The genomic organization of the mouse CD94 C‐type lectin gene

The mouse natural killer (NK) gene complex is located on chromosome 6 and contains a number of genes encoding C‐type lectin receptors which have been found to regulate NK cell function. Among these are CD94 and four NKG2 genes. Like its human counterpart, the mouse CD94 protein associates with different NKG2 isoforms and recognizes the atypical MHC class I molecule Qa‐1^b. Here, the genomic organization of the mouse CD94 gene was determined by analysing a BAC clone containing the CD94 gene. The mouse CD94 gene contains six exons separated by five introns. Exons I and II encode the 5′ untranslated region (UTR) and the transmembrane domain. Exon III encodes the stalk region and exons IV–VI encode the carbohydrate recognition domain (CRD). Furthermore, we cloned and sequenced the CD94 promoter region, and putative regulatory DNA elements were identified. Further studies on the CD94 promoter region may help to elucidate the restricted expression pattern of CD94 in NK cells and a subpopulation of T cells.     

Two genes in the rat homologous to human NKG2

Two different lectin-like receptors for MHC class I molecules have so far been identified on natural killer (NK) cells, the Ly-49 homodimeric receptors in mice and the NKG2/CD94 heterodimeric receptors in humans. The recent identification of a rat CD94 orthologue implied that NK cell receptors equivalent to NKG2/CD94 also exist in rodents. Here we describe the cDNA cloning of two rat genes homologous to members of the human NKG2 multigene family. The deduced rat NKG2A protein contains a cytoplasmic immunoreceptor tyrosine-based inhibition motif (ITIM), whereas the cytoplasmic tail of rat NKG2C lacks ITIM. The genes map to the rat NK gene complex and are selectively expressed by NK cells. The expression is strain dependent, with high expression in DA and low in PVG NK cells, correlating with the expression of rat CD94. Ly-49 genes have previously been identified in the rat, and the existence of rat NKG2 genes in addition to a CD94 orthologue suggests that NK cell populations utilize different C-type lectin receptors for MHC class I molecules in parallel.     

Cloning of murine NKG2A, B and C: second family of C-type lectin receptors on murine NK cells

Multiple NK cell receptors for MHC class I have been identified. They include killer inhibitory receptors and CD94/NKG2 heterodimers in humans and the Ly49 family in mice. Here we report the cloning of murine NKG2A, B and C. The deduced amino acid sequence of mouse NKG2A contains only one consensus cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). NKG2A from B6 and BALB/c mice differ by six amino acid residues in the extracellular domain. Murine NKG2B, like its human conterpart, appears to be a splice variant of NKG2A and lacks a large portion of the stalk region. Murine NKG2C lacks an ITIM in its cytoplasmic domain, a feature shared by human and rat NKG2C. However, unlike the human counterpart, the transmembrane domain of mouse NKG2C does not contain a charged amino acid residue. Mouse NKG2A mRNA was detected in IL-2-activated NK cells and spleen cells but not in other tissues. The NKG2A gene was localized on the distal portion of chromosome 6 where the NK complex has been located. These results further extend the repertoire of C-type lectin receptors on murine NK cells.     

Molecular characterization of human CD94: A type II membrane glycoprotein related to the C-type lectin superfamily

Natural killer (NK) cells preferentially express several genes of the C-type lectin superfamily which have been implicated in the regulation of NK cell function. We demonstrate that CD94 is a type II membrane protein encoded by a unique gene of the C-type lectin superfamily. While homology of CD94 with the NK cell-associated NKR-P1 and NKG2 C-type lectin genes is limited to the structural motifs conserved in the carbohydrate recognition domain, all of these genes are on human chromosome 12, the syntenic of mouse chromosome 6, where genes of the NK complex (NKR-P1 and Ly-49) are located. An unexpected feature of CD94 is the essential absence of a cytoplasmic domain, implying that association with other receptors may be necessary for the function of this molecule.     

Clonal analysis of NK cell development from bone marrow progenitors in vitro: orderly acquisition of receptor gene expression

In the mouse, two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2, have been identified on NK cells. Individual NK cells can express several Ly49 molecules as well as members of the CD94/NKG2 family. The expression of multiple receptors with different specificities for MHC class I is thus thought to generate NK cells with diverse recognition patterns. To delineate the mechanism by which NK cells begin to express different patterns of Ly49 and CD94/NKG2 molecules, we developed a clonal assay in which NK1.1(-), IL-2/ IL-15 receptor beta+ NK precursors generated by culture of multipotential Lin(-), c-kit+ progenitors in IL-7, stem cell factor and flt3 ligand are induced to differentiate into NK1.1+ , Ly49+ NK cells. Examination of the clonal populations thus generated revealed heterogeneity in the pattern of Ly49 and CD94/NKG2 gene expression. In addition, a distinct kinetic pattern of expression was observed. CD94, NKG2A, NKG2C and Ly49B were expressed first followed by Ly49G, then Ly49C and I and finally, Ly49A, D, E and F. The data suggest a stochastic but ordered acquisition of class I receptors on NK cells in which developing NK cells become capable of expressing distinct receptors at different times but show no absolute prerequisite to express the receptors that are acquired early in NK development for the expression of those that are acquired later.     

The role of CD94/NKG2 in innate and adaptive immunity

CD94/NKG2 is a heterodimer expressed on natural killer (NK) and a small subset of T cells. This receptor varies in function as an inhibitor or activator depending on which isoform of NKG2 is expressed. The ligand for CD94/NKG2 is HLA-E in human and its homolog, Qa1 in mouse, which are both nonclassical class I molecules that bind leader peptides from other class I molecules. Although <5% of CD8 T cells express the receptor in a naïve mouse, its expression is upregulated upon specific recognition of antigen. Similar to NK cells, most CD8 T cells that express high levels of CD94 co-express NKG2A, the inhibitory isoform. The engagement of this receptor can lead to a blocking of cytotoxicity. However, these receptors have also been implicated in the cell survival of both NK and CD8T cells. The level of CD94 expression is inversely correlated with the level of apoptosis in culture. Thus, CD94/NKG2 receptors may regulate effector functions and cell survival of NK cells and CD8 T cells, thereby playing a crucial role in the innate and adaptive immune response to a pathogen.     

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.     

CD69-triggered ERK activation and functions are negatively regulated by CD94 / NKG2-A inhibitory receptor

CD69 represents a functional triggering molecule on activated NK and T cells, capable of inducing cytotoxic activity and costimulating cytokine production. It belongs to the C-lectin type superfamily, and its gene maps in the NK gene complex, close to other genes coding for NK receptors. CD94 / NKG2-A complex is the inhibitory receptor for the non classical MHC class I molecule HLA-E on human NK cells. To investigate CD69-initiated signal transduction pathways, and to evaluate CD94 / NKG2-A interference on CD69 triggering ability, we have generated transfectants expressing both receptors in the RBL cell line. Here we report that CD69 engagement leads to the activation of extracellular signal-regulated kinase (ERK) enzymes belonging to the MAPK family, and that this event is required for CD69-mediated cell degranulation. Moreover, we show that the co-engagement of CD94 / NKG2-A inhibitory receptor effectively suppresses both CD69-triggered cell degranulation in RBL transfectants, through the inhibition of ERK activation, and CD69-induced cytotoxicity in human NK cells. Thus, here we provide new information on the molecular mechanisms initiated by CD69 activation receptor, and show that CD69-initiated signaling pathways and functional activity are negatively regulated by CD94 / NKG2-A inhibitory complex.     

The Qa-1b molecule binds to a large subpopulation of murine NK cells

Recent studies on human NK cells have demonstrated that the NK cell CD94/NKG2 receptors bind to the nonclassical MHC class I molecule HLA-E. A functional CD94/NKG2 complex has not yet been identified in rodents, but cDNA encoding rat and mouse CD94 and NKG2 have recently been cloned, suggesting that CD94/NKG2 receptors may exist in species other than man. The mouse nonclassical MHC class I molecule Qa-1 shares several features with HLA-E. This suggests that Qa-1 may be similarly recognized by murine NK cells. To study the ability of Qa-1 to bind to murine NK cells, we have produced a soluble tetrameric form of Qa-1^b . In the present study, we demonstrate that Qa-1^b tetramers distinctly bind to a large subset of fresh or IL-2-activated NK1.1⁺ /CD3⁻ splenocytes independently of the expression of Ly49 inhibitory receptors. Binding occurs whether NK cells have evolved in an MHC class I-expressing or in an MHC class I-deficient environment. Our data suggest the existence of a Qa-1-recognizing structure on a large subpopulation of murine NK cells that may be similar to the human CD94/NKG2 heterodimeric complex.     

A novel cluster of lectin-like receptor genes expressed in monocytic, dendritic and endothelial cells maps close to the NK receptor genes in the human NK gene complex.

The NK gene complex is a region on human chromosome 12 containing several families of lectin-like genes including the CD94 and NKG2 NK receptor genes. We report here that the region telomeric of CD94 contains in addition to the LOX-1 gene the novel human DECTIN-1 and the CLEC-1 and CLEC-2 genes within about 100 kb. Sequence similarities and chromosomal arrangement suggest that these genes form a separate subfamily of lectin-like genes within the NK gene complex. DECTIN-1 is selectively expressed in dendritic cells and to a lower extent in monocytes and macrophages. mRNA forms with and without a stalk exon are observed. During functional maturation of dendritic cells the level of DECTIN-1 mRNA is down-regulated several-fold. CLEC-1 is found to be not only expressed in dendritic cells, but also in endothelial cells and in the latter aspect resembles the LOX-1 gene. Whereas recombinant full-length DECTIN-1 and LOX-1 are transported to the cell surface, CLEC-1 proteins accumulate in perinuclear compartments. We propose that this family of lectin-like genes encodes receptors with important immune and/or scavenger functions in monocytic, dendritic and endothelial cells.     

The CD94/NKG2 C-type lectin receptor complex

A multigene family of human Ig-SF receptors and members of the murine Ly49 C-type lectin family are involved in natural killer (NK) cell-mediated recognition of MHC class I molecules. The human CD94 glycoprotein covalently assembles with different C-type lectins of the NKG2 family. By functional criteria, the CD94/ NKG2-A (kp43) receptor complex appears also involved in NK cell-mediated recognition of different HLA class I allotypes. Similarly to the other NK inhibitory receptors, NKG2-A contains cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). By contrast, NK clones bearing a different receptor complex (CD94/ p39) are triggered upon ligation by CD94-specific monoclonal antibodies (MAbs); the p39 subunit is likely encoded by other member(s) of the NKG2 family. Expression of the different CD94/ NKG2 complexes is warranted to precisely assess their specific interaction with HLA class I molecules, and the molecular basis for their divergent functional properties.     

Identification of a human member of the Ly-49 multigene family

Three classes of multigene family-encoded receptors enable NK cells to discriminate between polymorphic MHC class I molecules: Ly-49 homodimers, CD94/NKG2 heterodimers and the killer cell inhibitory receptors (KIR). Of these, CD94/NKG2 has been characterized in both rodents and humans. In contrast, Ly-49 family members have hitherto been found only in rodents, and KIR molecules only in the human. In this report, we describe a human cDNA, termed Ly-49L, that constitutes the first human member of the Ly-49 multigene family. Compared with rodent Ly-49 molecules, the Ly-49L sequence contains a premature stop codon and predicts a truncated protein that lacks the distal part of a C-terminal lectin domain. Evidence is presented that the premature stop codon results from incomplete excision of the intron between the first two lectin domain exons. Splice variants predicting a full-size Ly-49L protein were not detected. As demonstrated by Northern blot analysis, Ly-49L was transcribed by IL-2-activated NK cells, but not by freshly isolated B or T cells. PCR screening of a 22-clone yeast artificial chromosome contig localized the LY49L locus to the human NK gene complex on chromosome 12p12-p13. Southern blot analysis of genomic DNA showed a simple pattern with a full-length Ly-49L probe at low stringency hybridization conditions, suggesting that Ly-49L may be the only human member of the Ly-49 multigene family.     

Molecular characterization of a gene in the rat homologous to human CD94

Three classes of major histocompatibility (MHC) class I binding receptors on natural killer (NK) cells have so far been described: CD94/NKG2 heterodimeric receptors and killer cell inhibitory receptors in the human, and Ly-49 homodimers in rodents. CD94, NKG2 and Ly-49 belong to the C-type lectin super-family. As yet, CD94 and NKG2 molecules have not been detected in rodents or Ly-49 in humans. It has therefore been proposed that the two receptors represent functional equivalents in these species. The present study describes the cDNA cloning of a novel rat gene encoding a protein of 179 amino acids, 54.2 % identical to human CD94. The single-copy Cd94 gene is localized to the rat NK gene complex (NKC), within 50 kb from Nkrp2, between the Nkrp1 and Ly49 gene clusters. By Northern blot analysis, we showed that rat CD94 is selectively expressed by NK cells and a small subset of T cells, similar to the human ortho-logue. This expression is strain dependent, with high expression in DA NK cells and low in PVG NK cells. Evidence is presented that this difference is not due to receptor repertoire shaping by MHC-encoded ligands, but is controlled by genetic elements residing within the NKC. The identification of a rat CD94 orthologue suggests that NK cell populations utilize two different C-type lectin receptors for MHC class I molecules in parallel.     

Divergent functions of lectin-like receptors on NK cells

Summary: NK cells express a superfamily of surface proteins that share a common structure: dimeric type II integral membrane proteins whose extracellular domains have structural features of C-type (calcium-dependent) lectins. These receptors are encoded in a single genetic region called the NK complex (NKC). The NKC encompasses several families of genes, including Ly-49 (in mice and rats). NKR-Pl (in mice, rats, and humans). NKG2 (in humans and rats), and CD94 (in humans). Different NKC receptors have been shown to activate or to inhibit NK function, and different receptors within the same family can have opposing functions. In this review, we discuss the molecular pathways by which NK cells are activated, and the mechanisms by which inhibitory receptors interrupt activation. By studying the inhibitory receptor Ly-49A, we have demonstrated that inhibition utilizes the cytoplasmic phosphatase. SHP-1, which binds to a motif in the receptor cytoplasmic domain, termed an immunoreceptor tyrosine-based inhibitory motif (ITIM). In this regard, the lectin-like receptors are functionally similar to the immunoglobulin-like killer inhibitory receptors (KIRs) on human NK cells. The presence of an ITIM generally correlates with inhibitory activity among NKC lectin-like receptors, as demonstrated by the human NKG2 receptor family Lanier and his colleagues have recently shown that NKG2 receptors can form heterodimers with the invariant lectin-like receptor CD94. Selective association of CD94 with different NKG2 receptors may explain functional differences for CD94 in different NK clones.     

Molecular characterization of two novel C-type lectin-like receptors, one of which is selectively expressed in human dendritic cells

We have identified two human C-type lectin-like receptors, CLEC-1 and CLEC-2. Both display a single carbohydrate recognition domain and a cytoplasmic tyrosine-based motif. They are homologous to the NK cell receptors NKG2s and CD94 and also to the oxidized low-density lipoprotein receptor 1. CLEC-1 and CLEC-2 are preferentially transcribed in dendritic cells (DC) and in the liver, respectively. Following transient transfection in COS cells, CLEC-1 is expressed intracellularly, perhaps requiring an associated chain to reach the cell surface. CLEC-2 is expressed on the surface of transfected cells as a protein of approximately 33 kDa. CLEC-1 and CLEC-2 genes map to human chromosome 12, most likely in linkage with the NK gene complex (NKC). Thus, the NKC may encode C-type lectin-like receptors expressed not only in NK cells but also in other cells, and at least one of these is of potential importance in regulating DC function.