Honors & Awards

  • Dean's Postdoctoral Fellowship, Stanford School of Medicine (2013)

Professional Education

  • Master of Science, Leiden University (2002)
  • Doctor of Philosophy, Universiteit Utrecht (2011)

Stanford Advisors


All Publications

  • The production of KIR-Fc fusion proteins and their use in a multiplex HLA class I binding assay JOURNAL OF IMMUNOLOGICAL METHODS Hilton, H. G., Moesta, A. K., Guethlein, L. A., Blokhuis, J., Parham, P., Norman, P. J. 2015; 425: 79-87


    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.

    View details for DOI 10.1016/j.jim.2015.06.012

    View details for Web of Science ID 000363819600011

  • Co-evolution of the MHC class I and KIR gene families in rhesus macaques: ancestry and plasticity. Immunological reviews de Groot, N. G., Blokhuis, J. H., Otting, N., Doxiadis, G. G., Bontrop, R. E. 2015; 267 (1): 228-45


    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.

    View details for DOI 10.1111/imr.12313

    View details for PubMedID 26284481

  • Unravelling the T-cell-mediated autoimmune attack on CNS myelin in a new primate EAE model induced with MOG34-56 peptide in incomplete adjuvant EUROPEAN JOURNAL OF IMMUNOLOGY Jagessar, S. A., Heijmans, N., Blezer, E. L., Bauer, J., Blokhuis, J. H., Wubben, J. A., Drijfhout, J. W., van den Elsen, P. J., Laman, J. D., 't Hart, B. A. 2012; 42 (1): 217-227


    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.

    View details for DOI 10.1002/eji.201141863

    View details for Web of Science ID 000298596600024

    View details for PubMedID 21928277

  • The extreme plasticity of killer cell Ig-like receptor (KIR) haplotypes differentiates rhesus macaques from humans EUROPEAN JOURNAL OF IMMUNOLOGY Blokhuis, J. H., van der Wiel, M. K., Doxiadis, G. G., Bontrop, R. E. 2011; 41 (9): 2719-2728


    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.

    View details for DOI 10.1002/eji.201141621

    View details for Web of Science ID 000295260800037

    View details for PubMedID 21710469

  • Genomic plasticity of the MHC class I A region in rhesus macaques: extensive haplotype diversity at the population level as revealed by microsatellites IMMUNOGENETICS Doxiadis, G. G., de Groot, N., Otting, N., Blokhuis, J. H., Bontrop, R. E. 2011; 63 (2): 73-83


    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.

    View details for DOI 10.1007/s00251-010-0486-4

    View details for Web of Science ID 000287140500002

    View details for PubMedID 20949353

  • The mosaic of KIR haplotypes in rhesus macaques IMMUNOGENETICS Blokhuis, J. H., van der Wiel, M. K., Doxiadis, G. G., Bontrop, R. E. 2010; 62 (5): 295-306


    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.

    View details for DOI 10.1007/s00251-010-0434-3

    View details for Web of Science ID 000277024800004

    View details for PubMedID 20204612

  • Evidence for balancing selection acting on KIR2DL4 genotypes in rhesus macaques of Indian origin IMMUNOGENETICS Blokhuis, J. H., van der Wiel, M. K., Doxiadis, G. G., Bontrop, R. E. 2009; 61 (7): 503-512


    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.

    View details for DOI 10.1007/s00251-009-0379-6

    View details for Web of Science ID 000267783000003

    View details for PubMedID 19506858

  • A splice site mutation converts an inhibitory killer cell Ig-like receptor into an activating one MOLECULAR IMMUNOLOGY Blokhuis, J. H., Doxiadis, G. G., Bontrop, R. E. 2009; 46 (4): 640-648


    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.

    View details for DOI 10.1016/j.molimm.2008.08.270

    View details for Web of Science ID 000263429500016

    View details for PubMedID 19019442

  • Site-directed mutagenesis of the nidovirus replicative endoribonuclease NendoU exerts pleiotropic effects on the arterivirus life cycle JOURNAL OF VIROLOGY Posthuma, C. C., Nedialkova, D. D., Zevenhoven-Dobbe, J. C., Blokhuis, J. H., Gorbalenya, A. E., Snijder, E. J. 2006; 80 (4): 1653-1661


    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.

    View details for DOI 10.1128/JVI.80.4.1653-1661.2006

    View details for Web of Science ID 000235248500005

    View details for PubMedID 16439522