Sigbjørn Fossum

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.

Identification of lectin-like receptors expressed by antigen presenting cells and neutrophils and their mapping to a novel gene complex

In an experimental rat model, we recently mapped an arthritis susceptibility locus to the distal part of Chromosome 4 containing genes predicted to encode C-type lectin superfamily (CLSF) receptors. Here we report the cDNA cloning and positional arrangement of these receptor genes, which represent rat orthologues to human Mincle and DCIR and to mouse MCL and Dectin-2, as well as four novel receptors DCIR2, DCIR3, DCIR4 and DCAR1, not previously reported in other species. We furthermore report the cDNA cloning of human Dectin-2 and MCL, and of the mouse orthologues to the novel rat receptors. Similar to the killer-cell lectin-like receptors (KLR) some of these receptors exhibit structural features suggesting that they regulate leukocyte reactivity; e.g., human DCIR and rodent DCIR1 and DCIR2 carry an immunoreceptor tyrosine-based inhibitory motif (ITIM), predicting inhibitory function, and conversely, in all three species Mincle has a positively charged amino acid in the transmembrane region, suggesting activating function. Sequence comparisons show that the receptors form a discrete family, more closely related to group II CLSF receptors than to the group V KLR. Their distance to the KLR is underscored by their preservation of evolutionary conserved calcium/saccharide binding residues, present in group II and lacking in group V CLSF and their cellular expression patterns, with most of the genes preferentially expressed by professional antigen-presenting cells (dendritic cells, macrophages and B cells) and neutrophils. In all three species, the genes map together, forming an evolutionary conserved gene complex, which we call the antigen presenting lectin-like receptor complex (APLEC).

Cutting Edge: Molecular Cloning of a Killer Cell Ig-Like Receptor in the Mouse and Rat

We report the molecular cloning of a KIR3DL1 receptor in the mouse and the rat, between 37.4 and 45.4% identical with primate killer cell Ig-like receptors (KIRs/CD158). Both mouse and rat molecules contain a pair of immunoreceptor tyrosine-based inhibition motifs in their cytoplasmic regions, suggesting an inhibitory function. Southern blot analysis indicated a single KIR gene in the rat, whereas the mouse genome contains more than one KIR-related element. The rat Kir3dl1 locus was mapped to the leukocyte receptor gene complex on chromosome 1, whereas mouse Kir3dl1 was localized to the X chromosome. RT-PCR demonstrated that KIR3DL1 was selectively expressed by NK cells in both rat and mouse. An epitope-tagged expression construct of mouse KIR3DL1 transfected into 293T cells induced expression of a ∼55-kDa protein. Our data indicate that KIR receptors may contribute to the NK cell receptor repertoire in rodents, alongside the Ly-49 family.

The genes and gene organization of the Ly49 region of the rat natural killer cell gene complex

We here report the cDNA sequences of 11 new rat Ly49 genes with full and three with incomplete open reading frames. Although obtained from different inbred rat strains, these as well as six previously published cDNA represent non‐allelic genes matching different loci in the Brown Norway (BN) rat genome, which is predicted to contain 34 Ly49 loci distributed over the distal part of the NK cell gene complex. Some of the cloned genes appear to be mutated to non‐function in the BN genome, which harbors additional genes with full open reading frames, suggesting at least 26 non‐allelic functional Ly49 genes in the rat. Of the encoded receptors, 13 are predicted to be inhibitory, eight to be activating, whereas five may be both (‘bifunctional’). Phylogenetic analysis bears evidence of a highly dynamic genetic region, in which only the most distally localized Ly49 gene has a clear‐cut mouse ortholog. In phylograms, the majority of the genes cluster into three subgroups with the genes mapping together, defining three chromosomal regions that seem to have undergone recent expansions. When comparing the lectin‐like domains, the receptors form smaller subgroups, most containing at least one inhibitory and one activating or ‘bifunctional’ receptor, where close sequence similarities suggest recent homogenization events.

Rat NKp46 activates natural killer cell cytotoxicity and is associated with FcεRIγ and CD3ζ

NKp46 has been identified in the human, rat, mouse, monkey, and cattle. We have generated a monoclonal antibody, WEN23, against rat NKp46. By flow cytometry, NKp46 is expressed by all natural killer (NK) cells but not by T cells, B cells, granulocytes, monocytes, dendritic cells, or macrophages. Thus, NKp46/WEN23 is the first NK cell‐specific marker in the rat. In a redirected lysis assay, preincubation of the effector cells with WEN23 augmented lysis of the Fc receptor (FcR)+ murine tumor target cells, indicating that NKp46 is an activating NK cell receptor. Moreover, preincubation of the effector cells with WEN23 F(ab′)2 fragments reduced killing of target cells, confirming the activating function of NKp46 and indicating that the mouse tumor target cells express a ligand for rat NKp46. Lysis of FcR− mouse and human tumor target cells was reduced after incubation of effector cells with WEN23, suggesting that rat NKp46 recognizes a ligand that is conserved between primates and rodents. By Western blot and immunoprecipitation using WEN23, NKp46 is expressed as a monomer of ∼47 kDa in interleukin‐2‐activated NK cells. The immunoreceptor tyrosine‐based activation motif bearing adaptor proteins CD3ζ and the γ chain of FcRI for IgE (FcɛRIγ) with NKp46 from lysates of NK cells, indicating that rat NKp46 activates NK cell cytotoxicity by similar pathways as CD16.

Mincle, the receptor for mycobacterial cord factor, forms a functional receptor complex with MCL and FcεRI-γ.

Upon receptor activation, the myeloid C‐type lectin receptor Mincle signals via the Syk‐CARD9‐Bcl10‐MALT1 pathway. It does so by recruiting the ITAM‐bearing FcεRI‐γ. The related receptor macrophage C‐type Lectin (MCL) has also been shown to be associated with Syk and to be dependent upon this signaling axis. We have previously shown that MCL co‐precipitates with FcεRI‐γ, but were unable to show a direct association, suggesting that MCL associates with FcεRI‐γ via another molecule. Here, we have used rat primary cells and cell lines to investigate this missing link. A combination of flow cytometric and biochemical analysis showed that Mincle and MCL form heteromers on the cell surface. Furthermore, association with MCL and FcεRI‐γ increased Mincle expression and enhanced phagocytosis of Ab‐coated beads. The results presented in this paper suggest that the Mincle/MCL/FcεRI‐γ complex is the functionally optimal form for these C‐type lectin receptors on the surface of myeloid 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.

Two major groups of rat NKR-P1 receptors can be distinguished based on chromosomal localization, phylogenetic analysis and Clr ligand binding.

A major subset of non‐alloreactive NK cells in PVG strain rats is generally low in Ly49 receptors, but expresses the rat NKR‐P1BPVG receptor (previously termed NKR‐P1C). The NKR‐P1B+ 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+ 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.

Development and function of CD94-deficient natural killer cells.

The CD94 transmembrane-anchored glycoprotein forms disulfide-bonded heterodimers with the NKG2A subunit to form an inhibitory receptor or with the NKG2C or NKG2E subunits to assemble a receptor complex with activating DAP12 signaling proteins. CD94 receptors expressed on human and mouse NK cells and T cells have been proposed to be important in NK cell tolerance to self, play an important role in NK cell development, and contribute to NK cell-mediated immunity to certain infections including human cytomegalovirus. We generated a gene-targeted CD94-deficient mouse to understand the role of CD94 receptors in NK cell biology. CD94-deficient NK cells develop normally and efficiently kill NK cell-susceptible targets. Lack of these CD94 receptors does not alter control of mouse cytomegalovirus, lymphocytic choriomeningitis virus, vaccinia virus, or Listeria monocytogenes. Thus, the expression of CD94 and its associated NKG2A, NKG2C, and NKG2E subunits is dispensable for NK cell development, education, and many NK cell functions.

The lectin-like receptor KLRE1 inhibits natural killer cell cytotoxicity.

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.