The evolution of the natural killer complex; a comparison between mammals using new high-quality genome assemblies and targeted annotation.
ABSTRACT: Natural killer (NK) cells are a diverse population of lymphocytes with a range of biological roles including essential immune functions. NK cell diversity is in part created by the differential expression of cell surface receptors which modulate activation and function, including multiple subfamilies of C-type lectin receptors encoded within the NK complex (NKC). Little is known about the gene content of the NKC beyond rodent and primate lineages, other than it appears to be extremely variable between mammalian groups. We compared the NKC structure between mammalian species using new high-quality draft genome assemblies for cattle and goat; re-annotated sheep, pig, and horse genome assemblies; and the published human, rat, and mouse lemur NKC. The major NKC genes are largely in the equivalent positions in all eight species, with significant independent expansions and deletions between species, allowing us to propose a model for NKC evolution during mammalian radiation. The ruminant species, cattle and goats, have independently evolved a second KLRC locus flanked by KLRA and KLRJ, and a novel KLRH-like gene has acquired an activating tail. This novel gene has duplicated several times within cattle, while other activating receptor genes have been selectively disrupted. Targeted genome enrichment in cattle identified varying levels of allelic polymorphism between the NKC genes concentrated in the predicted extracellular ligand-binding domains. This novel recombination and allelic polymorphism is consistent with NKC evolution under balancing selection, suggesting that this diversity influences individual immune responses and may impact on differential outcomes of pathogen infection and vaccination.
Project description:The natural killer complex (NKC) is a genetic region of highly linked genes encoding several receptors involved in the control of NK cell function. The NKC is highly polymorphic, and allelic variability of various NKC loci has been demonstrated in inbred mice. Making use of BALB.B6-Cmv1r congenic mice, in which the NKC from disease-susceptible C57BL/6 mice has been introduced into the disease-resistant BALB/c background, we show here that during murine malaria infection, the NKC regulates a range of pathophysiological syndromes such as cerebral malaria, pulmonary edema, and severe anemia, which contribute to morbidity and mortality in human malaria. Parasitemia levels were not affected by the NKC genotype, indicating that control of malarial fatalities by the NKC cells does not operate through effects on parasite growth rate. Parasite-specific antibody responses and the proinflammatory gene transcription profile, as well as the TH1/TH2 balance, also appeared to be influenced by NKC genotype, providing evidence that this region, known to control innate immune responses via NK and/or NK T-cell activation, can also significantly regulate acquired immunity to infection. To date, NKC-encoded innate system receptors have been shown mainly to regulate viral infections. Our data provide evidence for critical NKC involvement in the broad immunological responses to a protozoan parasite.
Project description:The natural killer (NK) receptor gene complex (NKC) encodes a large number of C-type lectin-like receptors, which are expressed on NK and other immune-related cells. These receptors play an important role in regulating NK-cell cytolytic activity, protecting cells against virus infection and tumorigenesis. To understand the evolutionary history of the NKC, we characterized the C-type lectin-like NKC genes and their organization from four major orders of placental mammals, primates (human), rodents (mouse and rat), carnivores (dog), and artiodactyls (cattle) and then conducted phylogenetic analysis of these genes. The results indicate that the NKC of placental mammals is highly heterogeneous in terms of the gene content and rates of birth and death of different gene lineages, but the NKC is also remarkably conserved in its gene organization and persistence of orthologous gene lineages. Among the 28 identified NKC gene lineages, 4, KLRA1, KLRB1, CLEC2D, and CLEC4A/B/C, have expanded rapidly in rodents only. The high birth and death rate of these 4 gene families might be due to functional differentiation driven by positive selection. Identification of putative NKC sequences in opossum and chicken genomes implies that the expansion of the NKC gene families might have occurred before the radiation of placental mammals but after the divergence of birds from mammals.
Project description:The natural killer gene complex (NKC) on chromosome 6 contains clusters of genes that encode both activation and inhibitory receptors expressed on mouse natural killer (NK) cells. NKC genes, particularly belonging to the Nkrp1 and Ly49 gene families, display haplotype differences between different mouse strains and allelic polymorphisms of individual genes, as previously revealed by conventional analysis in a small number of inbred mouse strains. Herein we used array-based comparative genomic hybridization (aCGH) to efficiently compare the NKC in 21 mouse strains to the reference C57BL/6 strain. By using unsupervised clustering methods, we could sort these variations into the same groups as determined by previous RFLP analyses of Nkrp1 and Ly49 genes. Prospective analyses of aCGH and RFLP data validated these relationships. Moreover, aCGH data predicted monoclonal antibody reactivity with an allospecific determinant on molecules expressed by NK cells. Taken together, these data demonstrate the structural variation in the NKC between mouse strains as well as the usefulness of aCGH in analysis of complex, polymorphic gene clusters.
Project description:The natural killer (NK) gene complex (NKC) encodes numerous C-type lectin-like receptors that govern the activity of NK cells. Although some of these receptors (Ly49s, NKG2D, CD94/NKG2A) recognize MHC or MHC-like molecules, others (Nkrp1, NKRP1A, NKp80, NKp65) instead bind C-type lectin-like ligands to which they are genetically linked in the NKC. To understand the basis for this recognition, we determined the structure of human NKp65, an activating receptor implicated in the immunosurveillance of skin, bound to its NKC-encoded ligand keratinocyte-associated C-type lectin (KACL). Whereas KACL forms a homodimer resembling other C-type lectin-like dimers, NKp65 is monomeric. The binding mode in the NKp65-KACL complex, in which a monomeric receptor engages a dimeric ligand, is completely distinct from those used by Ly49s, NKG2D, or CD94/NKG2A. The structure explains the exceptionally high affinity of the NKp65-KACL interaction compared with other cell-cell interaction pairs (KD = 6.7 × 10(-10) M), which may compensate for the monomeric nature of NKp65 to achieve cell activation. This previously unreported structure of an NKC-encoded receptor-ligand complex, coupled with mutational analysis of the interface, establishes a docking template that is directly applicable to other genetically linked pairs in the NKC, including Nkrp1-Clr, NKRP1A-LLT1, and NKp80-AICL.
Project description:Many well-known immune-related C-type lectin-like receptors (CTLRs) such as NKG2D, CD69, and the Ly49 receptors are encoded in the natural killer gene complex (NKC). Recently, we characterized the orphan NKC gene CLEC2A encoding for KACL, a further member of the human CLEC2 family of CTLRs. In contrast to the other CLEC2 family members AICL, CD69, and LLT1, KACL expression is mostly restricted to skin. Here we show that KACL is a non-disulfide-linked homodimeric surface receptor and stimulates cytotoxicity by human NK92MI cells. We identified the corresponding activating receptor on NK92MI cells that is encoded adjacently to the CLEC2A locus and binds KACL with high affinity. This CTLR, termed NKp65, stimulates NK cytotoxicity and release of proinflammatory cytokines upon engagement of cell-bound KACL. NKp65, a distant relative of the human activating NK receptor NKp80, possesses an amino-terminal hemITAM that is required for NKp65-mediated cytotoxicity. Finally, we show that KACL expression is mainly restricted to keratinocytes. Freshly isolated keratinocytes express KACL and are capable of stimulating NKp65-expressing cells in a KACL-dependent manner. Thus, we report a unique NKC-encoded receptor-ligand system that may fulfill a dedicated function in the immunobiology of human skin.
Project description:Killer cell immunoglobulin-like receptor/HLA class I (KIR/HLA-I) combinations are associated with disease risk, implicating functional roles for NK cells (NKCs) or KIR(+) T cells. KIR/HLA-I interactions can act through inhibition of NKC activation by target cells and NKC licensing for greater intrinsic responsiveness. We compared licensing conferred by the weaker, HLA-C group 1/KIR2DL3, and the stronger, HLA-C group 2/KIR2DL1, inhibitory combinations. The "rheostat model" predicts weaker licensing by HLA-C1/KIR2DL3 interactions than HLA-C2/KIR2DL1. We analyzed degranulation in NKC subsets expressing single and multiple receptors for HLA-I. NKG2A had the strongest licensing impact, while KIR2DL3, KIR2DL1, and KIR3DL1 were weaker, and not significantly different to each other. Presence of one or two matched HLA-C allotypes did not alter licensing of KIR2DL3(+) and KIR2DL1(+) NKC. Coexpression of activating KIR2DS1 disarmed KIR2DL3(+) and KIR2DL1(+) NKC to a similar extent. KIR3DL1 and NKG2A combined for more enhanced licensing of double-positive NKC than the combination of KIR2DL3 and KIR2DL1. Thus, KIR2DL3 and KIR2DL1 have similar capacity to license NKC, suggesting that inhibitory signal strength and amount of available HLA-C ligands do not correlate with NKC licensing. Altogether, our results show that the basis for disease associations of HLA-C and KIR2DL likely encompasses factors other than licensing.
Project description:Ly49-mediated recognition of MHC-I molecules on host cells is considered vital for natural killer (NK)-cell regulation and education; however, gene-deficient animal models are lacking because of the difficulty in deleting this large multigene family. Here, we describe NK gene complex knockdown (NKC(KD)) mice that lack expression of Ly49 and related MHC-I receptors on most NK cells. NKC(KD) NK cells exhibit defective killing of MHC-I-deficient, but otherwise normal, target cells, resulting in defective rejection by NKC(KD) mice of transplants from various types of MHC-I-deficient mice. Self-MHC-I immunosurveillance by NK cells in NKC(KD) mice can be rescued by self-MHC-I-specific Ly49 transgenes. Although NKC(KD) mice display defective recognition of MHC-I-deficient tumor cells, resulting in decreased in vivo tumor cell clearance, NKG2D- or antibody-dependent cell-mediated cytotoxicity-induced tumor cell cytotoxicity and cytokine production induced by activation receptors was efficient in Ly49-deficient NK cells, suggesting MHC-I education of NK cells is a single facet regulating their total potential. These results provide direct genetic evidence that Ly49 expression is necessary for NK-cell education to self-MHC-I molecules and that the absence of these receptors leads to loss of MHC-I-dependent "missing-self" immunosurveillance by NK cells.
Project description:<h4>Background</h4>Duplicated sequences are an important source of gene innovation and structural variation within mammalian genomes. We performed the first systematic and genome-wide analysis of segmental duplications in the modern domesticated cattle (Bos taurus). Using two distinct computational analyses, we estimated that 3.1% (94.4 Mb) of the bovine genome consists of recently duplicated sequences (>or= 1 kb in length, >or= 90% sequence identity). Similar to other mammalian draft assemblies, almost half (47% of 94.4 Mb) of these sequences have not been assigned to cattle chromosomes.<h4>Results</h4>In this study, we provide the first experimental validation large duplications and briefly compared their distribution on two independent bovine genome assemblies using fluorescent in situ hybridization (FISH). Our analyses suggest that the (75-90%) of segmental duplications are organized into local tandem duplication clusters. Along with rodents and carnivores, these results now confidently establish tandem duplications as the most likely mammalian archetypical organization, in contrast to humans and great ape species which show a preponderance of interspersed duplications. A cross-species survey of duplicated genes and gene families indicated that duplication, positive selection and gene conversion have shaped primates, rodents, carnivores and ruminants to different degrees for their speciation and adaptation. We identified that bovine segmental duplications corresponding to genes are significantly enriched for specific biological functions such as immunity, digestion, lactation and reproduction.<h4>Conclusion</h4>Our results suggest that in most mammalian lineages segmental duplications are organized in a tandem configuration. Segmental duplications remain problematic for genome and assembly and we highlight genic regions that require higher quality sequence characterization. This study provides insights into mammalian genome evolution and generates a valuable resource for cattle genomics research.
Project description:Killer cell immunoglobulin-like receptor (KIR) genes encode for activating and inhibitory surface receptors, which are correlated with the regulation of Natural Killer (NK) cell cytotoxic activity. Reduced NK cell cytotoxic activity has been consistently reported in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) patients, and KIR haplotypes and allelic polymorphism remain to be investigated. The aim of this article was to conduct a pilot study to examine KIR genotypes, haplotypes, and allelic polymorphism in CFS/ME patients and nonfatigued controls (NFCs). Comparison of KIR and allelic polymorphism frequencies revealed no significant differences between 20 CFS/ME patients and 20 NFCs. A lower frequency of the telomeric A/B motif (P < 0.05) was observed in CFS/ME patients compared with NFCs. This pilot study is the first to report the differences in the frequency of KIR on the telomeric A/B motif in CFS/ME patients. Further studies with a larger CFS/ME cohort are required to validate these results.
Project description:Natural Killer (NK) cells can recognize and kill MHC-incompatible normal bone marrow-derived 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 allogeneic 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 DA NK cells, in contrast to high expression in alloreactivity-competent PVG, (DA X PVG)F1, and PVG.1AVI 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.1AVI 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.