoglobulin superfamily surface protein, is a ligand for CD48. J Exp Med 188:2083, 1998. Latchman Y, McKay PF, Reiser H: Identification of the 2B4 molecule as a counter-receptor for CD48. J Immunol 161:5809, 1998. Kato K, Koyanagi M, Okada H, Takanashi T, Wong YW, Williams AF, Okumura K, Yagita H: CD48 is a counter-receptor for mouse CD2 and is involved in T cell activation. J Exp Med 176:1241, 1992. Gonzalez-Cabrero J, Wise CJ, Latchman Y, Freeman GJ, Sharpe AH, Reiser H: CD48-deficient mice have a pronounced defect in CD4⫹ T cell activation. Proc Nat Acad Sci USA 96:1019, 1999. Killeen N, Stuart SG, Littman DR: Development and function of T cells in mice with a disrupted CD2 gene. EMBO J 11:4329, 1992. Brown MG, Fulmek S, Matsumoto K, Cho R, Lyons PA, Levy ER, Scalzo AA, Yokoyama WM: A 2-Mb YAC contig and physical map of the natural killer gene complex on mouse chromosome 6. Genomics 42:16, 1997. Westgaard IH, Berg SF, Orstavik S, Fossum S, Dissen E: Identification of a human member of the Ly-49 multigene family. Eur J Immunol 28:1839, 1998.

25

51. Barten R, Trowsdale J: The human Ly-49L gene. Immunogenetics 49:731, 1999. 52. Olcese L, Lang P, Vely F, Cambiaggi A, Marguet D, Blery M, Hippen KL, Biassoni R, Moretta A, Moretta L, Cambier JC, Vivier E: Human and mouse killer-cell inhibitory receptors recruit PTP1C and PTP1D protein tyrosine phosphatases. J Immunol 156:4531, 1996. 53. Nakamura MC, Niemi EC, Fisher MJ, Shultz LD, Seaman WE, Ryan JC: Mouse Ly49A interrupts early signaling events in NK cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase. J Exp Med 185:673, 1997. 54. Lazetic S, Chang C, Houchins JP, Lanier LL, Phillips JH: Human NK cell receptors involved in MHC class I recognition are disulfide-linked heterodimers of CD94 and NKG2 subunits. J Immunol 157:4741, 1996. 55. Carretero M, Cantoni C, Bellon T, Bottino C, Biassoni R, Rodriguez A, Perez-Villar JJ, Moretta L, Moretta A, Lopez-Botet M: The CD94 and NKG2-A C type lectins covalently assemble to form a natural killer cell inhibitory receptor for HLA class I molecules. Eur J Immunol 27:563, 1997. 56. Brooks AG, Posch PE, Scorzelli CJ, Borrego F, Coligan JE: NKG2A complexed with CD94 defines a novel inhibitory NK cell receptor. J Exp Med 185:795, 1997. 57. Le Drean E, Vely F, Olcese L, Cambiaggi A, Guia S, Krystal G, Gervois N, Moretta A, Jotereau F, Vivier E: Inhibition of antigen-induced T cell response and antibody-induced NK cell cytotoxicity by NKG2A: association of NKG2A with SHP-1 and SHP-2 protein-tyrosine phosphatase. Eur J Immunol 28:264, 1998. 58. Braud VM, Allan DSJ, O’Callaghan CA, Soderstrom K, D’Andrea A, Ogg GS, Lazetic S, Young NT, Bell JI, Phillips JH, Lanier LL, McMichael AJ: HLA-E binds to natural killer cell receptors CD94/NKG2A, B, and C. Nature 391:795, 1998. 59. Borrego F, Ulbrecht M, Weiss EH, Coligan JE, Brooks AG: Recognition of human histocompatibility leukocyte antigen (HLA)-E complexed with HLA class I signal sequence-derived peptides by CD94/NKG2 confers protection from natural killer cell-mediated lysis. J Exp Med 187:813, 1998. 60. Lee N, Llano M, Carretero M, Ishitani A, Navarro F, Lopez-Botet M, Geraghty DE: HLA-E is a major ligand for the NK inhibitory receptor CD94/NKG2A. Proc Natl Acad Sci USA 95:5199, 1998. 61. Aldrich CJ, DeCloux A, Woods AS, Cotter RJ, Soloski MJ, Forman J: Identification of a TAP-dependent leader peptide recognized by alloreactive T cells specific for a class Ib antigen. Cell 79:649, 1994. 62. Vance RE, Kraft JR, Altman JD, Jensen PE, Raulet DH: Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b). J Exp Med 188:1841, 1998.

26

63. Cantoni C, Verdiani S, Falco M, Pessino A, Conte R, Pende D, Ponte M, Moretta L, Biassoni R: p49, a putative HLA class I-specific inhibitory NK receptor belonging to the immunoglobulin superfamily. Eur J Immunol 28:1980, 1998. 64. Rajagopalan S, Long EO: An HLA-G-specific receptor expressed on all natural killer cells. J Exp Med 189: 1093, 1999. 65. Samaridis J, Colonna M: Cloning of novel immunoglobulin superfamily receptors expressed on human myeloid and lymphoid cells: structural evidence for a new stimulatory and inhibitory pathways. Eur J Immunol 27:660, 1997. 66. Wagtmann N, Rojo S, Eichler E, Mohrenweiser H, Long EO: A new human gene complex encodes the killer cell inhibitory receptors and a related family of monocyte/ macrophage receptors. Cur Biol 7:615, 1997. 67. Arm JP, Nwankwo C, Austen KF: Molecular identification of a novel family of human Ig superfamily members that possess immunoreceptor tyrosine-based inhibition motifs and homology to the mouse gp49B1 inhibitory receptor. J Immunol 159:2342, 1997. 68. Cosman D, Fanger N, Borges L, Kibin M, Chin W, Peterson L, Hsu M-L: A novel immunoglobulin superfamily receptor for cellular and viral MHC class I molecules. Immunity 7:273, 1997. 69. Colonna M, Navarro F, Bellon T, Llano M, Garcia P, Samaridis J, Angman L, Cella M, Lopez-Botet M: A common inhibitory receptor for major histocompatibility complex class I molecules on human lymphoid and myelomonocytic cells. J Exp Med 186:1809, 1997. 70. Colonna M, Samaridis J, Cella M, Angman L, Allen RL, O’Callaghan CA, Dunbar R, Ogg GS, Cerundolo V, Rollick A: Human myelomonocytic cells express an inhibitory receptor for classical and nonclassical MHC class I molecules. J Immunol 160:3096, 1998. 71. Navarro F, Llano M, Bellon T, Colonna M, Geraghty DE, Lopez-Botet M: The ILT2(LIR1) and CD94/ NKG2A NK cell receptors respectively recognize HLA-G1 and HLA-E molecules co-expressed on target cells. Eur J Immunol 29:277, 1999. 72. Borges L, Hsu M-L, Fanger N, Kubin M, Cosman D: A family of human lymphoid and myeloid Ig-like receptors, some of which bind to MHC class I molecules. J Immunol 159:5192, 1997. 73. Kubagawa H, Burrows PD, Cooper MD: A novel pair of immunoglobulin-like receptors expressed by B cells and myeloid cells. Proc Natl Acad Sci USA 94:5261, 1997. 74. Hayami K, Fukuta D, Nishikawa Y, Yamashita Y, Inui M, Ohyama Y, Hikida M, Ohmori H, Takai T: Molecular cloning of a novel murine cell-surface glycoprotein homologous to killer cell inhibitory receptors. J Biol Chem 272:7320, 1997. 75. Dupont B, Selvakumar A, Steffens U: The killer cell

A. B. H. Bakker et al.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

inhibitory receptor genomic region on human chromosome 19q13.4. Tiss Antigens 49:557, 1997. Maeda A, Kuorsaki M, Kurosaki T: Paired immunoglobulin-like receptor (PIR)-A is involved in activating mast cells through its association with Fc receptor ␥ chain. J Exp Med 188:991, 1998. Yamashita Y, Ono M, Takai T: Inhibitory and stimulatory functions of paired Ig-like receptor (PIR) family in RBL-2H3 cells. J Immunol 161:4042, 1998. Nakajima H, Samaridis J, Angman L, Colonna M: Human myeloid cells express an activating ILT receptor (ILT1) that associates with Fc receptor ␥-chain. J Immunol 162:5, 1999. Biassoni R, Cantoni C, Falco M, Verdiani S, Bottino C, Vitale M, Conte R, Poggi A, Moretta A, Moretta L: The human leukocyte antigen (HLA)-C specific “activatory” or “inhibitory” natural killer cell receptors display highly homologous extracellular domains but differ in their transmembrane and intracytoplasmic portions. J Exp Med 183:645, 1996. Houchins JP, Lanier LL, Niemi E, Phillips JH, Ryan JC: Natural killer cell cytolytic activity is inhibited by NKG2-A and activated by NKG2-C. J Immunol 158: 3603, 1997. Mason LH, Anderson SK, Yokoyama WM, Smith HRC, Winkler-Pickett R, Ortaldo JR: The Ly-49D receptor activates murine natural killer cells. J Exp Med 184: 2119, 1996. Lanier LL, Corliss BC, Wu J, Leong C, Phillips JH: Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 391:703, 1998. Olcese L, Cambiaggi A, Semenzato G, Bottino C, Moretta A, Vivier E: Human killer cell activatory receptors for MHC class I molecules are included in a multimeric complex expressed by natural killer cells. J Immunol 158:5083, 1997. Campbell KS, Cella M, Carretero M, Lopez-Botet M, Colonna M: Signaling through human killer cell activating receptors triggers tyrosine phosphorylation of an associated protein complex. Eur J Immunol 28:599, 1998. McVicar DW, Taylor LS, Gosselin P, Willette-Brown J, Mikhael AI, Geahlen RL, Nakamura MC, Linnemeyer P, Seaman WE, Anderson SK, Ortaldo JR, Mason LH: DAP12-mediated signal transduction in natural killer cells. A dominant role for the syk protein-tyrosine kinase. J Biol Chem 273:32934, 1998. Mason LH, Willette-Brown J, Anderson SK, Gosselin P, Shores EW, Love PE, Ortaldo JR, McVicar DW: Characterization of an associated 16 kDA tyrosine phosphoprotein require for Ly-49D signal transduction. J Immunol 160:4148, 1998. Smith KM, Wu J, Bakker ABH, Phillips JH, Lanier LL:

NK Cell Activation: Stimulatory Pathways and Inhibitory Signals

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

Ly49D and Ly49H associate with mouse DAP12 and form activating receptors: J Immunol 161:7, 1998. Lanier LL, Corliss B, Wu J, Phillips JH: Association of DAP12 with activating CD94/NKG2C NK cell receptors. Immunity 8:693, 1998. Biassoni R, Pessino A, Malaspina A, Cantoni C, Bottino C, Sivori S, Moretta L, Moretta A: Role of amino acid position 70 in the binding affinity of p50.1 and p58.1 receptors for HLA-Cw4 molecules. Eur J Immunol 27: 3095, 1997. Winter CC, Gumperz JE, Parham P, Long EO, Wagtmann N: Direct binding and functional transfer of NK cell inhibitory receptors reveal novel patterns of HLA-C allotype recognition. J Immunol 161:571, 1998. Valiante NM, Uhrberg M, Shilling HG, Lienert-Weidenbach K, Arnett KL, D’Andrea A, Phillips JH, Lanier LL, Parham P: Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors. Immunity 7:739, 1997. Uhrberg M, Valiante NM, Shum BP, Shilling HG, Lienert-Weidenbach K, Corliss B, Tyan D, Lanier LL, Parham P: Human diversity in killer cell inhibitory receptor (KIR) genes. Immunity 7:753, 1997. Cantoni C, Biassoni R, Pende D, Sivori S, Accame L, Pareti L, Semenzato G, Moretta L, Moretta A, Bottino C: The activating form of CD94 receptor complex: CD94 covalently associated with the Kp39 protein that represents the product of the NKG2-C gene. Eur J Immunol 28:327, 1998. Marincola FM, Shamamian P, Alexander RB, Gnarra JR, Turetskaya RL, Nedospasov SA, Simonis TB, Taubenberger JK, Yannelli J, Mixon A, et al.: Loss of HLA haplotype and B locus down-regulation in melanoma cell lines. J Immunol 153:1225, 1994. Cohen GB, Gandhi RT, Davis DM, Mandelboim O, Chem BK, Strominger JL, Baltimore D: The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells. Immunity 10:661, 1999. Vitale M, Bottino C, Sivori S, Sanseverino L, Castriconi R, Marcenaro E, Augugliaro R, Moretta L, Moretta A: NKp44, a novel triggering surface molecule specifically expressed by activated natural killer cells, is involved in non-major histocompatibility complex-restricted tumor cell lysis. J Exp Med 187:2065, 1998. Cantoni C, Bottino C, Vitale M, Pessino A, Augugliaro R, Malaspina A, Parolini S, Moretta L, Moretta A, Biassoni R: NKp44, a triggering receptor involved in tumor cell lysis by activated human natural killer cells, is a novel member of the immunoglobulin superfamily. J Exp Med 189:787, 1999. Sivori S, Vitale M, Morelli L, Sanseverino L, Augugliaro R, Bottino C, Moretta L, Moretta A: p46, a novel natural

27

99.

100.

101.

102.

103.

104.

105.

106.

107.

108.

109.

killer cell-specific surface molecule that mediates cell activation. J Exp Med 186:1129, 1997. Pessino A, Sivori S, Bottino C, Malaspina A, Morelli L, Moretta L, Biassoni R, Moretta A: Molecular cloning of NKp46: a novel member of the immunoglobulin superfamily involved in triggering of natural cytotoxicity. J Exp Med 188:953, 1998. Sivori S, Pende D, Bottino C, Marcenaro E, Pessino A, Biassoni R, Moretta L, Moretta A: NKp46 is the major triggering receptor involved in the natural cytotoxicity of fresh or cultured human NK ceils. Correlation between surface density of NKp46 and natural cytotoxicity against autologous, allogeneic or xenogeneic target cells. Eur J Immunol 29:1656, 1999. Tomasello E, Olcese L, Vely F, Geourgeon C, Blery M, Moqrich A, Gautheret D, Djabali M, Mattei M-G, Vivier E: Gene structure, expression pattern, and biological activity of mouse killer cell activating receptorassociated protein (KARAP)/DAP-12. J Biol Chem 273: 34115, 1998. Bakker ABH, Baker E, Sutherland GR, Phillips JH, Lanier LL: Myeloid DAP12-associating Lectin (MDL)-1 is a novel cell surface receptor involved in the activation of myeloid cells. Proc Natl Acad Sci USA 96:9792, 1999. Wu J, Song Y, Bakker ABH, Bauer S, Spies T, Lanier LL, Phillips JH: An activating immunoreceptor complex formed by NKG2D and DAP10. Science 285:730, 1999. Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ, et al.: SH2 domains recognize specific phosphopeptide sequences, Cell 72:767, 1993. Houchins JP, Yabe T, McSherry C, Bach FH: DNA sequence analysis of NKG2, a family of related cDNA clones encoding type II integral membrane proteins on human natural killer cells. J Exp Med 173:1017, 1991. Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL, Spies T: Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285:727, 1999. Groh V, Bahram S, Bauer S, Herman A, Beauchamp M, Spies T: Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium. Proc Natl Acad Sci USA 93:12445, 1996. Groh V, Steinle A, Bauer S, Spies T: Recognition of stress-induced MHC molecules by intestinal epithelial ␥␦ T cells. Science 279:1737, 1998. Groh V, Rhinehart R, Secrist H, Bauer S, Grabstein KH, Spies T: Broad tumor-associated expression and recognition by tumor-derived gammadelta T cells of MICA and MICB. Proc Natl Acad Sci USA 96:6879, 1999.