A major subset of non-alloreactive NK cells in PVG strain rats is generally low in Ly49 receptors, but expresses the rat NKR-P1B PVG receptor (previously termed NKR-P1C). The NKR-P1B 1 NK subset is inhibited by a non-polymorphic target cell ligand, which we have shown here to be a C-type lectin-related molecule (Clr). Clr11 ligates two divergent NKR-P1B alleles as judged by an NFAT-driven reporter assay, and inhibits NK-cell cytotoxicity of NKR-P1B 1 NK cells. Clr11 also interacts with the prototypic NKR-P1A receptor and exerts a stimulatory influence on NK lysis. NKR-P1A and B are encoded by adjacent genes in the proximal part of the NK gene complex and show close sequence homology in their extracellular region. They diverge from another pair, NKR-P1F and -G, which is encoded by a second, distal Nkrp1 gene cluster. NKR-P1F and -G bind an overlapping panel of Clr ligands, but not Clr11. Rat Clr molecules appear to be constitutively expressed by hematopoietic cells; expression in tumor cell lines is more variable. The data show the existence of two phylogenetic groups of NKR-P1 molecules, which demonstrate conservation of ligand-binding properties independent of signaling function.Key words: Cytotoxicity . NK cells . Receptor . Rodent Introduction NK cells express several families of killer cell lectin-like receptors (KLR). These are type II transmembrane proteins encoded by the NK gene complex (NKC), which is located on chromosome 4 in the rat [1,2]. The NKR-P1 molecules (KLRB or CD161) were among the first KLR to be characterized, but their function long remained elusive [3][4][5]. Although the NKR-P1 family has expanded to include both activating and inhibitory members in rodents, it consists of only one member in several other mammalian species, including in human [6]. The nature of their ligands has been controversial [7,8], but it has recently been shown that members of another KLR-related receptor family, the C-type lectin-related (Clr) molecules [9], also termed C-type lectin 2D (CLEC2D), can act as ligands for certain NKR-P1 molecules [10,11]. The Clr gene family also shows considerable expansion in rodents and the Clr genes are interspersed with the Nkrp1 genes in the proximal (centromeric) part of the NKC. A recent analysis concluded that there are 11 Clr/Clec2d genes in the rat BN strain genome, which were numbered consecutively from proximal to distal. One of these (Clr8) is most likely only a gene fragment, which could have been excluded, but we will nevertheless adhere to the Clr numbering suggested by Hao et al. [6].In the mouse, the inhibitory NKR-P1B and -D receptors both bind Clrb (also termed Ocil). Clrb is constitutively expressed by hematopoietic cells and inhibits cytolytic activity of NKR-P1B/ D-expressing NK cells [10,11]. The activating NKR-P1F receptor has been shown to bind another Clr molecule, Clrg, by the use of a reporter assay and soluble recombinant proteins [11] functional consequence of this interaction remains obscure. In human, the NKR-P1A receptor binds a Clr orthologue term...

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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 multi-gene 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.

We report the cloning and functional characterization in the mouse and the rat of a novel natural killer (NK) cell receptor termed KLRE1. The receptor is a type II transmembrane protein with a COOH-terminal lectin-like domain, and constitutes a novel KLR family. Rat Klre1 was mapped to the NK gene complex. By Northern blot and flow cytometry using newly generated monoclonal antibodies, KLRE1 was shown to be expressed by NK cells and a subpopulation of CD3+ cells, with pronounced interstrain variation. Western blot analysis indicated that KLRE1 can be expressed on the NK cell surface as a disulphide-linked dimer. The predicted proteins do not contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or a positively charged amino acid in the transmembrane domain. However, in a redirected lysis assay, the presence of whole IgG, but not of F(ab′)2 fragments of a monoclonal anti-KLRE1 antibody inhibited lysis of Fc-receptor bearing tumor target cells. Moreover, the tyrosine phosphatase SHP-1 was coimmunoprecipitated with KLRE1 from pervanadate-treated interleukin 2–activated NK cells. Together, our results indicate that KLRE1 may form a functional heterodimer with an as yet unidentified ITIM-bearing partner that recruits SHP-1 to generate an inhibitory receptor complex.

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 superfamily. 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 orthologue. 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.

The gene for a rat NK lectin-like receptor (NKLLR), named NKR-P2, has been cloned and characterized. Sequence analysis shows that it represents the orthologue of human NKG2D and that the two molecules form a distinct NKLLR family, no more related to NKG2A/B, -C or -E than to other NKLLR families. Nkrp2 is a single-copy gene containing seven introns, mapping to the rat NK gene complex. Rat NKR-P2 differs from the human orthologue in that its cytoplasmic tail contains 13 additional amino acids, encoded by a separate exon. Splice variants lacking this exon were not detected in T cells or NK cells. NKR-P2 is strongly expressed by NK cells. In contrast to other NKLLR, it is also strongly expressed by resting thoracic duct CD4+ and CD8+ T cells, but not by thymocytes or other hemopoietic cells.

We here report the molecular cloning of a novel family of killer-cell lectin-like (KLR) receptors in the rat and the mouse, termed KLRI. In both species, there are two members, KLRI1 and KLRI2. While the extracellular lectin-like domains of KLRI1 and KLRI2 are similar [74% (rat) and 83% (mouse) amino acid identity], they differ intracellularly. KLRI1 has two immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic domain, suggesting an inhibitory function. KLRI2 has no ITIM, but a positively charged lysine residue in the transmembrane region, suggesting association with activating adapter molecules. Klri1 and Klri2 are localized within the natural killer (NK) cell gene complex on rat chromosome 4 and mouse chromosome 6. By RT-PCR and Northern blot analysis KLRI1 and KLRI2 were selectively expressed by NK cells in both rat and mouse. Epitope-tagged expression constructs of rat KLRI1 and rat KLRI2 induced surface expression of a nondisulphide-linked protein of M(r) 36,000/39,000 and M(r) 34,000, respectively.

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.