Erik Dissen

NKp46 defines a subset of bovine leukocytes with natural killer cell characteristics

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 FcγR+ target cell line P815 after preincubation with antibody against NKp46. Together, these data indicate that bovine NKp46 is anactivating receptor and demonstrate the existence of a subset of leukocytes in cattle that, in terms of surface markers, morphology and function, represent NK cells.

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).

Natural killer cell receptors in cattle: a bovine killer cell immunoglobulin-like receptor multigene family contains members with divergent signaling motifs

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.

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.

Microbial colonization induces oligoclonal expansions of intraepithelial CD8 T cells in the gut

Two populations of CD8+ IEL generally express restricted, but apparently random and non‐overlapping TCR repertoires. Previous studies in mice suggested that this could be explained by a dual origin of CD8+ IEL, i.e. that CD8αβ+ IEL derive from a few peripheral CD8+ T cell lymphoblasts stimulated by microbial antigens in gut‐associated lymphoid tissue, whereas CD8αα+ IEL descend from an inefficient intestinal maturation pathway. We show here that the gut mucosa, instead, becomes seeded with surprisingly broad and generally non‐overlapping CD8 IEL repertoires and that oligoclonality is induced locally after microbial colonization. In germ‐free (GF) rats, both CD8αβ+ and CD8αα+ IEL displayed surprisingly diverse TCR Vβ repertoires, although β‐chain diversity tended to be somewhat restricted in the CD8αα+ subset. CDR3 length displays in individual Vβ‐Cβ and Vβ‐Jβ combinations generally revealed polyclonal distributions over 6–11 different lengths, similar to CD8+ lymph node T cells, and CDR3β sequencing provided further documentation of repertoire diversity. By contrast, in ex‐GF rats colonized with normal commensal microflora, both CD8αβ+ and CD8αα+ IEL displayed oligoclonal CDR3 length distributions for most of the Vβ genes analyzed. Our data suggest that microbial colonization induces apparently random clonal expansions of CD8αβ+ and CD8αα+ IEL locally in the gut.