Jeroen H. Blokhuis

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.

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 (MOG34‐56) in incomplete Freunds 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 MOG34–56 displayed high production of IL‐17A and specific lysis of MOG34–56‐pulsed EBV B‐lymphoblastoid cells as typical hallmarks. Cytotoxicity was directed at the epitope MOG40–48 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 MOG34–56, 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.

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.

Class I HLA haplotypes form two schools that educate NK cells in different ways

Individuals heterozygous for HLA haplotypes have increased NK cell diversity because of distinct education pathways. NK cell immunity gets schooled Population-level genetic analysis can give us clues as to which factors guide immune evolution. Now, Horowitz et al. have applied this analysis to polymorphisms in HLA that affect NK cell education. They find that the HLA-B haplotype −21M that delivers functional peptides to the conserved CD94/NKG2A receptor rarely encodes ligands for the more diverse killer cell immunoglobulin-like receptors (KIRs), in contrast to the haplotype −21T, which does not deliver functional peptides. Individuals homo- or heterozygous for −21M are more likely to have more diverse CD94/NKG2A+ NK cells, suggesting that these HLA haplotypes may have specialized to either the KIR or CD94/NKG2A school through complementary coevolution. Natural killer (NK) cells are lymphocytes that have vital functions in innate and adaptive immunity, as well as placental reproduction. Polymorphic human leukocyte antigen (HLA) class I educates NK cells through interactions with killer cell immunoglobulin-like receptors (KIRs) and by supplying peptides that bind HLA-E to form ligands for CD94/NKG2A receptors on NK cells. HLA-B dimorphism in the leader peptide modulates this latter function: −21methionine (−21M) delivers functional peptides, but −21threonine (−21T) does not. Genetic analysis of human populations worldwide showed that haplotypes with −21M HLA-B rarely encoded the KIR ligands Bw4+HLA-B and C2+HLA-C. Thus, there are two fundamental forms of HLA haplotype: one preferentially supplying CD94/NKG2A ligands and the other preferentially supplying KIR ligands. This −21 HLA-B dimorphism divides the human population into three groups: M/M, M/T, and T/T. Mass cytometry and assays of immune function demonstrated that M/M and M/T individuals have CD94/NKG2A+ NK cells that are better educated, phenotypically more diverse, and functionally more potent than those in T/T individuals. The KIR school of NK cell education evolved in the context of the much older CD94/NKG2A school, and this complementary coevolution may have facilitated the specialization of HLA haplotypes toward one school or the other.Natural killer (NK) cells are lymphocytes that have vital functions in innate and adaptive immunity, as well as placental reproduction. Polymorphic human leukocyte antigen (HLA) class I educates NK cells through interactions with killer cell immunoglobulin-like receptors (KIRs) and by supplying peptides that bind HLA-E to form ligands for CD94/NKG2A receptors on NK cells. HLA-B dimorphism in the leader peptide modulates this latter function: -21methionine (-21M) delivers functional peptides, but -21threonine (-21T) does not. Genetic analysis of human populations worldwide showed that haplotypes with -21M HLA-B rarely encoded the KIR ligands Bw4+HLA-B and C2+HLA-C. Thus, there are two fundamental forms of HLA haplotype: one preferentially supplying CD94/NKG2A ligands and the other preferentially supplying KIR ligands. This -21 HLA-B dimorphism divides the human population into three groups: M/M, M/T, and T/T. Mass cytometry and assays of immune function demonstrated that M/M and M/T individuals have CD94/NKG2A+ NK cells that are better educated, phenotypically more diverse, and functionally more potent than those in T/T individuals. The KIR school of NK cell education evolved in the context of the much older CD94/NKG2A school, and this complementary coevolution may have facilitated the specialization of HLA haplotypes toward one school or the other.

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.

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.

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.

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.

The production of KIR-Fc fusion proteins and their use in a multiplex HLA class I binding assay.

Soluble recombinant proteins that comprise the extracellular part of a surface expressed receptor attached to the Fc region of an IgG antibody have facilitated the determination of ligand specificity for an array of immune system receptors. Among such receptors is the family of killer cell immunoglobulin-like receptors (KIR) that recognize HLA class I ligands. These receptors, expressed on natural killer (NK) cells and T cells, play important roles in both immune defense and placental development in early pregnancy. Here we describe a method for the production of two domain KIR-Fc fusion proteins using baculovirus infected insect cells. This method is more scalable than traditional mammalian cell expression systems and produces efficiently folded proteins that carry posttranslational modifications found in native KIR. We also describe a multiplex binding assay using the Luminex platform that determines the avidity and specificity of two domain KIR-Fc for a panel of microbeads, each coated with one of 97 HLA class I allotypes. This assay is simple to perform, and represents a major improvement over the assays used previously, which were limited in the number of KIR and HLA class I combinations that could be assayed at any one time. The results obtained from this assay can be used to predict the response of NK cell and T cells when their KIR recognize HLA class I.

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

Summary 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.