MAFA-L,an ITIM-containing receptor encoded by the human NK cell gene complex and expressed by basophils and NK cells

The natural killer cell gene complex on human chromosome 12p12-13 encodes several C-type lectin receptor genes expressed by NK cells and other hematopoietic cells. We have identified a novel receptor gene in this region encoding a putative type II transmembrane glycoprotein. The product is 54 % identical to the rat mast cell function-associated antigen (MAFA), which inhibits mast cell activation by IgE. The human MAFA-like receptor (MAFA-L) and the rat MAFA protein are expressed by basophils and both have an immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic tail, consistent with an inhibitory role in basophil activation. Unlike rat MAFA, expression of the MAFA-L gene is not limited to mast cells and basophils. In common with other genes in the NK cell gene complex MAFA-L is also expressed by natural killer cells as well as the monocyte-like cell-line U937. Expression in NK cells is restricted to peripheral blood NK cells, decidual NK cells do not express MAFA-L. While MAFA-L and rat MAFA might have a similar role in basophils, the expression of MAFA-L in other cell types implies additional functions for this molecule. The presence of the MAFA-L gene in the human NK cell complex indicates that this locus encodes C-type lectin receptors expressed by a variety of cells important in host defense.     

Human KLRF1, a novel member of the killer cell lectin-like receptor gene family: molecular characterization, genomic structure, physical mapping to the NK gene complex and expression analysis

The human NK gene complex localized on chromosome 12p12.3 - p13.2 codes for several lectin-like receptor genes expressed by NK cells as well as by other hematopoietic cells. In this study, by using the expressed sequence tag database we identified a novel receptor gene, designated as killer cell lectin-like receptor, subfamily F, member 1 (KLRF1), encoding a putative type II transmembrane glycoprotein. The KLRF1 gene has been localized on the high-resolution physical map of chromosome 12p. The genomic structure of the KLRF1 gene and the existence of one spliced variant are also described. KLRF1 was expressed at the mRNA level in peripheral blood leukocytes, activated NK cells, monocytes and NK and myeloid cell lines. The presence of two immunoreceptor tyrosine-based inhibitory-like motifs within the cytoplasmic tail of KLRF1 suggests an inhibitory role in NK cell and monocyte activity.     

Genomic organization and allelic polymorphism of the human killer cell inhibitory receptor gene KIR 103

Killer cell inhibitory receptors (KIR) belong to the immunoglobulin super-family of molecules and are expressed on natural killer (NK) cells. The KIRs of the p58/p50 family have two immunoglobulin domains and are ligands for HLA-Cw antigens, whereas the p70/p70Δ family has three immunoglobulin domains and comprises ligands for HLA-B antigens and possibly some HLA-A antigens. Members of a third KIR family, KIR103, have two immunoglobulin domains but have highest nucleotide sequence homology to the p70 family. The ligands for KIR 103 on target cells are currently unknown. We here report the complete genomic organization of KIR103. It spans about 12 kb of DNA and consists of eight exons of which exon 1 and exon 2 encode the leader sequence. Exon 3 encodes the first immunoglobulin domain (γ1), and exon 4 encodes the main part of the second immunoglobulin domain (γ3), which also contains sequences contributed by exon 5 and exon 6. Exon 6 encodes the transmembrane domain, whereas exons 7 and 8 encode most of the cytoplasmic domain. KIR 103 is polymorphic, and two alleles, 103AS and 103LR, are defined in this study. Additional full-length cDNA clones for KIR 103 have been isolated and are shown to be formed by alternative mRNA splicing with exon skipping. Some of these truncated KIR 103 cDNA could encode shorter transmembrane molecules, whereas others lack the transmembrane domain and are candidate genes for secreted KIR products. KIR 103 is localized to the KIR genetic region on chromosome 19q13.4.     

p49, a putative HLA class I-specific inhibitory NK receptor belonging to the immunoglobulin superfamily

NK cells display several killer inhibitory receptors (KIR) specific for different alleles of MHC class I molecules. A family of KIR are represented by type I transmembrane proteins belonging to the immunoglobulin superfamily (Ig-SF). Besides cDNA encoding for these KIR, additional cDNA have been identified which encode for Ig-SF receptors with still undefined specificity. Here we analyze one of these cDNA, termed cl.15.212, which encodes a type I transmembrane protein characterized by two extracellular Ig-like domains and a 115-amino acid cytoplasmic tail containing a single immuno-receptor tyrosine-based inhibitory motif (ITIM) which is typical of KIR. cl.15.212 cDNA displays approximately 50 % sequence homology with other Ig-SF members. Different from the other KIR, cl.15.212 mRNA is expressed by all NK cells and by a fraction of KIR⁺ T cell clones. cl.15.212 cDNA codes for a membrane-bound receptor displaying an apparent molecular mass of 49 kDa, thus termed p49. To determine the specificity of the cl.15.212-encoded receptor, we generated soluble fusion proteins consisting of the ectodomain of p49 and the Fc portion of human IgG1. Soluble molecules bound efficiently to 221 cells transfected with HLA-G1, -A3, -B46 alleles and weakly to -B7 allele. On the other hand, they did not bind to 221 cells either untransfected or transfected with HLA-A2, -B51, -Cw3 or -Cw4. The binding specificity of soluble p49-Fc was confirmed by competition experiments using an anti-HLA class I-specific monoclonal antibody. Finally, different cDNA encoding for molecules homologous to cl.15.212 cDNA have been isolated, two of which lack the sequence encoding the transmembrane portion, thus suggesting they may encode soluble molecules.     

Signaling through human killer cell activating receptors triggers tyrosine phosphorylation of an associated protein complex

Our understanding of the biology of human natural killer (NK) cells has significantly advanced in recent years upon identification of a family of NK cell-expressed genes that encode killer cell inhibitory receptors (KIR). Individual KIR can selectively bind various HLA class I allotypes and consequently transduce inhibitory signals that block NK cell lysis of ligand-bearing target cells. A distinct subset of related and linked genes express truncated versions of KIR that are otherwise highly homologous in amino acid sequence. Interestingly, these receptors appear to transmit stimulatory signals into NK cells and have been termed killer cell activating receptors (KAR). In this report, we demonstrate that recognition of HLA-Cw3 by the p50 KAR, NKAT8, can potentiate the cytotoxic response of appropriate NK cell clones. Specific cross-linking of this KAR with a monoclonal antibody resulted in intracellular calcium mobilization, protein tyrosine phosphorylation, and phosphorylation of the MAP kinases, ERK1 and ERK2. In addition, we identified a KAR-associated disulfide-linked dimer of a 13-kDa protein that was absent in the Jurkat T cell line and is predicted to participate in these activation signaling events. Upon treatment of NK cells with pervanadate, the disulfide-linked p13 and additional proteins of 25, 30, 37 and 50 – 95 kDa were identified as KAR-associated tyrosine phosphoproteins. Importantly, p13 was inducibly tyrosine phosphorylated upon cross-linking of NKAT8, which strongly suggests that the associated p13 provides KAR with appropriate cytoplasmic structure to couple with tyrosine kinase-mediated signaling effectors.     

Polymorphism and domain variability of human killer cell inhibitory receptors

Summary: Thirty-two different full-length cDNA clones for human killer cell inhibitory receptors (KIR) have been identified. They all belong to the immunoglobulin superfamily and encode mature proteins with one, two or three extracellular Ig domains. The inhibitory receptors have nearly identical transmembrane domains and cytoplasmic domains ranging in length from 76 to 95 amino-acid residues. The activating receptors have a characteristic transmembrane domain with a charged lysine residue and a short cytoplasmic tail without the protein tyrosine phosphatase binding motif I/VXYXXL present in the inhibitory receptors. Sequence analysis demonstrates that three clusters correspond to the inhibitory receptors of 58 kDa, while two clusters encode activating receptors of 50 kDa. TWD other clusters correspond to the inhibitory receptors of 70 kDa and one cluster encodes genes with sequence homology to one of the two p70 clusters but contains the transmembrane and cytoplasmic domains characteristic of activating receptors. The data are consistent with a genomic organization of the KIR genetic region containing at least three KIR genes encoding receptors for each of the gene products of the HLA class I loci. Alternative mRNA splicing could be responsible for generation of activating or inhibitory receptors with the same extracellular domains. Separate genes encoding receptors with opposite function is, however, an equally likely possibility.     

NK cell receptor gene of the KIR family with two IG domains but highest homology to KIR receptors with three IG domains

The killer cell inhibitory receptors (KIRs) are surface glycoproteins expressed by natural killer (NK) cells and some T cells. They recognize polymorphic human HLA class I molecules. Two families of KIRs have been identified and named p58 and p70. The p58 family of genes encode type I membrane proteins with two extracellular immunoglobulin (Ig) domains, while the p70 genes have three Ig domains. We here report the cloning and characterization of a novel KIR cDNA obtained from tumor cell lines with NK reactivity (YT and NK-92). This gene is also expressed in the normal cell line NK 3.3 and in NK cells obtained from some but not all normal donors. The clone, KIR103AS, has an open reading frame consistent with a KIR with two extracellular Ig domains, a transmembrane region and a 114 amino acid long cytoplasmic domain containing a single src homology 2 (SH2) binding motif. The membrane distal Ig domain of KIR 103AS has highest homology with the first Ig domain of p70 KIRs and differs significantly from the first Ig domain of p58 KIRs. The second, membrane proximal Ig domain of KIR103AS has similar and high homology with the membrane proximal Ig domains of both p70 and p58 KIRs. The extracellular domains of KIR 103AS therefore share characteristic features with both p70 and p58 genes: the domain structure is identical to p58 KIRs but the sequence homology matches closely with p70 KIRs. The putative transmembrane and cytoplasmic domains are distinctly different from all previously reported KIR cDNAs.     

A novel surface molecule homologous to the p58/p50 family of receptors is selectively expressed on a subset of human natural killer cells and induces both triggering of cell functions and proliferation

Human natural killer (NK) cells express inhibitory (p58) or activatory (p50) receptors for HLA-C alleles. Here, we describe a novel member of the p58/p50 family that is expressed by a subset of NK cells in about one third of donors. This molecule, termed p50.3, mediates NK cell triggering as revealed by the induction of intracellular free calcium mobilization, cytokine release and cytotoxicity. In addition, anti-p50.3 monoclonal antibody (mAb) induced a selective, strong proliferation of p50.3⁺ NK cells in peripheral blood lymphocytes. Although p50.3 molecules do not appear to display an obvious HLA class I specificity, they are usually coexpressed with known inhibitory receptors for HLA class I alleles. mAb-mediated cross-linking of these receptors leads to inhibition of the anti-p50.3 mAb-induced NK cell activation and proliferation. Surface p50.3 molecules are glycoproteins of ∼ 55–58 kDa which, upon deglycosylation, display a relative molecular mass of 36 kDa, similar to that of deglycosylated (activatory) p50 receptors. Analysis of the two-dimensional peptide maps of the 50.3 molecules revealed a high homology with the other HLA-C-specific p58/p50 receptors. The use of a set of oligodeoxynucleotide primers, previously shown to amplify the activatory (p50) forms of HLA-C-specific receptors, consistently amplified in p50.3⁺ clones a cDNA sequence termed KKA3. This sequence belongs to the p58/p50 multigene family, that encodes for a transmembrane protein specifically stained by anti-p50.3 mAb in cell transfectants. Similar to p50 molecules, the KKA3-encoded molecules are characterized by two extracellular immunoglobulin-like domains, by the presence of a lysine in the transmembrane region and a short (39 amino acids) cytoplasmic tail which does not contain immune receptor tyrosine-based activation motifs (ITAM)-like sequences.     

NK受体KIR生物学功能的研究进展

杀伤细胞免疫球蛋白样受体(Killer Ig-like receptor,KIR)为免疫球蛋白超家族成员,表达于NK细胞和某些T细胞亚群。KIR包括抑制型和激活型受体,其通过识别表达于靶细胞上的人类白细胞抗原1(HLA-Ⅰ)类分子,调节NK细胞的杀伤活性,在自身免疫性疾病、妊娠、移植等生理病理过程中起重要的作用。本文综述了近年来关于KIR研究的最新进展,主要包括KIR的结构、其特异性配体、KIR介导的信号传导和KIR的生物学功能。     

The centromeric part of the human natural killer (NK) receptor complex: lectin-like receptor genes expressed in NK, dendritic and endothelial cells

Summary: The human natural killer (NK) receptor complex encompasses a region of about 2 Mb on the short arm of chromosome 12. It contains at least 18 lectin-like receptor genes, of which some are expressed in NK and NK/T cells and function as NK receptors. Close to the CD94 and NKG2 NK receptor genes in the centromeric part, a novel family of genes, expressed in myeloid, dendritic and/or endothelial cells, recently became evident. These genes encode a receptor for oxidized low density lipoprotein in endothelial cells and three other receptors potentially serving regulatory functions in dendritic cells. Although the overall structure of the human NK receptor complex is similar to the syntenic rodent regions, the centromeric part lacks the cluster of Ly49 genes. This supports the notion that recognition of MHC class Ia molecules has evolved separately in rodents and humans in the lectin-like Ly49 and the killer immunoglobulin-like receptors, respectively. In the telomeric part, other lectin-like genes expressed in different hematopoietic lineages are found. The receptors of the NK receptor complex apparently serve important functions in several leukocytes and in endothelial cells, and the exact role of these receptors, their ligands, and their distinct and co-ordinate regulation in different cell lineages warrants further investigation.( )
This work was supported by grants of the Austrian National Bank and the European Commission.     

HLA and KIR polymorphisms affect NK-cell anti-tumor activity

Killer cell immunoglobulin-like receptors (KIRs) and their cognate human leukocyte antigen (HLA) ligands are key to the maintenance of natural killer (NK) cell tolerance. The gene complexes encoding both KIRs and HLA ligands are extremely polymorphic. Because the extent of NK cell inhibition varies with the allelic forms expressed, NK cell tolerance can be broken more easily in some individuals than in others. This explains why particular combinations of KIR and HLA genes are associated with an increased risk of autoimmune diseases or with more efficient antiviral responses. Breaking of NK cell tolerance might be prerequisite to kill leukemic blasts. At present, there are ample indications that NK cells can eradicate acute myeloid leukemia blasts in patients with a favorable combination of HLA and KIR genes. Selecting these individuals for clinical trials should give insight into the feasibility of anti-tumor therapy mediated through NK cells.     

The molecular basis of Natural Killer (NK) cell recognition and function

Natural Killer cells are likely to play an important role in the host defenses because they kill virally infected or tumor cells but spare normal self-cells. The molecular mechanism that explains why NK cells do not kill indiscriminately has recently been elucidated. It is due to several specialized receptors that recognize major histocompatibility complex (MHC) class I molecules expressed on normal cells. The lack of expression of one or more HLA class I alleles leads to NK-mediated target cell lysis. Different types of receptors specific for groups of HLA-C, HLA-B, and, very recently, HLA-A alleles have been identified. While in most instances, they function as inhibitory receptors, an activatory form of the HLA-C-specific receptors has been identified in some donors. Molecular cloning of HLA-C-, HLA-B- or HLA-A-specific receptors has revealed new members of the immunoglobulin superfamily with two or three Ig-like domains, respectively, in their extracellular portion. While the inhibitory form is characterized by a long cytoplasmic tail associated with a non-polar transmembrane portion, the activatory one has a short tail asociated with a Lys-containing transmembrane portion. Thus, these human NK receptors are different from the murine Ly49, that is a type II transmembrane protein characterized by a C-type lectin domain. A subset of activated T lymphocytes expresses NK-type class I-specific receptors. These receptors exert an inhibiting activity on T cell receptor-mediated functions and may provide an important mechanism of downregulation of T cell responses.     

自然杀伤细胞活化性受体的研究进展

自然杀伤细胞（NK）是机体固有免疫系统的重要效应细胞,其不仅能杀死病毒感染细胞和肿瘤细胞,还参与调节固有免疫应答和适应性免疫应答.NK细胞对靶细胞的杀伤效应取决于NK细胞抑制性受体和活化性受体与其配体相互作用的整合,而NK细胞不杀伤正常组织是因为抑制性受体对HLA-Ⅰ类分子的优势识别.NK细胞抑制性受体研究比较成熟,近几年NK细胞活化性受体研究进展很快.     

Nucleotide and amino acid sequence alignment for human killer cell inhibitory receptors (KIR), 1998

Abstract: We present alignments of nucleotide sequences and deduced amino acid sequences for all currently identified killer cell inhibitory receptors (KIR). The genes for these receptors have been localized to human chromosome 19q13.4 and constitute a large gene family. They all encode structures typical of type I transmembrane molecules belonging to the immunoglobulin superfamily (Ig-SF). Extensive polymorphism exists within the genes and many of the currently identified cDNA clones represent alternatively spliced forms of previously reported full-length clones. The sequence alignments have been posted on the World Wide Web and are accessible at the Tissue Antigens web site at: http://www.tissue-antigens.dk/kir-align.html . The alignments will be updated at intervals.     

Cytotoxic activity of natural killer cells lacking killer-inhibitory receptors for self-HLA class I molecules against autologous hematopoietic stem cells in healthy individuals

Killer-inhibitory receptors (KIR) are receptors for self-HLA class I molecules, which are expressed on natural killer (NK) cells and small subsets of T-lymphocytes. KIR receptors that do not bind to self-HLA class I have been implicated in the pathogenesis of pure red-cell aplasia and other autoimmune diseases. However, NK cells whose inhibitory receptors lack any apparent self-ligand can also be found in healthy individuals. We therefore tested whether these NK cells are capable of exerting cytotoxic activity against autologous CD34(+) hematopoietic precursors. We detected NK cells whose sole inhibitory receptors were CD94/NKG2-A and that had no affinity for autologous HLA-C molecules. In vitro, such cells were able to kill autologous CD34(+) stem cells that expressed MHC class I antigen at a high level in about 50% of the cases of HLA-C group 2 donors. Two individual clones derived from this NK subpopulation were stimulated by autologous HLA-Cw5/6-positive stem cells, but not by allogeneic HLA-Cw7-positive stem cells. Our findings demonstrate the presence of potentially autoreactive natural killer cells in otherwise healthy individuals.     

The human TREM gene cluster at 6p21.1 encodes both activating and inhibitory single IgV domain receptors and includes NKp44

We have characterized a cluster of single immunoglobulin variable (IgV) domain receptors centromeric of the major histocompatibility complex (MHC) on human chromosome 6. In addition to triggering receptor expressed on myeloid cells (TREM)-1 and TREM2, the cluster contains NKp44, a triggering receptor whose expression is limited to NK cells. We identified three new related genes and two gene fragments within a cluster of approximately 200 kb. Two of the three new genes lack charged residues in their transmembrane domain tails. Further, one of the genes contains two potential immunotyrosine Inhibitory motifs in its cytoplasmic tail, suggesting that it delivers inhibitory signals. The human and mouse TREM clusters appear to have diverged such that there are unique sequences in each species. Finally, each gene in the TREM cluster was expressed in a different range of cell types.