KIR2DS5 allotypes that recognize the C2 epitope of HLA-C are common among Africans and absent from Europeans.

INTRODUCTION: KIR2DS5 is an activating human NK cell receptor of lineage III KIR. These include both inhibitory KIR2DL1, 2 and 3 and activating KIR2DS1 that recognize either the C1 or C2 epitope of HLA-C. In Europeans KIR2DS5 is essentially monomorphic, with KIR2DS5*002 being predominant. Pioneering investigations showed that KIR2DS5*002 has activating potential, but cannot recognize HLA-A, -B, or -C. Subsequent studies have shown that KIR2DS5 is highly polymorphic in Africans, and that KIR2DS5*006 protects pregnant Ugandan women from preeclampsia. Because inhibitory C2-specific KIR2DL1 correlates with preeclampsia, whereas activating C2-specific KIR2DS1 protects, this association pointed to KIR2DS5*006 being an activating C2-specific receptor. To test this hypothesis we made KIR-Fc fusion proteins from all ten KIR2DS5 allotypes and tested their binding to a representative set of HLA-A, -B and -C allotypes. RESULTS: Six African-specific KIR2DS5 bound to C2+ HLA-C but not to other HLA class I. Their avidity for C2 is ∼20% that of C2-specific KIR2DL1 and ∼40% that of C2-specific KIR2DS1. Among the African C2 receptors is KIR2DS5*006, which protected a cohort of pregnant Ugandans from pre-eclampsia. Three African KIR2DS5 allotypes and KIR2DS5*002, bound no HLA-A, -B or -C. As a group the C2-binding KIR2DS5 allotypes protect against pre-eclampsia compared to the non-binding KIR2DS5 allotypes. Natural substitutions that contribute to loss or reduction of C2 receptor function are at positions 127, 158, and 176 in the D2 domain. CONCLUSIONS: KIR2DS5*005 has the KIR2DS5 consensus sequence, is the only allele found at both centromeric and telomeric locations of KIR2DS5, and is likely the common ancestor of all KIR2DS5 alleles. That KIR2DS5*005 has C2 receptor activity, points to KIR2DS5*002, and other allotypes lacking C2 receptor function, being products of attenuation, a characteristic feature of most KIR B haplotype genes. Alleles encoding attenuated and active KIR2DS5 are present in both centromeric and telomeric locations.

The Intergenic Recombinant HLA-B∗46:01 Has a Distinctive Peptidome that Includes KIR2DL3 Ligands.

HLA-B∗46:01 was formed by an intergenic mini-conversion, between HLA-B∗15:01 and HLA-C∗01:02, in Southeast Asia during the last 50,000 years, and it has since become the most common HLA-B allele in the region. A functional effect of the mini-conversion was introduction of the C1 epitope into HLA-B∗46:01, making it an exceptional HLA-B allotype that is recognized by the C1-specific natural killer (NK) cell receptor KIR2DL3. High-resolution mass spectrometry showed that HLA-B∗46:01 has a low-diversity peptidome that is distinct from those of its parents. A minority (21%) of HLA-B∗46:01 peptides, with common C-terminal characteristics, form ligands for KIR2DL3. The HLA-B∗46:01 peptidome is predicted to be enriched for peptide antigens derived from Mycobacterium leprae. Overall, the results indicate that the distinctive peptidome and functions of HLA-B∗46:01 provide carriers with resistance to leprosy, which drove its rapid rise in frequency in Southeast Asia.

Resurrecting KIR2DP1: A Key Intermediate in the Evolution of Human Inhibitory NK Cell Receptors That Recognize HLA-C.

KIR2DP1 is an inactive member of the human lineage III KIR family, which includes all HLA-C-specific receptor genes. The lethal, and only, defect in KIR2DP1 is a nucleotide deletion in codon 88. Fixed in modern humans, the deletion is also in archaic human genomes. KIR2DP1 is polymorphic, with dimorphism at specificity-determining position 44. By repairing the deletion, we resurrected 11 alleles of KIR2DP1F , the functional antecedent of KIR2DP1 We demonstrate how K44-KIR2DP1F with lysine 44 recognized C1+HLA-C, whereas T44-KIR2DP1F recognized C2+HLA-C. Dimorphisms at 12 other KIR2DP1F residues modulate receptor avidity or signaling. KIR2DP1 and KIR2DL1 are neighbors in the centromeric KIR region and are in tight linkage disequilibrium. Like KIR2DL1, KIR2DP1 contributed to CenA and CenB KIR haplotype differences. Encoded on CenA, C1-specific K44-KIR2DP1F were stronger receptors than the attenuated C2-specific T44-KIR2DP1F encoded on CenB The last common ancestor of humans and chimpanzees had diverse lineage III KIR that passed on to chimpanzees but not to humans. Early humans inherited activating KIR2DS4 and an inhibitory lineage III KIR, likely encoding a C1-specific receptor. The latter spawned the modern family of HLA-C receptors. KIR2DP1F has properties consistent with KIR2DP1F having been the founder gene. The first KIR2DP1F alleles encoded K44-C1 receptors; subsequently KIR2DP1F alleles encoding T44-C2 receptors evolved. The emergence of dedicated KIR2DL2/3 and KIR2DL1 genes encoding C1 and C2 receptors, respectively, could have led to obsolescence of KIR2DP1F Alternatively, pathogen subversion caused its demise. Preservation of KIR2DP1F functional polymorphism was a side effect of fixation of the deletion in KIR2DP1F by micro gene conversion.

Complex MHC Class I Gene Transcription Profiles and Their Functional Impact in Orangutans.

MHC haplotypes of humans and the African great ape species have one copy of the MHC-A, -B, and -C genes. In contrast, MHC haplotypes of orangutans, the Asian great ape species, exhibit variation in the number of gene copies. An in-depth analysis of the MHC class I gene repertoire in the two orangutan species, Pongo abelii and Pongo pygmaeus, is presented in this article. This analysis involved Sanger and next-generation sequencing methodologies, revealing diverse and complicated transcription profiles for orangutan MHC-A, -B, and -C. Thirty-five previously unreported MHC class I alleles are described. The data demonstrate that each orangutan MHC haplotype has one copy of the MHC-A gene, and that the MHC-B region has been subject to duplication, giving rise to at least three MHC-B genes. The MHC-B*03 and -B*08 lineages of alleles each account for a separate MHC-B gene. All MHC-B*08 allotypes have the C1-epitope motif recognized by killer cell Ig-like receptor. At least one other MHC-B gene is present, pointing to MHC-B alleles that are not B*03 or B*08. The MHC-C gene is present only on some haplotypes, and each MHC-C allotype has the C1-epitope. The transcription profiles demonstrate that MHC-A alleles are highly transcribed, whereas MHC-C alleles, when present, are transcribed at very low levels. The MHC-B alleles are transcribed to a variable extent and over a wide range. For those orangutan MHC class I allotypes that are detected by human monoclonal anti-HLA class I Abs, the level of cell-surface expression of proteins correlates with the level of transcription of the allele.

Co-evolution of the MHC class I and KIR gene families in rhesus macaques: ancestry and plasticity.

Researchers dealing with the human leukocyte antigen (HLA) class I and killer immunoglobulin receptor (KIR) multi-gene families in humans are often wary of the complex and seemingly different situation that is encountered regarding these gene families in Old World monkeys. For the sake of comparison, the well-defined and thoroughly studied situation in humans has been taken as a reference. In macaques, both the major histocompatibility complex class I and KIR gene families are plastic entities that have experienced various rounds of expansion, contraction, and subsequent recombination processes. As a consequence, haplotypes in macaques display substantial diversity with regard to gene copy number variation. Additionally, for both multi-gene families, differential levels of polymorphism (allelic variation), and expression are observed as well. A comparative genetic approach has allowed us to answer questions related to ancestry, to shed light on unique adaptations of the species' immune system, and to provide insights into the genetic events and selective pressures that have shaped the range of these gene families.

Unravelling the T-cell-mediated autoimmune attack on CNS myelin in a new primate EAE model induced with MOG34-56 peptide in incomplete adjuvant.

Induction of experimental autoimmune encephalomyelitis (EAE) has been documented in common marmosets using peptide 34-56 from human myelin/oligodendrocyte glycoprotein (MOG(34-56) ) in incomplete Freund's adjuvant (IFA). Here, we report that this EAE model is associated with widespread demyelination of grey and white matter. We performed an in-depth analysis of the specificity, MHC restriction and functions of the activated T cells in the model, which likely cause EAE in an autoantibody-independent manner. T-cell lines isolated from blood and lymphoid organs of animals immunized with MOG(34-56) displayed high production of IL-17A and specific lysis of MOG(34-56) -pulsed EBV B-lymphoblastoid cells as typical hallmarks. Cytotoxicity was directed at the epitope MOG(40-48) presented by the non-classical MHC class Ib allele Caja-E, which is orthologue to HLA-E and is expressed in non-inflamed brain. In vivo activated T cells identified by flow cytometry in cultures with MOG(34-56,) comprised CD4(+) CD56(+) and CD4(+) CD8(+) CD56(+) T cells. Furthermore, phenotypical analysis showed that CD4(+) CD8(+) CD56(+) T cells also expressed CD27, but CD16, CD45RO, CD28 and CCR7 were absent. These results show that, in the MOG34-56/IFA marmoset EAE model, a Caja-E-restricted population of autoreactive cytotoxic T cells plays a key role in the process of demyelination in the grey and white matter.

The extreme plasticity of killer cell Ig-like receptor (KIR) haplotypes differentiates rhesus macaques from humans.

NK cells are essential in shaping immune responses and play an important role during pregnancy and in controlling infections. Killer cell immunoglobulin-like receptors (KIRs) educate the NK cell and determine its state of activation. Our goal was to determine how the KIR repertoire of the rhesus macaque (Macaca mulatta) has been shaped during evolution. The presence or absence of 22 KIR gene groups was determined in 378 animals. Some unexpected observations were made in an outbred colony comprising animals of different origins. For instance, the KIR region appears to be highly plastic, and an unprecedented number of genotypes and haplotypes was observed. In contrast to humans, there is no distinction between group A and B haplotypes in the rhesus macaque, suggesting that different selective forces may be operative. Moreover, specific genes appear to be either present or absent in animals of different geographic origins. This extreme plasticity may have been propelled by co-evolution with the rhesus macaque MHC class I region, which shows signatures of expansion. The mosaic-like complexity of KIR genotypes as observed at the population level may represent an effective strategy for surviving epidemic infections.

Genomic plasticity of the MHC class I A region in rhesus macaques: extensive haplotype diversity at the population level as revealed by microsatellites.

The Mamu-A genes of the rhesus macaque show different degrees of polymorphism, transcription level variation, and differential haplotype distribution. Per haplotype, usually one "major" transcribed gene is present, A1 (A7), in various combinations with "minor" genes, A2 to A6. In silico analysis of the physical map of a heterozygous animal revealed the presence of similar Mamu-A regions consisting of four duplication units, but with dissimilar positions of the A1 genes on both haplotypes, and in combination with different minor genes. Two microsatellites, D6S2854 and D6S2859, have been selected as potential tools to characterize this complex region. Subsequent analysis of a large breeding colony resulted in the description of highly discriminative patterns, displaying copy number variation in concert with microsatellite repeat length differences. Sequencing and segregation analyses revealed that these patterns are unique for each Mamu-A haplotype. In animals of Indian, Burmese, and Chinese origin, 19, 15, or 9 haplotypes, respectively, could be defined, illustrating the occurrence of differential block duplications and subsequent rearrangements by recombination. The haplotypes can be assigned to 12 unique combinations of genes (region configurations). Although most configurations harbor two transcribed A genes, one or three genes per haplotype are also present. Additionally, haplotypes lacking an A1 gene or with an A1 duplication appear to exist. The presence of different transcribed A genes/alleles in monkeys from various origins may have an impact on differential disease susceptibilities. The high-throughput microsatellite technique will be a valuable tool in animal selection for diverse biomedical research projects.

The mosaic of KIR haplotypes in rhesus macaques.

To further refine and improve biomedical research in rhesus macaques, it is necessary to increase our knowledge concerning both the degree of allelic variation (polymorphism) and diversity (gene copy number variation) in the killer cell immunoglobulin-like receptor (KIR) gene cluster. Pedigreed animals in particular should be studied, as segregation data will provide clues to the linkage of particular KIR genes/alleles segregating on a haplotype and to its gene content as well. A dual strategy allowed us to screen the presence and absence of genes and the corresponding transcripts, as well as to track differences in transcription levels. On the basis of this approach, 14 diverse KIR haplotypes have been described. These haplotypes consist of multiple inhibitory and activating Mamu-KIR genes, and any gene present on one haplotype may be absent on another. This suggests that the cost of accelerated evolution by recombination may be the loss of certain framework genes on a haplotype.

Evidence for balancing selection acting on KIR2DL4 genotypes in rhesus macaques of Indian origin.

The interaction of killer-cell immunoglobulin-like receptors (KIR) and their respective major histocompatibility complex (MHC) ligands can alter the activation state of the natural killer (NK) cell. In both humans and rhesus macaques, particular types of non-classical MHC class I molecules are predominantly expressed on the trophoblast. In humans, human leukocyte antigen G has been demonstrated to act as a ligand for KIR2DL4, present on all NK cells, whereas Mamu-AG may execute a similar function in rhesus macaques. During primate evolution, orthologues of KIR2DL4 appear to have been highly conserved, suggesting strong purifying selection. A cohort of 112 related and unrelated rhesus macaques of mostly Indian origin were selected to study their KIR2DL4 genes for the occurrence of polymorphism. Comparison of the proximal region provided evidence for strong conservative selection acting on the exons encoding the Ig domains. As is found in humans, in the Indian rhesus macaque population, two different KIR2DL4 entities are encountered, which differ for their intra-cellular signalling motifs. One genotype contains a complex mutation in the distal region of exon 9, which negates a serine/threonine kinase site. Furthermore, both allelic entities are present in a distribution, which suggests that balancing selection is operating on these two distinct forms of KIR2DL4.

A splice site mutation converts an inhibitory killer cell Ig-like receptor into an activating one.

The killer cell Ig-like receptor (KIR) 3DH protein in rhesus macaques (Macaca mulatta) is thought to be an activating one because it contains a charged arginine in its transmembrane domain and has a truncated cytoplasmic domain. MmKIR3DH has thus far been characterized by an analysis of cDNA. Its presence and polymorphism has been further investigated by examining mRNA transcripts and genomic sequences in families. Multiple copies of MmKIR3DH are present per animal, suggesting that the gene has been duplicated on some haplotypes. All transcripts are truncated and lack exon 8. Investigation of the gene itself shows that exon 8 is present, intact, and homologous to MmKIR2DL4. However, there is a mutation in the donor splice site of intron 8, which is absent in MmKIR2DL4 genomic sequences. This mutation introduces a frameshift, subsequently resulting in a premature stopcodon. To further verify this mutation, a cohort of unrelated animals from different geographical locations was examined, and both exon 8 and the splice site mutation were seen to be present in their MmKIR3DH genes. The data suggest that the splice site mutation causes the truncation of the MmKIR3DH transcript and the subsequent loss of its inhibitory motifs further downstream. Loss of inhibitory potential through different mutations is observed in other primate species as well, suggesting convergent evolution; however, this is the first report to document that a mutation in an intron produces a similar effect.

Site-directed mutagenesis of the Nidovirus replicative endoribonuclease NendoU exerts pleiotropic effects on the arterivirus life cycle.

The highly conserved NendoU replicative domain of nidoviruses (arteriviruses, coronaviruses, and roniviruses) belongs to a small protein family whose cellular branch is prototyped by XendoU, a Xenopus laevis endoribonuclease involved in nucleolar RNA processing. Recently, sequence-specific in vitro endoribonuclease activity was demonstrated for the NendoU-containing nonstructural protein (nsp) 15 of several coronaviruses. To investigate the biological role of this novel enzymatic activity, we have characterized a comprehensive set of arterivirus NendoU mutants. Deleting parts of the NendoU domain from nsp11 of equine arteritis virus was lethal. Site-directed mutagenesis of conserved residues exerted pleiotropic effects. In a first-cycle analysis, replacement of two conserved Asp residues in the C-terminal part of NendoU rendered viral RNA synthesis and virus production undetectable. In contrast, mutagenesis of other conserved residues, including two putative catalytic His residues that are absolutely conserved in NendoU and cellular homologs, produced viable mutants displaying reduced plaque sizes (20 to 80% reduction) and reduced yields of infectious progeny of up to 5 log units. A more detailed analysis of these mutants revealed a moderate reduction in RNA synthesis, with subgenomic RNA synthesis consistently being more strongly affected than genome replication. Our data suggest that the arterivirus nsp11 is a multifunctional protein with a key role in viral RNA synthesis and additional functions in the viral life cycle that are as yet poorly defined.