Structure of the murine CD94-NKG2A receptor in complex with Qa-1b presenting an MHC-I leader peptide.

The heterodimeric natural killer cells antigen CD94 (CD94)-NKG2-A/NKG2-B type II integral membrane protein (NKG2A) receptor family expressed on human and mouse natural killer (NK) cells monitors global major histocompatibility complex (MHC) class I cell surface expression levels through binding to MHC class Ia-derived leader sequence peptides presented by HLA class I histocompatibility antigen, alpha chain E (HLA-E; in humans) or H-2 class I histocompatibility antigen, D-37 (Qa-1b; in mice). Although the molecular basis underpinning human CD94-NKG2A recognition of HLA-E is known, the equivalent interaction in the murine setting is not. By determining the high-resolution crystal structure of murine CD94-NKG2A in complex with Qa-1b presenting the Qa-1 determinant modifier peptide (QDM), we resolved the mode of binding. Compared to the human homologue, the murine CD94-NKG2A-Qa-1b-QDM displayed alterations in the distribution of interactions across CD94 and NKG2A subunits that coincide with differences in electrostatic complementarity of the ternary complex and the lack of cross-species reactivity. Nevertheless, we show that Qa-1b could be modified through W65R + N73I mutations to mimic HLA-E, facilitating binding with both human and murine CD94-NKG2A. These data underscore human and murine CD94-NKG2A cross-species heterogeneity and provide a foundation for humanising Qa-1b in immune system models.

Thermal-exchange HLA-E multimers reveal specificity in HLA-E and NKG2A/CD94 complex interactions.

There is growing interest in HLA-E-restricted T-cell responses as a possible novel, highly conserved, vaccination targets in the context of infectious and malignant diseases. The developing field of HLA multimers for the detection and study of peptide-specific T cells has allowed the in-depth study of TCR repertoires and molecular requirements for efficient antigen presentation and T-cell activation. In this study, we developed a method for efficient peptide thermal exchange on HLA-E monomers and multimers allowing the high-throughput production of HLA-E multimers. We optimized the thermal-mediated peptide exchange, and flow cytometry staining conditions for the detection of TCR and NKG2A/CD94 receptors, showing that this novel approach can be used for high-throughput identification and analysis of HLA-E-binding peptides which could be involved in T-cell and NK cell-mediated immune responses. Importantly, our analysis of NKG2A/CD94 interaction in the presence of modified peptides led to new molecular insights governing the interaction of HLA-E with this receptor. In particular, our results reveal that interactions of HLA-E with NKG2A/CD94 and the TCR involve different residues. Altogether, we present a novel HLA-E multimer technology based on thermal-mediated peptide exchange allowing us to investigate the molecular requirements for HLA-E/peptide interaction with its receptors.

Nonameric Peptide Orchestrates Signal Transduction in the Activating HLA-E/NKG2C/CD94 Immune Complex as Revealed by All-Atom Simulations.

The innate immune system's natural killer (NK) cells exert their cytolytic function against a variety of pathological challenges, including tumors and virally infected cells. Their activation depends on net signaling mediated via inhibitory and activating receptors that interact with specific ligands displayed on the surfaces of target cells. The CD94/NKG2C heterodimer is one of the NK activating receptors and performs its function by interacting with the trimeric ligand comprised of the HLA-E/ß2m/nonameric peptide complex. Here, simulations of the all-atom multi-microsecond molecular dynamics in five immune complexes provide atomistic insights into the receptor-ligand molecular recognition, as well as the molecular events that facilitate the NK cell activation. We identify NKG2C, the HLA-Eα2 domain, and the nonameric peptide as the key elements involved in the molecular machinery of signal transduction via an intertwined hydrogen bond network. Overall, the study addresses the complex intricacies that are necessary to understand the mechanisms of the innate immune system.

Higher-Order Structure Characterization of NKG2A/CD94 Protein Complex and Anti-NKG2A Antibody Binding Epitopes by Mass Spectrometry-Based Protein Footprinting Strategies.

NK group 2 member A (NKG2A), an immune checkpoint inhibitor, is an emerging therapeutic target in immuno-oncology. NKG2A forms a heterodimer with CD94 on the cell surface of NK and a subset of T cells and recognizes the nonclassical human leukocyte antigen (HLA-E) in humans. Therapeutic blocking antibodies that block the ligation between HLA-E and NKG2A/CD94 have been shown to enhance antitumor immunity in mice and humans. In this study, we illustrate the practical utilities of mass spectrometry (MS)-based protein footprinting in areas from reagent characterization to antibody epitope mapping. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) in the higher-order structure characterization of NKG2A in complex with CD94 provides novel insights into the conformational dynamics of NKG2A/CD94 heterodimer. To fully understand antibody/target interactions, we employed complementary protein footprinting methods, including HDX-MS and fast photochemical oxidation of proteins (FPOP)-MS, to determine the binding epitopes of therapeutic monoclonal antibodies targeting NKG2A. Such a combination approach provides molecular insights into the binding mechanisms of antibodies to NKG2A with high specificity, demonstrating the blockade of NKG2A/HLA-E interaction.

HLAs, TCRs, and KIRs, a Triumvirate of Human Cell-Mediated Immunity.

In all human cells, human leukocyte antigen (HLA) class I glycoproteins assemble with a peptide and take it to the cell surface for surveillance by lymphocytes. These include natural killer (NK) cells and γδ T cells of innate immunity and αß T cells of adaptive immunity. In healthy cells, the presented peptides derive from human proteins, to which lymphocytes are tolerant. In pathogen-infected cells, HLA class I expression is perturbed. Reduced HLA class I expression is detected by KIR and CD94:NKG2A receptors of NK cells. Almost any change in peptide presentation can be detected by αß CD8+ T cells. In responding to extracellular pathogens, HLA class II glycoproteins, expressed by specialized antigen-presenting cells, present peptides to αß CD4+ T cells. In comparison to the families of major histocompatibility complex (MHC) class I, MHC class II and αß T cell receptors, the antigenic specificity of the γδ T cell receptors is incompletely understood.

The murine CD94/NKG2 ligand, Qa-1b, is a high-affinity, functional ligand for the CD8αα homodimer.

The immune co-receptor CD8 molecule (CD8) has two subunits, CD8α and CD8ß, which can assemble into homo or heterodimers. Nonclassical (class-Ib) major histocompatibility complex (MHC) molecules (MHC-Ibs) have recently been identified as ligands for the CD8αα homodimer. This was demonstrated by the observation that histocompatibility 2, Q region locus 10 (H2-Q10) is a high-affinity ligand for CD8αα which also binds the MHC-Ib molecule H2-TL. This suggests that MHC-Ib proteins may be an extended source of CD8αα ligands. Expression of H2-T3/TL and H2-Q10 is restricted to the small intestine and liver, respectively, yet CD8αα-containing lymphocytes are present more broadly. Therefore, here we sought to determine whether murine CD8αα binds only to tissue-specific MHC-Ib molecules or also to ubiquitously expressed MHC-Ib molecules. Using recombinant proteins and surface plasmon resonance-based binding assays, we show that the MHC-Ib family furnishes multiple binding partners for murine CD8αα, including H2-T22 and the CD94/NKG2-A/B-activating NK receptor (NKG2) ligand Qa-1b We also demonstrate a hierarchy among MHC-Ib proteins with respect to CD8αα binding, in which Qa-1b > H2-Q10 > TL. Finally, we provide evidence that Qa-1b is a functional ligand for CD8αα, distinguishing it from its human homologue MHC class I antigen E (HLA-E). These findings provide additional clues as to how CD8αα-expressing cells are controlled in different tissues. They also highlight an unexpected immunological divergence of Qa-1b/HLA-E function, indicating the need for more robust studies of murine MHC-Ib proteins as models for human disease.

Between Innate and Adaptive Immune Responses: NKG2A, NKG2C, and CD8⁺ T Cell Recognition of HLA-E Restricted Self-Peptides Acquired in the Absence of HLA-Ia.

On healthy cells the non-classical HLA class Ib molecule HLA-E displays the cognate ligand for the NK cell receptor NKG2A/CD94 when bound to HLA class I signal peptide sequences. In a pathogenic situation when HLA class I is absent, HLA-E is bound to a diverse set of peptides and enables the stimulatory NKG2C/CD94 receptor to bind. The activation of CD8⁺ T cells by certain p:HLA-E complexes illustrates the dual role of this low polymorphic HLA molecule in innate and adaptive immunity. Recent studies revealed a shift in the HLA-E peptide repertoire in cells with defects in the peptide loading complex machinery. We recently showed that HLA-E presents a highly diverse set of peptides in the absence of HLA class Ia and revealed a non-protective feature against NK cell cytotoxicity mediated by these peptides. In the present study we have evaluated the molecular basis for the impaired NK cell inhibition by these peptides and determined the cell surface stability of individual p:HLA-E complexes and their binding efficiency to soluble NKG2A/CD94 or NKG2C/CD94 receptors. Additionally, we analyzed the recognition of these p:HLA-E epitopes by CD8⁺ T cells. We show that non-canonical peptides provide stable cell surface expression of HLA-E, and these p:HLA-E complexes still bind to NKG2/CD94 receptors in a peptide-restricted fashion. Furthermore, individual p:HLA-E complexes elicit activation of CD8⁺ T cells with an effector memory phenotype. These novel HLA-E epitopes provide new implications for therapies targeting cells with abnormal HLA class I expression.

Evidence of functional Cd94 polymorphism in a free-living house mouse population.

The CD94 receptor, expressed on natural killer (NK) and CD8+ T cells, is known as a relatively non-polymorphic receptor with orthologues in humans, other primates, cattle, and rodents. In the house mouse (Mus musculus), a single allele is highly conserved among laboratory strains, and reports of allelic variation in lab- or wild-living mice are lacking, except for deficiency in one lab strain (DBA/2J). The non-classical MHC-I molecule Qa-1b is the ligand for mouse CD94/NKG2A, presenting alternative non-americ fragment of leader peptides (Qa-1 determinant modifier (Qdm)) from classical MHC-I molecules. Here, we report a novel allele identified in free-living house mice captured in Norway, living among individuals carrying the canonical Cd94 allele. The novel Cd94LocA allele encodes 12 amino acid substitutions in the extracellular lectin-like domain. Flow cytometric analysis of primary NK cells and transfected cells indicates that the substitutions prevent binding of CD94 mAb and Qa-1b/Qdm tetramers. Our data further indicate correlation of Cd94 polymorphism with the two major subspecies of house mice in Europe. Together, these findings suggest that the Cd94LocA/NKG2A heterodimeric receptor is widely expressed among M. musculus subspecies musculus, with ligand-binding properties different from mice of subspecies domesticus, such as the C57BL/6 strain.

The Egyptian Rousette Genome Reveals Unexpected Features of Bat Antiviral Immunity.

Bats harbor many viruses asymptomatically, including several notorious for causing extreme virulence in humans. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of Rousettus aegyptiacus, a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We found an expanded and diversified KLRC/KLRD family of natural killer cell receptors, MHC class I genes, and type I interferons, which dramatically differ from their functional counterparts in other mammals. Such concerted evolution of key components of bat immunity is strongly suggestive of novel modes of antiviral defense. An evaluation of the theoretical function of these genes suggests that an inhibitory immune state may exist in bats. Based on our findings, we hypothesize that tolerance of viral infection, rather than enhanced potency of antiviral defenses, may be a key mechanism by which bats asymptomatically host viruses that are pathogenic in humans.

CD94 of tongue sole Cynoglossus semilaevis binds a wide arrange of bacteria and possesses antibacterial activity.

In this study, we examined the expression patterns and the functions of the tongue sole Cynoglossus semilaevis CD94, CsCD94. CsCD94 is composed of 209 amino acid residues and shares 43.0-50.2% overall identities with known teleost CD94 sequence. CsCD94 has a C-type lectin-like domain. Expression of CsCD94 occurred in multiple tissues and was upregulated during bacterial infection. Recombinant CsCD94 (rCsCD94) exhibited apparent binding and agglutinating activities against both Gram-positive and Gram-negative bacteria in a Ca2+-dependent manner. Treatment of bacteria with rCsCD94 enhanced phagocytosis of the bacteria by peripheral blood leukocytes. Furthermore, incubation of rCsCD94 with bacteria reduced the survival of the bacteria in vitro. Taken together, these results indicate that rCsCD94 is a key factor in the bactericidal and phagocytic effects of tongue sole, and reveal for the first time an essential role of fish CD94 in antibacterial immunity, thereby adding insight into the function of CD94.

High diversification of CD94 by alternative splicing in New World primates.

CD94 forms heterodimers with NKG2A, -C, or -E to constitute lectin-like natural killer cell receptors for MHC-E. Its structure differs from other C-type lectins in that the second α-helix is replaced by a loop that forms the interacting interface with the NKG2 molecules. Although CD94 has remained highly conserved mammals, several alternative splicing variants have been detected in some species. To evaluate the prevalence and significance of this phenomenon, we have cloned and sequenced CD94 cDNAs in six species of New World primates from the Cebidae and Atelidae families. Full-length sequences had a mean similarity of 96 % amongst New World primates and of 90 % to the human orthologue, with little variation in the residues interacting with NKG2 or MHC-E molecules. Despite this high conservation, a total of 14 different splice variants were identified, half of which were shared by two or more primate species. Homology-based modeling of the C-type lectin domain showed that most isoforms folded stably, although they had modifications that prevented its interaction with NKG2 and MHC-E. Two isoforms were predicted to replace the typical CD94 loop by a second α-helix, evidencing a domain fold transition from a CD94 structure to a canonical C-type lectin. These two structures were more similar to members of the CLEC lectin family than to the native CD94. Thus, CD94 has remained conserved in primates to maintain functional interactions with NKG2 and MHC-E, while at the same time has diversified by alternative splicing potentially providing additional functional scenarios.

A structural basis for antigen presentation by the MHC class Ib molecule, Qa-1b.

The primary function of the monomorphic MHC class Ib molecule Qa-1(b) is to present peptides derived from the leader sequences of other MHC class I molecules for recognition by the CD94-NKG2 receptors expressed by NK and T cells. Whereas the mode of peptide presentation by its ortholog HLA-E, and subsequent recognition by CD94-NKG2A, is known, the molecular basis of Qa-1(b) function is unclear. We have assessed the interaction between Qa-1(b) and CD94-NKG2A and shown that they interact with an affinity of 17 µM. Furthermore, we have determined the structure of Qa-1(b) bound to the leader sequence peptide, Qdm (AMAPRTLLL), to a resolution of 1.9 Å and compared it with that of HLA-E. The crystal structure provided a basis for understanding the restricted peptide repertoire of Qa-1(b). Whereas the Qa-1(b-AMAPRTLLL) complex was similar to that of HLA-E, significant sequence and structural differences were observed between the respective Ag-binding clefts. However, the conformation of the Qdm peptide bound by Qa-1(b) was very similar to that of peptide bound to HLA-E. Although a number of conserved innate receptors can recognize heterologous ligands from other species, the structural differences between Qa-1(b) and HLA-E manifested in CD94-NKG2A ligand recognition being species specific despite similarities in peptide sequence and conformation. Collectively, our data illustrate the structural homology between Qa-1(b) and HLA-E and provide a structural basis for understanding peptide repertoire selection and the specificity of the interaction of Qa-1(b) with CD94-NKG2 receptors.

Binding affinities of NKG2D and CD94 to sialyl Lewis X-expressing N-glycans and heparin.

Lectin-like receptors natural killer group 2D (NKG2D) and CD94 on natural killer (NK) cells bind to α2,3-NeuAc-containing N-glycans and heparin/heparan sulfate (HS). Using recombinant glutathione S-transferase-fused extracellular lectin-like domains of NKG2D (rGST-NKG2Dlec) and CD94 (rGST-CD94lec), we evaluated their binding affinities (K(d)) to high sialyl Lewis X (sLeX)-expressing transferrin secreted by HepG2 cells (HepTf) and heparin-conjugated bovine serum albumin (Heparin-BSA), using quartz crystal microbalance (QCM) and enzyme immunoassay (EIA) microplate methods. K(d) values obtained by linear reciprocal plots revealed good coincidence between the two methods. K(d) values of rGST-NKG2Dlec obtained by QCM and EIA, respectively, were 1.19 and 1.11 µM for heparin-BSA >0.30 and 0.20 µM for HepTf, while those of rGST-CD94lec were 1.31 and 1.45 µM for HepTf >0.37 and 0.36 µM for heparin-BSA. These results suggested that these glycans can interact with NKG2D and CD94 to modulate NK cell-dependent cytotoxicity.

A novel system of polymorphic and diverse NK cell receptors in primates.

There are two main classes of natural killer (NK) cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR) and the structurally unrelated killer cell lectin-like receptors (KLR). While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2) rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in "higher" primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire.

NKG2D and CD94 bind to heparin and sulfate-containing polysaccharides.

Killer lectin-like receptors NKG2D and CD94 on natural killer cells trigger cytotoxicity through binding of glycans on target cells including sialyl Lewis X antigen. We previously reported that NKG2D and CD94 recognize alpha2,3-linked NeuAc on multi-antennary N-glycans. Here we further investigated polysaccharide binding by these receptors, using glutathione-S-transferase-fused extracellular domains of NKG2D AA 73-216 (rNKG2Dlec) and CD94 AA 68-179 (rCD94lec). We found that rNKG2Dlec and rCD94lec bind in a dose-dependent manner to plates coated with heparin-conjugated bovine serum albumin (heparin-BSA). Binding to heparin-BSA was suppressed by soluble sulfate-containing polysaccharides, but minimally impacted by 2-O-, 6-O-, and 2-N-desulfated heparin. Mutagenesis revealed that (152)Y and (199)Y of NKG2D and (144)F, (160)N, and (166)C of CD94 were critical for binding to heparin-BSA. The present manuscript provides the first evidence that NKG2D and CD94 bind to heparin and sulfate-containing polysaccharides.

NKG2D and CD94 bind to multimeric alpha2,3-linked N-acetylneuraminic acid.

Killer lectin-like receptors on natural killer cells mediate cytotoxicity through glycans on target cells including the sialyl Lewis X antigen (sLeX). We investigated whether NK group 2D (NKG2D) and CD94 can bind to sialylated N-linked glycans, using recombinant glutathione S-transferase-fused extracellular lectin-like domains of NKG2D (rNKG2Dlec) and CD94 (rCD94lec). Both rNKG2Dlec and rCD94lec bound to plates coated with high-sLeX-expressing transferrin secreted by HepG2 cells (HepTF). The binding of rNKG2Dlec and rCD94lec to HepTF was markedly suppressed by treatment of HepTF with neuraminidase and in the presence of N-acetylneuraminic acid. Moreover, rNKG2Dlec and rCD94lec bound to alpha2,3-sialylated human alpha(1)-acid glycoprotein (AGP) but not to alpha2,6-sialylated AGP. Mutagenesis revealed that (152)Y of NKG2D and (144)F and (160)N of CD94 were critical for HepTF binding. This is the first report that NKG2D and CD94 bind to alpha2,3-sialylated but not to alpha2,6-sialylated multi-antennary N-glycans.

Structural basis for NKG2A/CD94 recognition of HLA-E.

The NKG2x/CD94 (x = A, C, E) natural killer-cell receptors perform an important role in immunosurveillance by binding to HLA-E complexes that exclusively present peptides derived from MHC class I leader sequences, thereby monitoring MHC class I expression. We have determined the crystal structure of the NKG2A/CD94/HLA-E complex at 4.4-A resolution, revealing two critical aspects of this interaction. First, the C-terminal region of the peptide, which displays the most variability among class I leader sequences, interacts entirely with CD94, the invariant component of these receptors. Second, residues 167-170 of NKG2A/C account for the approximately 6-fold-higher affinity of the inhibitory NKG2A/CD94 receptor compared to its activating NKG2C/CD94 counterpart. These residues do not contact HLA-E or peptide directly but instead form part of the heterodimer interface with CD94. An evolutionary analysis across primates reveals that whereas CD94 is evolving under purifying selection, both NKG2A and NKG2C are evolving under positive selection. Specifically, residues at the CD94 interface have evolved under positive selection, suggesting that the evolution of these genes is driven by an interaction with pathogen-derived ligands. Consistent with this possibility, we show that NKG2C/CD94, but not NKG2A/CD94, weakly but specifically binds to the CMV MHC-homologue UL18. Thus, the evolution of the NKG2x/CD94 family of receptors has likely been shaped both by the need to bind the invariant HLA-E ligand and the need to avoid subversion by pathogen-derived decoys.

CD94-NKG2A recognition of human leukocyte antigen (HLA)-E bound to an HLA class I leader sequence.

The recognition of human leukocyte antigen (HLA)-E by the heterodimeric CD94-NKG2 natural killer (NK) receptor family is a central innate mechanism by which NK cells monitor the expression of other HLA molecules, yet the structural basis of this highly specific interaction is unclear. Here, we describe the crystal structure of CD94-NKG2A in complex with HLA-E bound to a peptide derived from the leader sequence of HLA-G. The CD94 subunit dominated the interaction with HLA-E, whereas the NKG2A subunit was more peripheral to the interface. Moreover, the invariant CD94 subunit dominated the peptide-mediated contacts, albeit with poor surface and chemical complementarity. This unusual binding mode was consistent with mutagenesis data at the CD94-NKG2A-HLA-E interface. There were few conformational changes in either CD94-NKG2A or HLA-E upon ligation, and such a "lock and key" interaction is typical of innate receptor-ligand interactions. Nevertheless, the structure also provided insight into how this interaction can be modulated by subtle changes in the peptide ligand or by the pairing of CD94 with other members of the NKG2 family. Differences in the docking strategies used by the NKG2D and CD94-NKG2A receptors provided a basis for understanding the promiscuous nature of ligand recognition by NKG2D compared with the fidelity of the CD94-NKG2 receptors.

The heterodimeric assembly of the CD94-NKG2 receptor family and implications for human leukocyte antigen-E recognition.

The CD94-NKG2 receptor family that regulates NK and T cells is unique among the lectin-like receptors encoded within the natural killer cell complex. The function of the CD94-NKG2 receptors is dictated by the pairing of the invariant CD94 polypeptide with specific NKG2 isoforms to form a family of functionally distinct heterodimeric receptors. However, the structural basis for this selective pairing and how they interact with their ligand, HLA-E, is unknown. We describe the 2.5 A resolution crystal structure of CD94-NKG2A in which the mode of dimerization contrasts with that of other homodimeric NK receptors. Despite structural homology between the CD94 and NKG2A subunits, the dimer interface is asymmetric, thereby providing a structural basis for the preferred heterodimeric assembly. Structure-based sequence comparisons of other CD94-NKG2 family members, combined with extensive mutagenesis studies on HLA-E and CD94-NKG2A, allows a model of the interaction between CD94-NKG2A and HLA-E to be established, in which the invariant CD94 chain plays a more dominant role in interacting with HLA-E in comparison to the variable NKG2 chain.

Complexity in the cattle CD94/NKG2 gene families.

Natural killer cell responses are controlled to a large extent by the interaction of an array of inhibitory and activating receptors with their ligands. The mostly nonpolymorphic CD94/NKG2 receptors in both humans and mice were shown to recognize a single nonclassical MHC class I molecule in each case. In this paper, we describe the CD94/NKG2 gene family in cattle. NKG2 and CD94 sequences were amplified from cDNA derived from four animals. Four CD94 sequences, ten NKG2A, and three NKG2C sequences were identified in total. In contrast to human, we show that cattle have multiple distinct NKG2A genes, some of which show minor allelic variation. All of the sequences designated NKG2A have two tyrosine-based inhibitory motifs in the cytoplasmic domain and one putative gene has, in addition, a charged residue in the transmembrane domain. NKG2C appears to be essentially monomorphic in cattle. All of the NKG2A sequences are similar apart from NKG2A-01, which, in contrast, shares the majority of its carbohydrate recognition domain with NKG2-C. Most of the genes appear to generate multiple alternatively spliced forms. These findings suggest that the CD94/NKG2A heterodimers in cattle, in contrast to other species, are binding several different ligands. Because NKG2C is not polymorphic, this raises questions as to the combined functional capacity of the CD94/NKG2 gene families in cattle.