MHC class I recognition by Ly49 natural killer cell receptors

Natural killer (NK) cell function is regulated by NK receptors that bind either classical MHC class I (MHC-I) molecules or their structural relatives (MICA, RAE-1 and H-60). Two distinct families of NK receptors have been identified: the C-type lectin-like family (Ly49, NKG2D and CD94/NKG2) and the immunoglobulin-like family (KIRs and LIRs). Here, we describe the crystal structure of the C-type lectin-like NK receptor (Ly49A), bound to its MHC-I ligand (H-2D(d)). We also discuss results from recent mutagenesis studies of the Ly49A/H-2D(d) interaction in the context of the complex structure.     

Structure of the Ly49 family of natural killer (NK) cell receptors and their interaction with MHC class I molecules

Natural killer (NK) cells are an essential component of the innate immunity toward tumors and virally infected cells. The function of NK cells is regulated by a precise balance between inhibitory and activating signals. These signals are mediated by NK cell receptors that bind either classical MHC class I molecules or their structural relatives such as MICA, ULBP, RAE-1, and H-60. Two separate families of NK cell receptors have been identified: the immunoglobulin-like family (KIR, LIR) and C-type lectin-like family (Ly49, NKG2D, and CD94/NKG2). Here we summarize the structure of Ly49 C-type lectin-like proteins hitherto solved (Ly49A, Ly49C and Ly49I) and their interaction with MHC class I molecules as determined by the co-crystal structure of Ly49A/H-2Dd and Ly49C/H-2Kb.     

Cis association of Ly49A with MHC class I restricts natural killer cell inhibition

Natural killer (NK) cell function is negatively regulated by inhibitory receptors interacting with major histocompatibility complex class I molecules expressed on target cells. Here we show that the inhibitory Ly49A NK cell receptor not only binds to its H-2D(d) ligand expressed on potential target cells (in trans) but also is constitutively associated with H-2D(d) in cis (on the same cell). Cis association and trans interaction occur through the same binding site. Consequently, cis association restricts the number of Ly49A receptors available for binding of H-2D(d) on target cells and reduces NK cell inhibition through Ly49A. By lowering the threshold at which NK cell activation exceeds NK cell inhibition, cis interaction allows optimal discrimination of normal and abnormal host cells.     

H-2 allele specificity of the NK cell C-type lectin-like MHC class I receptor Ly49A visualized by soluble Ly49A tetramer

Ly49A is a C-type lectin-like receptor on NK cells that recognizes MHC class I ligands, H-2D(d) and D(k). The engagement of Ly49A with the ligands inhibits activation of NK cells and protects target cells from lysis by NK cells. Here we express the extracellular region of Ly49A with an N-terminal biotinylation tag in Escherichia coli to obtain soluble Ly49A (sLy49A) after refolding. sLy49A is indistinguishable from native Ly49A expressed on NK cells serologically and in the ability to specifically bind H-2D(d) after tetramerization with R-phycoerythrin-coupled streptavidin. The fluorescently labeled tetramer of sLy49A is applied to explore MHC class I haplotype specificity of Ly49A. We demonstrate the hierarchical reactivity of Ly49A with H-2 of various alleles in the order of d > k, r > p > v > q > s > z. Reactivity of sLy49A tetramer to spleen lymphocytes from B10.QBR mice (H-2K(b), I(b), D(q), Qa-1/Tla(b)) but not from C57BL/10 mice (H-2(b)) identifies H-2D(q) and L(q) as candidates for a Ly49A ligand. Binding of sLy49A tetramer to H-2D(q)- or L(q)-transfected cell lines demonstrates that the two highly related MHC class I molecules, H-2D(q) and L(q), are ligands for Ly49A. sLy49A tetramer staining also demonstrates preferential expression of Ly49A ligand on a subset of B cells in P/J mice. These results provide the basis to examine the molecular mechanism by which Ly49A discriminates polymorphic MHC class I molecules.     

Recognition of H-2K(b) by Ly49Q suggests a role for class Ia MHC regulation of plasmacytoid dendritic cell function

Ly49Q is a member of the polymorphic Ly49 family of NK cell receptors that displays both a high degree of conservation and a unique expression pattern restricted to myeloid lineage cells, including plasmacytoid dendritic cells (pDC). The function and ligand specificity of Ly49Q are unknown. Here, we use reporter cell analysis to demonstrate that a high-affinity ligand for Ly49Q is present on H-2(b), but not H-2(d), H-2(k), H-2(q), or H-2(a)-derived tumor cells and normal cells ex vivo. The ligand is peptide-dependent and MHC Ia-like, as revealed by its functional absence on cells deficient in TAP-1, beta(2)m, or H-2K(b)D(b) expression. Furthermore, Ly49Q is specific for H-2K(b), as the receptor binds peptide-loaded H-2K(b) but not H-2D(b) complexes, and Ly49Q recognition can be blocked using anti-K(b) but not anti-D(b) mAb. Greater soluble H-2K(b) binding to ligand-deficient pDC also suggests cis interactions of Ly49Q and H-2K(b). These results demonstrate that Ly49Q efficiently binds H-2K(b) ligand, and suggest that pDC function, like that of NK cells, is regulated by classical MHC Ia molecules. MHC recognition capability by pDC has important implications for the role of this cell type during innate immune responses.     

Structural basis of MHC class I recognition by natural killer cell receptors

Summary: Natural killer (NK)-cell function is regulated by NK receptors that recognize MHC class I (MHC-I) molecules on target cells. Two structurally distinct families of NK receptors have been identified, the immunoglobulin-like family (killer cell immunoglobulin-like receptors (KIRs), leukocyte immunoglobulin-like receptors (LIRs)) and the C-type lectin-like family (Ly49, CD94/NKG2A, NKG2D, CD69). Recently, the three-dimensional structures of several NK receptors were determined, in free form or bound to MHC-I. These include those of unbound KIRs, NKG2D, CD69, LIR-1 and the CD94 subunit of the CD94/NKG2A heterodimer. Together, these structures define the basic molecular architecture of both the immunoglobulin-like and C-type lectin-like families of NK receptors. In addition, crystal structures have been reported for the complex between Ly49A and H-2D^d, and for KIR2DL2 bound to HLA-Cw3. The complex structures provide a framework for understanding MHC-I recognition by NK receptors from both families and reveal striking differences in the nature of this recognition, despite the receptors' functional similarity.
This research was supported, in part, by National Institutes of Health grants R01 AI47900 and R37 36900 (RAM) and a fellowship from the Cancer Research Institute (MWS). We are grateful to DW Wolan and IA Wilson for providing coordinates of NKG2D prior to publication, and to members of our laboratories for encouragement.     

Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors.

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.     

Ly49i2 is an inhibitory rat natural killer cell receptor for an MHC class Ia molecule (RT1-A1c)

We have exploited strain-specific differences in the NK allorecognition repertoires to generate rat monoclonal antibodies against receptors involved in the control of allogeneic responses by rat NK cells. The monoclonal antibody STOK2 binds to a homodimeric glycoprotein that has been implicated as an inhibitory receptor for an MHC molecule in the PVG strain. In the present study, we haveidentified this glycoprotein as a novel rat Ly49 receptor (Ly49i2) containing an immunoreceptor tyrosine-based inhibitory motif. Ligation of the Ly49i2 receptor induces inhibitory signals, and Ly49i2 coprecipitates with the inhibitory tyrosine phosphatase SHP-1 in stably transfected RNK-16 cells. Moreover, it inhibits natural killing of lymphoblast targets and transfected fibroblast targets expressingthe classical MHC class Ia allele RT1-A1(c). Ly49i2, therefore, is an inhibitory receptor for specific MHC class Ia molecules, similar to inhibitory members of the mouse Ly49 family.     

Reciprocal age related change in natural killer cell receptors for MHC class I

Natural killer (NK) cells are essential for healthy aging. NK cell activation is controlled by MHC class I-specific CD94/NKG2 receptors and killer immunoglobulin-like receptors (KIR). To assess NK cytotoxic function in isolation from MHC receptor engagement, we measured the ability of purified NK cells to kill mouse P815 target cells in the presence of anti-CD16 mAb. CD16-mediated cytotoxicity did not change with age, indicating that NK activation and cytotoxic granule release remained functional. We then investigated MHC class I receptor expression on NK cells. There was an age related decrease in CD94 and NKG2A expression and a reciprocal age related increase in KIR expression. NKG2A expression also declined with age on CD56(+) T cells. CD94/NKG2A receptor function was proportional to expression, indicating that NK cell inhibitory signaling pathways were intact. NKG2A and KIR expression were complementary, suggesting that CD94/NKG2A function could substitute for inhibitory KIR function during polyclonal NK cell development in both young and elderly adults. The distinct roles of CD94/NKG2A and KIR receptors suggest that shifting MHC class I receptor expression patterns reflect age related changes in NK cell and CD56(+) T cell turnover and function in vivo.     

Major histocompatibility complex class I-dependent skewing of the natural killer cell Ly49 receptor reportoire

Subsets of mouse natural killer (NK) cells express receptors encoded by the Ly49 gene family that recognize allelic determinants on major histocompatibility complex (MHC) class I molecules. Recognition of self class I molecules typically inhibits NK cell lytic function. The presence of NK cell subsets expressing receptors which are able to discriminate class I alleles raises the possibility that there exist mechanisms to coordinate the NK cell receptor repertoire with the class I molecules of the host. In the present study, we determined the effects of class I gene expression on the frequencies of NK cells expressing three different Ly49 receptors defined by monoclonal antibodies. We show here an MHC-dependent skewing of NK cell subsets expressing multiple Ly49 receptors with specificity for self MHC. The results provide the first evidence that the frequencies of NK cells expressing different Ly49 receptors are determined by the host's MHC molecules. The results also extend previous findings that MHC class I expression influences the cell surface levels of each Ly49 receptor, suggesting an additional mechanism by which MHC molecules may influence the effective specificity of NK cells. Models to account for self tolerance and MHC-controlled repertoire differences are discussed.     

Natural killer cell activation and inhibition by receptors for MHC class I.

Several recent advances have been made in our understanding of the mechanisms which human natural killer cells recognize MHC class I molecules. Three are of special relevance: the identification of a novel molecule (DAP12) with a key role in the activation pathways; the observation that certain immunoglobulin-like receptors for HLA class I molecules are also utilized by other leucocyte lineages; and the definition of MHC class Ib proteins (i.e. HLA-E and Qa-1b) as specific ligands for the phylogenetically conserved CD94-NKG2 lectin-like receptors.     

Specificity, tolerance and developmental regulation of natural killer cells defined by expression of class I-specific Ly49 receptors

Summary: Natural killer cells in the mouse express class 1 MHC-specific inhibitory receptors of the Ly49 protein family. The receptors mediate inhibition of the lysis of tumor cells and normal cells, and mediate the specificity of bone-marrow graft rejection by NK cells in vivo. The function of these receptors may be to confer upon NK cells the capacity to distinguish normal self cells from cells that have down-regulated expression of some or all self-class I molecules, Ly49 receptors discriminate between different class I molecules, and are distributed in expression to overlapping subsets of NK cells. The receptors appear to interact with class I-MHC residues and associated N-glycans, with little or no discrimination of the class I- bound peptide. The Ly49 receptor repertoire may be initially generated by a stochastic process that distributes receptors randomly to I different cells and treats the two alleles of a given Ly49 gene independently. However, class I-MHC-dependent "education" processes shape the functional repertoire. The education processes silence potentially auto-aggressive NK cells, probably by ensuring that each NK cell expresses at least one self-specific Ly49 receptor. In addition, NK cell clones that express multiple self-specific Ly49 receptors are disfavored by the education processes, perhaps to confer greater discrimination on to individual NK cells.     

A structural perspective on MHC class I recognition by killer cell immunoglobulin-like receptors

Killer cell immunoglobulin-like receptors (KIR) play a critical role in the regulation of natural killer (NK) cell activity through their recognition of class I MHC molecules expressed on target cells. KIR recognition provides vital information to NK cells about whether a target cell should be lysed or spared. Understanding the molecular mechanism of this recognition has remained a strong focus of investigation. This has resulted in the crystal structures of several members of the KIR family and more recently the determinations of the three dimensional structures of KIR2DL2 and KIR2DL1 complexed with their respective ligands, HLA-Cw3 and HLA-Cw4. A strong structural conservation has been revealed both in the receptor design and in the overall mode of KIR binding to class I molecules. Nevertheless, distinct differences in the receptor binding sites allow for high specificity between ligands. Furthermore, unexpected similarities with T-cell receptor (TCR) recognition of MHC molecules are also observed. The detailed interactions between KIR and HLA-C molecules and their functional implications will be reviewed here.     

Natural killing of MHC class I(-) lymphoblasts by NK cells from long-term bone marrow culture requires effector cell expression of Ly49 receptors.

NK cells from long-term bone marrow culture (LTBMC) were compared with IL-2-activated splenic NK cells [short-term spleen cell culture (STSC)] with regard to expression of inhibitory Ly49 receptors and cytotoxic function. In the LTBMC, the total number of NK cells expressing either one of the Ly49 molecules A, C/I and G2 was strongly reduced (10-15% of NK1.1(+) cells) compared to the STSC (80-90% of NK1.1(+) cells). With regard to cytotoxic function, we confirmed that LTBMC-derived NK cells efficiently killed the prototype NK target YAC-1. However, against other targets, killing was more variable. First, while STSC-derived NK cells clearly distinguished MHC class I(-) from MHC class I(+) tumor cell targets, LTBMC-derived NK cells did not; they either killed both targets equally well or not at all. Secondly, LTBMC-derived NK cells were largely incapable of killing lymphoblast targets deficient in MHC class I expression. To test whether this cytotoxic defect was due to the low number of Ly49(+) NK cells in the LTBMC, we separated Ly49(+) and Ly49(-) NK cells by cell sorting and tested them individually. This experiment showed that only Ly49(+) NK cells in the LTBMC were able to kill MHC class I(-) lymphoblasts (and to distinguish them from MHC class I(+)), despite good cytotoxicity against YAC-1 cells in both populations. These data suggest that certain modes of NK cell triggering are dependent on Ly49 receptor expression. From our results, we speculate that inhibitory receptors are expressed before triggering receptors for normal self cells during NK cell development, which may be an important mechanism to preserve self tolerance during the early stages of NK cell maturation.     

Molecular genotyping of the murine H-2K MHC class I allele

An increasing number of genetically modified mouse mutants are employed in immunological research. Many experiments require the crossbreeding of various mouse lines and screening for the genetic background of the offspring. Analysis for various immunological phenotypes such as the MHC class I haplotype is usually performed by FACS analysis of peripheral blood lymphocytes. Here, we report a PCR based technique for the differentiation of the MHC class I H-2K(k) and the H-2K(b) haplotype. Advantages of this method are cost efficiency and rapidity in particular when multiple genetic variables such as the disruption of a gene, transgenesis or the MHC haplotype are to be tested.     

Regulation of CR3 (CD11b/CD18)-dependent natural killer (NK) cell cytotoxicity by tumour target cell MHC class I molecules

Phagocyte and NK cell CR3 functions as both an adhesion molecule and an iC3b receptor mediating cytotoxic responses to microorganisms. Cytotoxic activation of iC3b receptor function requires ligation of both a CD11b I-domain site for iC3b and a lectin site located in the C-terminus of CD11b. Because tumours lack the CR3-binding polysaccharides of bacteria and fungi, iC3b-opsonized tumours do not stimulate CR3-dependent cytotoxicity. Previous studies showed that NK cells could be induced to kill iC3b-opsonized tumours with small soluble β-glucans that bound with high affinity to CR3, bypassing the absence of similar polysaccharides on tumour membranes. Because CR3 signalling requires several tyrosine phosphorylation events, it appeared possible that CR3-dependent killing of autologous tumour cells might be suppressed by NK cell inhibitory receptors for MHC class I (KIR and CD94/NKG2) whose action involves recruitment of SHP-1 and SHP-2 tyrosine phosphatases. In the current study, Epstein–Barr virus (EBV)-transformed B cells were used as targets following opsonization with iC3b. Soluble β-glucan primed CR3 for killing of iC3b-coated B cells, but autologous class I-bearing targets were 84% more resistant than class I-deficient Daudi cells. Blockade of target cell class I with a MoAb specific for a domain recognized by both KIR and CD94/NKG2 resulted in comparable killing of class I⁺ B cells. By contrast, another MoAb to class II had no effect on cytotoxicity. These data suggest that NK cell recognition of class I suppresses CR3/tyrosine kinase-dependent cytotoxicity in the same way as it suppresses cytotoxicity mediated by other tyrosine kinase-linked receptors such as FcγRIIIA (CD16).     

Clonal acquisition of inhibitory Ly49 receptors on developing NK cells is successively restricted and regulated by stromal class I MHC

We report an in vitro stroma-dependent system for the clonal growth and differentiation of natural killer (NK) cells from lymphoid-restricted bone marrow progenitors or bone marrow NK1.1+ cells. Strikingly, the potential to initiate expression of specific Ly49 receptors becomes increasingly restricted as NK cells develop. Moreover, when NK cells express a Ly49 receptor specific for stromal cell class I MHC, they are less likely to initiate expression of another Ly49 receptor in the clonal culture system. The results indicate multiple roles for stromal cells in NK cell development, in supporting clonal growth, in initiation of Ly49 receptor expression, and in formation of the NK cell receptor repertoire.     

Structure of killer cell immunoglobulin-like receptors and their recognition of the class I MHC molecules

Summary: The recognition of class I MHC molecules by killer cell immunoglobulin-like receptors (KIR) constitutes an integral part of immune surveillance by the innate immune system. To understand the molecular basis of this recognition, the structures of several members of this superfamily have been determined. Despite their functional diversity, members of this superfamily share many conserved structural features. A central question is how these receptors recognize their ligands. The recent determination of the crystal structure of KIR2DL2 in complex with HLA-Cw3 has revealed the molecular mechanisms underpinning this interaction, which ultimately modulates the cytolytic activity of natural killer cells. While the recognition of MHC molecules by KIR is characterized by a number of unique features, some unexpected similarities with T-cell receptor recognition of MHC molecules are also observed. The detailed interactions between KIR2DL2 and HLA-Cw3 and their functional implications will be reviewed here.