Natural killer (NK) cells have not previously been precisely identified or characterized in cattle or any other ruminant species. We have generated a monoclonal antibody against bovine NKp46, which is expressed exclusively by NK cells in man. NKp46 + cells comprised 1-10% of blood mononuclear cells in cattle, and did not stain with antibodies against CD3, CD4, TCR1, B cell or granulocyte markers. The majority of the NKp46 + cells expressed CD2, and a variable fraction also expressed CD8. The tissue distribution of NKp46 + cells in cattle was compatible with the tissue distribution of NK cells in other species. Bovine NKp46 + cells had typical, large granular lymphocyte morphology, and proliferated vigorously in response to bovine IL-2 for a limited number of cell divisions. IL-2-activated NKp46 + cells killed the bovine kidney cell line MDBK. This cytotoxicity was inhibited by preincubation with antibody against NKp46. In a redirected lysis assay, IL-2-activated NKp46 + cells killed the Fc + R + target cell line P815 after preincubation with antibody against NKp46. Together, these data indicate that bovine NKp46 is an activating receptor and demonstrate the existence of a subset of leukocytes in cattle that, in terms of surface markers, morphology and function, represent NK cells.

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).

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.Keywords: C-type lectin r Macrophage C-type Lectin (MCL) r Mincle r Myeloid cells r Signaling adaptor See accompanying Commentary by YamasakiAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionMacrophage inducible C-type lectin (Mincle) (also called CLEC4E) and macrophage C-type lectin (MCL) (also called CLEC4D) are single-pass transmembrane proteins that belong to the C-type lectin-like domain superfamily, and their genes lie adjacent to each other in the APLEC (antigen-presenting lectin-like complex) gene complex [1] in all species thus far examined. Mincle and MCL are expressed on cells of myeloid origin [2][3][4][5][6][7][8]. Mincle is normally expressed at low levels, but receptor levels are increased by exposure to different inflammatory signals [6,7,9]. Mincle has been shown to recognize the mycobacterial glycolipid trehalose-6,6-dimycolate (TDM, also called cord factor), present in the cell wall of some Mycobacterium species and considered as a virulence Correspondence: Dr. Michael R. Daws e-mail: m.r.daws@medisin.uio.no factor [10,11]. Moreover, Mincle-deficient mice show increased mycobacterial burden following challenge with Bacillus CalmetteGuérin (BCG), suggesting that Mincle has an important in vivo role in the immune response to mycobacteria [12]. In addition, Mincle recognizes a number of pathogenic fungi, particularly Malassezia spp. [7,8], and the endogenous ligand spliceosome-associated protein 130 released during cell necrosis [9]. We have been unable to identify a ligand for MCL, but it has recently been reported that MCL can also recognize TDM [13].The majority of activating C-type lectin receptors signal via associated adaptor proteins. Mincle has been shown to be associated with . MCL carries no known signaling motifs in its cytoplasmic region, and has no charged residues in its transmembrane domain, but it has been shown to activate spleen tyrosine kinase (Syk) [4]. We have recently shown that immunoprecipitation of MCL from a rat myeloid cel...

SummaryNatural Killer (NK) cells can recognize and kill MHC-incompatible normal bone marrowderived cells. Presently characterized MHC-binding receptors on NK cells, including the Ly-49 family in the mouse, transmit inhibitory signals upon binding to cognate class I MHC ligands. Here we study in vivo NK-mediated lysis of normal allogeneic lymphocytes in crosses between alloreactivity-competent PVG rats and alloreactivity-deficient DA rats. NK cells from both strains are able to lyse standard tumor targets. We identify an autosomal dominant locus, Nka, that controls NK-mediated alloreactivity. Individuals carrying the dominant PVG allele in single dose were fully competent in eliminating anogeneic target cells, suggesting that Nka encodes or regulates a gene product inducing or activating alloreactivity. By linkage analysis and pulsed field gel electrophoresis, a natural killer gene complex (NKC) on rat chromosome 4 is described that contains the rat NKR-P1 and Ly-49 multigene families plus a rat NKG2D homologue. Nka maps within the NKC, together with the most telomeric Ly-49 family members, but separate from NKG2D and the NKR-P1 family. The Nka-encoded response, moreover, correlates with the expression of transcripts for Ly-49 receptors in NK cell populations, as Northern blot analysis demonstrated low expression of Ly-49 genes in DANK cells, in contrast to high expression in alloreactivity-competent PVG, (DA • PVG)F1, and PVG.1AV1 NK cells. The low Ly-49 expression in DA is not induced by MHC haplotype, as demonstrated by high expression of Ly-49 in the DA MHC-congenic PVG.1AV1 strain. Finally, we have cloned and characterized the first four members of the rat Ly-49 gene family. Their cytoplasmic domains demonstrate substantial heterogeneity, consistent with the hypothesis that different Ly-49 family members may subserve different signaling functions.

Objective. To identify susceptibility genes in a rat model of rheumatoid arthritis (RA) and to determine whether the corresponding human genes are associated with RA.Methods. Genes influencing oil-induced arthritis (OIA) were position mapped by comparing the susceptibility of inbred DA rats with that of DA rats carrying alleles derived from the arthritis-resistant PVG strain in chromosomal fragments overlapping the quantitative trait locus Oia2. Sequencing of gene complementary DNA (cDNA) and analysis of gene messenger RNA (mRNA) expression were performed to attempt to clone a causal gene. Associations with human RA were evaluated by genotyping single-nucleotide polymorphisms (SNPs) in the corresponding human genes and by analyzing frequencies of alleles and haplotypes in RA patients and age-, sex-, and area-matched healthy control subjects.Results. Congenic DA rats were resistant to OIA when they carried PVG alleles for the antigenpresenting lectin-like receptor gene complex (APLEC), which encodes immunoregulatory C-type lectin-like receptors. Multiple differences in cDNA sequence and mRNA expression precluded cloning of a single causal gene. Five corresponding human APLEC genes were identified and targeted. The SNP rs1133104 in the dendritic cell immunoreceptor gene (DCIR), and a haplotype including that marker and 4 other SNPs in DCIR and its vicinity showed an indication of allelic association with susceptibility to RA in patients who were negative for antibodies to cyclic citrullinated peptide (anti-CCP), with respective odds ratios of 1.27 (95% confidence interval [95% CI] 1.06-1.52; uncorrected P ‫؍‬ 0.0073) and 1.37 (95% CI 1.12-1.67; uncorrected P ‫؍‬ 0.0019). Results of permutation testing supported this association of the haplotype with RA.

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.

Natural killer (NK) cells recognize and kill certain tumor cells, virally infected cells and MHC class I-disparate normal hematopoietic cells. NK cell cytotoxicity is regulated by a multitude of receptors with either activating or inhibitory signaling function. We here report the molecular cloning of bovine CD94 [killer cell lectin-like receptor (KLR)-D1] and NKp46 orthologues, four members of a bovine CD158 [killer cell immunoglobulin-like receptor (KIR)] family, and a novel KLR. This novel receptor was termed KLRJ1 and is most similar to Ly-49 (KLRA). The KLRD1 and KLRJ1 loci were mapped to a bovine NK gene complex on chromosome 5 by radiation hybrid mapping, whereas KIR2DL1 and NKP46 were localized to chromosome 18. Two of the bovine KIR (KIR2DL1 and KIR3DL1) contain immunoreceptor tyrosine-based inhibition motifs (ITIM), suggesting an inhibitory function. Bovine KIR2DS1 and KIR3DS1 lack ITIM but have an arginine-containing motif in their transmembrane domain, similar to primate KIR2DL4. Thus, KIR multigene families with divergent signaling motifs do not only exist in primates. Based on sequence comparison, it appears that the primate and bovine KIR multigene families may have evolved independently.

No abstract