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1.  The MICA-129Met/Val dimorphism affects plasma membrane expression and shedding of the NKG2D ligand MICA 
Immunogenetics  2015;68:109-123.
The MHC class I chain-related molecule A (MICA) is a ligand for the activating natural killer (NK) cell receptor NKG2D. A polymorphism causing a valine to methionine exchange at position 129 affects binding to NKG2D, cytotoxicity, interferon-γ release by NK cells and activation of CD8+ T cells. It is known that tumors can escape NKG2D-mediated immune surveillance by proteolytic shedding of MICA. Therefore, we investigated whether this polymorphism affects plasma membrane expression (pmMICA) and shedding of MICA. Expression of pmMICA was higher in a panel of tumor (n = 16, P = 0.0699) and melanoma cell lines (n = 13, P = 0.0429) carrying the MICA-129Val/Val genotype. MICA-129Val homozygous melanoma cell lines released more soluble MICA (sMICA) by shedding (P = 0.0015). MICA-129Met or MICA-129Val isoforms differing only in this amino acid were expressed in the MICA-negative melanoma cell line Malme, and clones with similar pmMICA expression intensity were selected. The MICA-129Met clones released more sMICA (P = 0.0006), and a higher proportion of the MICA-129Met than the MICA-129Val variant was retained in intracellular compartments (P = 0.0199). The MICA-129Met clones also expressed more MICA messenger RNA (P = 0.0047). The latter phenotype was also observed in mouse L cells transfected with the MICA expression constructs (P = 0.0212). In conclusion, the MICA-129Met/Val dimorphism affects the expression density of MICA on the plasma membrane. More of the MICA-129Met variants were retained intracellularly. If expressed at the cell surface, the MICA-129Met isoform was more susceptible to shedding. Both processes appear to limit the cell surface expression of MICA-129Met variants that have a high binding avidity to NKG2D.
doi:10.1007/s00251-015-0884-8
PMCID: PMC4728179  PMID: 26585323
Single nucleotide polymorphism; Major histocompatibility complex (MHC) class I chain-related molecules A (MICA); Plasma membrane expression; Proteolytic shedding; Tumor cells
2.  The MICA-129 dimorphism affects NKG2D signaling and outcome of hematopoietic stem cell transplantation 
EMBO Molecular Medicine  2015;7(11):1480-1502.
The MHC class I chain-related molecule A (MICA) is a highly polymorphic ligand for the activating natural killer (NK)-cell receptor NKG2D. A single nucleotide polymorphism causes a valine to methionine exchange at position 129. Presence of a MICA-129Met allele in patients (n = 452) undergoing hematopoietic stem cell transplantation (HSCT) increased the chance of overall survival (hazard ratio [HR] = 0.77, P = 0.0445) and reduced the risk to die due to acute graft-versus-host disease (aGVHD) (odds ratio [OR] = 0.57, P = 0.0400) although homozygous carriers had an increased risk to experience this complication (OR = 1.92, P = 0.0371). Overall survival of MICA-129Val/Val genotype carriers was improved when treated with anti-thymocyte globulin (HR = 0.54, P = 0.0166). Functionally, the MICA-129Met isoform was characterized by stronger NKG2D signaling, triggering more NK-cell cytotoxicity and interferon-γ release, and faster co-stimulation of CD8+ T cells. The MICA-129Met variant also induced a faster and stronger down-regulation of NKG2D on NK and CD8+ T cells than the MICA-129Val isoform. The reduced cell surface expression of NKG2D in response to engagement by MICA-129Met variants appeared to reduce the severity of aGVHD.
doi:10.15252/emmm.201505246
PMCID: PMC4644379  PMID: 26483398
cytotoxic T cells; graft-versus-host disease; NK-cell receptors; NK cells; single nucleotide polymorphism
3.  Targeting α4β7 integrin reduces mucosal transmission of SIV and protects GALT from infection 
Nature medicine  2014;20(12):1397-1400.
α4β7 integrin expressing CD4+ T cells preferentially traffic to gut-associated lymphoid tissues (GALT) and play a key role in HIV/SIV pathogenesis. The administration of an anti-α4β7 monoclonal antibody during acute infection protects macaques from transmission following repeated low-dose intra-vaginal challenges with SIVmac251. In treated animals that became infected the GALT was significantly protected and CD4+ T–cell numbers were maintained. Thus, targeting α4β7 reduces mucosal transmission of SIV in macaques.
doi:10.1038/nm.3715
PMCID: PMC4257865  PMID: 25419708
SIV; α4β7 monoclonal antibody; GI pathology; Repeated Low Dose challenge model; and rhesus macaques; mucosal transmission
4.  Human Induced Pluripotent Stem Cells Are Targets for Allogeneic and Autologous Natural Killer (NK) Cells and Killing Is Partly Mediated by the Activating NK Receptor DNAM-1 
PLoS ONE  2015;10(5):e0125544.
Human induced pluripotent stem cells (hiPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, iPSC-derived grafts are at risk of giving rise to teratomas in the host, if residuals of tumorigenic cells are not rejected by the recipient. We have analyzed the susceptibility of hiPSC lines to allogeneic and autologous natural killer (NK) cells. IL-2-activated, in contrast to resting NK cells killed hiPSC lines efficiently (P=1.69x10-39). Notably, the specific lysis of the individual hiPSC lines by IL-2-activated NK cells was significantly different (P=1.72x10-6) and ranged between 46 % and 64 % in 51Cr-release assays when compared to K562 cells. The hiPSC lines were killed by both allogeneic and autologous NK cells although autologous NK cells were less efficient (P=8.63x10-6). Killing was partly dependent on the activating NK receptor DNAM-1 (P=8.22x10-7). The DNAM-1 ligands CD112 and CD155 as well as the NKG2D ligands MICA and MICB were expressed on the hiPSC lines. Low amounts of human leukocyte antigen (HLA) class I proteins, which serve as ligands for inhibitory and activating NK receptors were also detected. Thus, the susceptibility to NK cell killing appears to constitute a common feature of hiPSCs. Therefore, NK cells might reduce the risk of teratoma formation even after autologous transplantations of pluripotent stem cell-derived grafts that contain traces of pluripotent cells.
doi:10.1371/journal.pone.0125544
PMCID: PMC4423859  PMID: 25950680
5.  Gibbon genome and the fast karyotype evolution of small apes 
Carbone, Lucia | Harris, R. Alan | Gnerre, Sante | Veeramah, Krishna R. | Lorente-Galdos, Belen | Huddleston, John | Meyer, Thomas J. | Herrero, Javier | Roos, Christian | Aken, Bronwen | Anaclerio, Fabio | Archidiacono, Nicoletta | Baker, Carl | Barrell, Daniel | Batzer, Mark A. | Beal, Kathryn | Blancher, Antoine | Bohrson, Craig L. | Brameier, Markus | Campbell, Michael S. | Capozzi, Oronzo | Casola, Claudio | Chiatante, Giorgia | Cree, Andrew | Damert, Annette | de Jong, Pieter J. | Dumas, Laura | Fernandez-Callejo, Marcos | Flicek, Paul | Fuchs, Nina V. | Gut, Marta | Gut, Ivo | Hahn, Matthew W. | Hernandez-Rodriguez, Jessica | Hillier, LaDeana W. | Hubley, Robert | Ianc, Bianca | Izsvák, Zsuzsanna | Jablonski, Nina G. | Johnstone, Laurel M. | Karimpour-Fard, Anis | Konkel, Miriam K. | Kostka, Dennis | Lazar, Nathan H. | Lee, Sandra L. | Lewis, Lora R. | Liu, Yue | Locke, Devin P. | Mallick, Swapan | Mendez, Fernando L. | Muffato, Matthieu | Nazareth, Lynne V. | Nevonen, Kimberly A. | O,Bleness, Majesta | Ochis, Cornelia | Odom, Duncan T. | Pollard, Katherine S. | Quilez, Javier | Reich, David | Rocchi, Mariano | Schumann, Gerald G. | Searle, Stephen | Sikela, James M. | Skollar, Gabriella | Smit, Arian | Sonmez, Kemal | Hallers, Boudewijn ten | Terhune, Elizabeth | Thomas, Gregg W.C. | Ullmer, Brygg | Ventura, Mario | Walker, Jerilyn A. | Wall, Jeffrey D. | Walter, Lutz | Ward, Michelle C. | Wheelan, Sarah J. | Whelan, Christopher W. | White, Simon | Wilhelm, Larry J. | Woerner, August E. | Yandell, Mark | Zhu, Baoli | Hammer, Michael F. | Marques-Bonet, Tomas | Eichler, Evan E. | Fulton, Lucinda | Fronick, Catrina | Muzny, Donna M. | Warren, Wesley C. | Worley, Kim C. | Rogers, Jeffrey | Wilson, Richard K. | Gibbs, Richard A.
Nature  2014;513(7517):195-201.
Gibbons are small arboreal apes that display an accelerated rate of evolutionary chromosomal rearrangement and occupy a key node in the primate phylogeny between Old World monkeys and great apes. Here we present the assembly and analysis of a northern white-cheeked gibbon (Nomascus leucogenys) genome. We describe the propensity for a gibbon-specific retrotransposon (LAVA) to insert into chromosome segregation genes and alter transcription by providing a premature termination site, suggesting a possible molecular mechanism for the genome plasticity of the gibbon lineage. We further show that the gibbon genera (Nomascus, Hylobates, Hoolock and Symphalangus) experienced a near-instantaneous radiation ~5 million years ago, coincident with major geographical changes in Southeast Asia that caused cycles of habitat compression and expansion. Finally, we identify signatures of positive selection in genes important for forelimb development (TBX5) and connective tissues (COL1A1) that may have been involved in the adaptation of gibbons to their arboreal habitat.
doi:10.1038/nature13679
PMCID: PMC4249732  PMID: 25209798
6.  MHC and KIR Polymorphisms in Rhesus Macaque SIV Infection 
Natural killer lymphocytes are essentially involved as the first line of defense against agents such as viruses and malignant cells. The activity of these cells is regulated via interaction of specific and diverse killer cell immunoglobulin-like receptors (KIR) with the highly polymorphic cognate MHC class I proteins on target cells. Genetic variability of both KIR and MHC-I ligands has been shown to be associated with resistance to many diseases, including infection with the immunodeficiency virus. Disease course and progression to AIDS after infection with human immunodeficiency virus-1 (HIV-1) is essentially influenced by the presence of the stimulatory KIR3DS1 receptor in combination with HLA-Bw4. Knowledge of such genetic interactions that contribute to not only disease resistance but also susceptibility are just as important. Such combined genetic factors were recently reported in the rhesus macaque AIDS model. Here, we review the rhesus macaque MHC class I and KIR gene systems and the role of their polymorphisms in the SIV infection model.
doi:10.3389/fimmu.2015.00540
PMCID: PMC4617107  PMID: 26557119
KIR; MHC class I; SIV infection; genetic association; rhesus macaque
7.  Differentiated adaptive evolution, episodic relaxation of selective constraints, and pseudogenization of umami and sweet taste genes TAS1Rs in catarrhine primates 
Frontiers in Zoology  2014;11:79.
Background
Umami and sweet tastes are two important basic taste perceptions that allow animals to recognize diets with nutritious carbohydrates and proteins, respectively. Until recently, analyses of umami and sweet taste were performed on various domestic and wild animals. While most of these studies focused on the pseudogenization of taste genes, which occur mostly in carnivores and species with absolute feeding specialization, omnivores and herbivores were more or less neglected. Catarrhine primates are a group of herbivorous animals (feeding mostly on plants) with significant divergence in dietary preference, especially the specialized folivorous Colobinae. Here, we conducted the most comprehensive investigation to date of selection pressure on sweet and umami taste genes (TAS1Rs) in catarrhine primates to test whether specific adaptive evolution occurred during their diversification, in association with particular plant diets.
Results
We documented significant relaxation of selective constraints on sweet taste gene TAS1R2 in the ancestral branch of Colobinae, which might correlate with their unique ingestion and digestion of leaves. Additionally, we identified positive selection acting on Cercopithecidae lineages for the umami taste gene TAS1R1, on the Cercopithecinae and extant Colobinae and Hylobatidae lineages for TAS1R2, and on Macaca lineages for TAS1R3. Our research further identified several site mutations in Cercopithecidae, Colobinae and Pygathrix, which were detected by previous studies altering the sensitivity of receptors. The positively selected sites were located mostly on the extra-cellular region of TAS1Rs. Among these positively selected sites, two vital sites for TAS1R1 and four vital sites for TAS1R2 in extra-cellular region were identified as being responsible for the binding of certain sweet and umami taste molecules through molecular modelling and docking.
Conclusions
Our results suggest that episodic and differentiated adaptive evolution of TAS1Rs pervasively occurred in catarrhine primates, most concentrated upon the extra-cellular region of TAS1Rs.
Electronic supplementary material
The online version of this article (doi:10.1186/s12983-014-0079-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12983-014-0079-4
PMCID: PMC4226867  PMID: 25389445
Catarrhine primates; TAS1Rs; Adaptive evolution; Positive selection; Episodic relaxation of selective constraints; Pseudogenization
8.  In Vivo Administration of a JAK3 Inhibitor during Acute SIV Infection Leads to Significant Increases in Viral Load during Chronic Infection 
PLoS Pathogens  2014;10(3):e1003929.
The studies reported herein are the first to document the effect of the in vivo administration of a JAK3 inhibitor for defining the potential role of NK cells during acute SIV infection of a group of 15 rhesus macaques (RM). An additional group of 16 MHC/KIR typed RM was included as controls. The previously optimized in vivo dose regimen (20 mg/kg daily for 35 days) led to a marked depletion of each of the major NK cell subsets both in the blood and gastro-intestinal tissues (GIT) during acute infection. While such depletion had no detectable effects on plasma viral loads during acute infection, there was a significant sustained increase in plasma viral loads during chronic infection. While the potential mechanisms that lead to such increased plasma viral loads during chronic infection remain unclear, several correlates were documented. Thus, during acute infection, the administration of the JAK3 inhibitor besides depleting all NK cell subsets also decreased some CD8+ T cells and inhibited the mobilization of the plasmacytoid dendritic cells in the blood and their localization to the GIT. Of interest is the finding that the administration of the JAK3 inhibitor during acute infection also resulted in the sustained maintenance during chronic infection of a high number of naïve and central memory CD4+ T cells, increases in B cells in the blood, but decreases in the frequencies and function of NKG2a+ NK cells within the GIT and blood, respectively. These data identify a unique role for JAK3 inhibitor sensitive cells, that includes NK cells during acute infection that in concert lead to high viral loads in SIV infected RM during chronic infection without affecting detectable changes in antiviral humoral/cellular responses. Identifying the precise mechanisms by which JAK3 sensitive cells exert their influence is critical with important implications for vaccine design against lentiviruses.
Author Summary
In efforts to define the potential role of innate immune effector mechanisms in influencing the course of SIV infection during the acute infection period, our lab utilized the in vivo daily administration of 20 mg/kg orally of a compound called Tofacitinib (a Janus kinase 3 inhibitor) to a group of 15 rhesus macaques starting at day −6 and until day 28 post intravenous SIVmac239 infection. An additional group of 16 similarly SIV infected rhesus macaques served as a placebo control. This drug targets the JAK/STAT pathway that is utilized by cells including the NK cell lineage, a major cell of the innate immune system. The dosage utilized was based on extensive previous PK studies that resulted in a marked depletion of the NK cells. Of interest while such drug administration had no effect on plasma viral loads during acute infection, such drug administration led to significant increases in plasma and gastro-intestinal tissues (GIT) viral loads during chronic infection. A series of phenotypic/functional studies were performed to determine the mechanisms for this delayed effect and the correlates identified. These data are the first to document the effect of JAK-3 inhibitor during acute SIV infection with implications for HIV vaccine design.
doi:10.1371/journal.ppat.1003929
PMCID: PMC3946395  PMID: 24603870
9.  Human-specific epigenetic variation in the immunological Leukotriene B4 Receptor (LTB4R/BLT1) implicated in common inflammatory diseases 
Genome Medicine  2014;6(3):19.
Background
Common human diseases are caused by the complex interplay of genetic susceptibility as well as environmental factors. Due to the environment’s influence on the epigenome, and therefore genome function, as well as conversely the genome’s facilitative effect on the epigenome, analysis of this level of regulation may increase our knowledge of disease pathogenesis.
Methods
In order to identify human-specific epigenetic influences, we have performed a novel genome-wide DNA methylation analysis comparing human, chimpanzee and rhesus macaque.
Results
We have identified that the immunological Leukotriene B4 receptor (LTB4R, BLT1 receptor) is the most epigenetically divergent human gene in peripheral blood in comparison with other primates. This difference is due to the co-ordinated active state of human-specific hypomethylation in the promoter and human-specific increased gene body methylation. This gene is significant in innate immunity and the LTB4/LTB4R pathway is involved in the pathogenesis of the spectrum of human inflammatory diseases. This finding was confirmed by additional neutrophil-only DNA methylome and lymphoblastoid H3K4me3 chromatin comparative data. Additionally we show through functional analysis that this receptor has increased expression and a higher response to the LTB4 ligand in human versus rhesus macaque peripheral blood mononuclear cells. Genome-wide we also find human species-specific differentially methylated regions (human s-DMRs) are more prevalent in CpG island shores than within the islands themselves, and within the latter are associated with the CTCF motif.
Conclusions
This result further emphasises the exclusive nature of the human immunological system, its divergent adaptation even from very closely related primates, and the power of comparative epigenomics to identify and understand human uniqueness.
doi:10.1186/gm536
PMCID: PMC4062055  PMID: 24598577
10.  Progression to AIDS in SIV-Infected Rhesus Macaques is Associated with Distinct KIR and MHC class I Polymorphisms and NK Cell Dysfunction 
Killer cell immunoglobulin-like receptors (KIR) regulate the activity of natural killer (NK) cells and have been shown to be associated with susceptibility to a number of human infectious diseases. Here, we analyzed NK cell function and genetic associations in a cohort of 52 rhesus macaques experimentally infected with SIVmac and subsequently stratified into high viral load (HVL) and low viral load (LVL) plasma viral loads at set point. This stratification coincided with fast (HVL) and slow (LVL) disease progression indicated by the disease course and critical clinical parameters including CD4+ T cell counts. HVL animals revealed sustained proliferation of NK cells but distinct loss of peripheral blood NK cell numbers and lytic function. Genetic analyses revealed that KIR genes 3DL05, 3DS05, and 3DL10 as well as 3DSW08, 3DLW03, and 3DSW09 are correlated, most likely due to underlying haplotypes. SIV-infection outcome associated with presence of transcripts for two inhibitory KIR genes (KIR3DL02, KIR3DL10) and three activating KIR genes (KIR3DSW08, KIR3DS02, KIR3DS05). Presence of KIR3DL02 and KIR3DSW08 was associated with LVL outcome, whereas presence of KIR3DS02 was associated with HVL outcome. Furthermore, we identified epistasis between KIR and MHC class I alleles as the transcript presence of the correlated genes KIR3DL05, KIR3DS05, and KIR3DL10 increased HVL risk when Mamu-B*012 transcripts were also present or when Mamu-A1*001 transcripts were absent. These genetic associations were mirrored by changes in the numbers, the level of proliferation, and lytic capabilities of NK cells as well as overall survival time and gastro-intestinal tissue viral load.
doi:10.3389/fimmu.2014.00600
PMCID: PMC4246914  PMID: 25506344
virus infection; killer cell immunoglobulin-like receptors; MHC class I ligands; genetic polymorphism; epistasis; rhesus macaque SIV infection model; AIDS
12.  Rhesus macaque KIR bind human MHC class I with broad specificity and recognize HLA-C more effectively than HLA-A and HLA-B 
Immunogenetics  2011;63(9):577-585.
Human killer cell immunoglobulin-like receptors (KIR) recognize A3/11, Bw4, C1 and C2 epitopes carried by mutually exclusive subsets of HLA-A, B, and C allotypes. Chimpanzee and orangutan have counterparts to HLA-A, B, and C, and KIR that recognize the A3/11, Bw4, C1 and C2 epitopes, either individually or in combination. Because rhesus macaque has counterparts of HLA-A and B, but not HLA-C, we expected that rhesus KIR would better recognize HLA-A and B, than HLA-C. Comparison of the interactions of nine rhesus KIR3D with 95 HLA isoforms, showed the KIR have broad specificity for HLA-A, B, and C, but vary in avidity. Considering both the strength and breadth of reaction, HLA-C was the major target for rhesus KIR, followed by HLA-B, then HLA-A. Strong reactions with HLA-A were restricted to the minority of allotypes carrying the Bw4 epitope, whereas strong reactions with HLA-B partitioned between allotypes having and lacking Bw4. Contrasting to HLA-A and B, every HLA-C allotype bound to the nine rhesus KIR. Sequence comparison of high- and low-binding HLA allotypes revealed the importance of polymorphism in the helix of the α1 domain and the peptide-binding pockets. At peptide position 9, nonpolar residues favor binding to rhesus KIR, whereas charged residues do not. Contrary to expectation, rhesus KIR bind more effectively to HLA-C, than to HLA-A and B. This property is consistent with MHC-C having evolved in hominids to be a generally superior ligand for KIR than MHC-A and MHC-B.
doi:10.1007/s00251-011-0535-7
PMCID: PMC3718024  PMID: 21614583
KIR receptors; MHC; rhesus macaque; NK cells
13.  Expression Patterns of Killer Cell Immunoglobulin-Like Receptors (KIR) of NK-Cell and T-Cell Subsets in Old World Monkeys 
PLoS ONE  2013;8(5):e64936.
The expression of killer cell immunoglobulin-like receptors (KIR) on lymphocytes of rhesus macaques and other Old World monkeys was unknown so far. We used our recently established monoclonal anti-rhesus macaque KIR antibodies in multicolour flow cytometry for phenotypic characterization of KIR protein expression on natural killer (NK) cells and T cell subsets of rhesus macaques, cynomolgus macaques, hamadryas baboons, and African green monkeys. Similar to human KIR, we found clonal expression patterns of KIR on NK and T cell subsets in rhesus macaques and differences between individuals using pan-KIR3D antibody 1C7 and antibodies specific for single KIR. Similar results were obtained with lymphocytes from the other studied species. Notably, African green monkeys show only a low frequency of KIR3D expressed on CD8+ αβT cells. Contrasting human NK cells are KIR-positive CD56bright NK cells and frequencies of KIR-expressing NK cells that are independent of the presence of their cognate MHC class I ligands in rhesus macaques. Interestingly, the frequency of KIR-expressing cells and the expression strength of KIR3D are correlated in γδ T cells of rhesus macaques and CD8+ αβT cells of baboons.
doi:10.1371/journal.pone.0064936
PMCID: PMC3661512  PMID: 23717676
14.  Human-specific CpG “beacons” identify loci associated with human-specific traits and disease 
Epigenetics  2012;7(10):1188-1199.
Regulatory change has long been hypothesized to drive the delineation of the human phenotype from other closely related primates. Here we provide evidence that CpG dinucleotides play a special role in this process. CpGs enable epigenome variability via DNA methylation, and this epigenetic mark functions as a regulatory mechanism. Therefore, species-specific CpGs may influence species-specific regulation. We report non-polymorphic species-specific CpG dinucleotides (termed “CpG beacons”) as a distinct genomic feature associated with CpG island (CGI) evolution, human traits and disease. Using an inter-primate comparison, we identified 21 extreme CpG beacon clusters (≥ 20/kb peaks, empirical p < 1.0 × 10−3) in humans, which include associations with four monogenic developmental and neurological disease related genes (Benjamini-Hochberg corrected p = 6.03 × 10−3). We also demonstrate that beacon-mediated CpG density gain in CGIs correlates with reduced methylation in these species in orthologous CGIs over time, via human, chimpanzee and macaque MeDIP-seq. Therefore mapping into both the genomic and epigenomic space the identified CpG beacon clusters define points of intersection where a substantial two-way interaction between genetic sequence and epigenetic state has occurred. Taken together, our data support a model for CpG beacons to contribute to CGI evolution from genesis to tissue-specific to constitutively active CGIs.
doi:10.4161/epi.22127
PMCID: PMC3469460  PMID: 22968434
epigenetics; epigenomics; CpG islands; gene regulation; evolution; human disease
15.  Characterisation of mouse monoclonal antibodies against rhesus macaque killer immunoglobulin-like receptors KIR3D 
Immunogenetics  2012;64(11):845-848.
Killer immunoglobulin-like receptors (KIRs) represent a highly polymorphic and diverse gene family in rhesus macaques. Analyses of the respective gene products have been hampered until now due to non-availability of specific monoclonal antibodies and failure of cross-reactivity of anti-human KIR antibodies. We utilised one activating (KIR3DSW08) and two inhibitory (KIR3DLW03 and KIR3DL05) rhesus macaque KIR-Fc fusion proteins for generation of monoclonal antibodies in mice. Besides broadly reacting ones, we obtained anti-rhesus macaque KIR antibodies with intermediate and with single specificity. These monoclonal antibodies were tested for binding to a panel of rhesus macaque KIR proteins after heterologous expression on transiently transfected cells. Epitope mapping identified two polymorphic regions that are located next to each other in the mature KIR proteins. The availability of monoclonal antibodies against rhesus macaque KIR proteins will enable future studies on KIR at the protein level in rhesus macaques as important animal models of human infectious diseases.
Electronic supplementary material
The online version of this article (doi:10.1007/s00251-012-0640-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s00251-012-0640-2
PMCID: PMC3470681  PMID: 22893031
Monoclonal antibodies; Killer immunoglobulin-like receptor (KIR); Epitope mapping; Rhesus macaque
16.  Genetic regulation of parasite infection: empirical evidence of the functional significance of an IL4 gene SNP on nematode infections in wild primates 
Background
Susceptibility to parasite infection affects fitness-related processes, such as mate choice and survival, yet its genetic regulation remains poorly understood. Interleukin-4 (IL4) plays a central role in the humoral immune defence against nematode parasite infections, inducing IgE switch and regulation of worm expulsion from the intestines. The evolutionary and functional significance of single nucleotide polymorphisms (SNPs) in IL4-genes is known, yet empirical information on the effect of IL4 SNPs on gastro-intestinal infections is lacking. Using samples from a population of wild red-fronted lemurs (Eulemur fulvus rufus, Primates: Lemuridae), from western Madagascar, we explored the association of IL4-gene promoter polymorphisms with nematode infections and investigated a possible functional role of the IL4 polymorphism on male reproductive success.
Results
Using sequence analyses of lemur DNA we detected a new SNP in the IL4 gene promoter area. Carriers of the genotype T/T showed higher nematode infection intensities than individuals of genotypes C/T and C/C. Genetic population analyses using data from more than 10 years, suggested higher reproductive success of T/T males than expected.
Conclusions
Our results suggest a regulatory effect of an IL4 gene promoter polymorphism on the intensity of parasite infections in a natural population of red-fronted lemurs, with a seemingly disadvantageous genotype represented in low frequencies. Long-term population analyses, however, point in the direction of a negative frequency-dependent association, giving a fitness advantage to the rare genotype. Due to low frequencies of the genotype in question conclusive evidence of a functional role of IL4 polymorphism cannot be drawn here; still, we suggest the use of IL4 polymorphism as a new molecular tool for quick assessment of individual genetic constitution with regard to nematode infection intensities, contributing to a better understanding of the actual components of the immune response that mediate protection against gastro-intestinal parasites.
doi:10.1186/1742-9994-8-9
PMCID: PMC3104205  PMID: 21501512
17.  Nuclear versus mitochondrial DNA: evidence for hybridization in colobine monkeys 
Background
Colobine monkeys constitute a diverse group of primates with major radiations in Africa and Asia. However, phylogenetic relationships among genera are under debate, and recent molecular studies with incomplete taxon-sampling revealed discordant gene trees. To solve the evolutionary history of colobine genera and to determine causes for possible gene tree incongruences, we combined presence/absence analysis of mobile elements with autosomal, X chromosomal, Y chromosomal and mitochondrial sequence data from all recognized colobine genera.
Results
Gene tree topologies and divergence age estimates derived from different markers were similar, but differed in placing Piliocolobus/Procolobus and langur genera among colobines. Although insufficient data, homoplasy and incomplete lineage sorting might all have contributed to the discordance among gene trees, hybridization is favored as the main cause of the observed discordance. We propose that African colobines are paraphyletic, but might later have experienced female introgression from Piliocolobus/Procolobus into Colobus. In the late Miocene, colobines invaded Eurasia and diversified into several lineages. Among Asian colobines, Semnopithecus diverged first, indicating langur paraphyly. However, unidirectional gene flow from Semnopithecus into Trachypithecus via male introgression followed by nuclear swamping might have occurred until the earliest Pleistocene.
Conclusions
Overall, our study provides the most comprehensive view on colobine evolution to date and emphasizes that analyses of various molecular markers, such as mobile elements and sequence data from multiple loci, are crucial to better understand evolutionary relationships and to trace hybridization events. Our results also suggest that sex-specific dispersal patterns, promoted by a respective social organization of the species involved, can result in different hybridization scenarios.
doi:10.1186/1471-2148-11-77
PMCID: PMC3068967  PMID: 21435245
18.  A Small, Variable and Irregular Killer Cell Ig-Like Receptor Locus Accompanies the Absence of MHC-C and MHC-G in Gibbons1 
The killer cell Ig-like receptors (KIR) of natural killer (NK) cells recognize major histocompatibility complex (MHC) class I ligands and function in placental reproduction and immune defense against pathogens. During the evolution of monkeys, great apes and humans, an ancestral KIR3DL gene expanded to become a diverse and rapidly evolving gene family of four KIR lineages. Characterising the KIR locus are three framework regions, defining two intervals of variable gene-content. By analysis of four KIR haplotypes from two species of gibbon, we find that the smaller apes do not conform to these rules. Although diverse and irregular in structure, the gibbon haplotypes are unusually small, containing only two to five functional genes. Comparison with the predicted ancestral hominoid KIR haplotype indicates that modern gibbon KIR haplotypes were formed by a series of deletion events, which created new hybrid genes as well as eliminating ancestral genes. Of the three framework regions, only KIR3DL3 (lineage V), defining the 5’ end of the KIR locus, is present and intact on all gibbon KIR haplotypes. KIR2DL4 (lineage I) defining the central framework region has been a major target for elimination or inactivation, correlating with the absence of its putative ligand, MHC-G, in gibbons. Similarly, the MHC-C driven expansion of lineage III KIR genes in great apes has not occurred in gibbons because they lack MHC-C. Our results indicate that the selective forces shaping the size and organisation of the gibbon KIR locus differed from those acting upon the KIR of other hominoid species.
doi:10.4049/jimmunol.0903016
PMCID: PMC3017126  PMID: 20026738
Comparative Immunology/Evolution; Reproductive Immunology; Natural Killer Cells; Cell Surface Molecules; MHC
19.  Expression Profiling of Major Histocompatibility and Natural Killer Complex Genes Reveals Candidates for Controlling Risk of Graft versus Host Disease 
PLoS ONE  2011;6(1):e16582.
Background
The major histocompatibility complex (MHC) is the most important genomic region that contributes to the risk of graft versus host disease (GVHD) after haematopoietic stem cell transplantation. Matching of MHC class I and II genes is essential for the success of transplantation. However, the MHC contains additional genes that also contribute to the risk of developing acute GVHD. It is difficult to identify these genes by genetic association studies alone due to linkage disequilibrium in this region. Therefore, we aimed to identify MHC genes and other genes involved in the pathophysiology of GVHD by mRNA expression profiling.
Methodology/Principal Findings
To reduce the complexity of the task, we used genetically well-defined rat inbred strains and a rat skin explant assay, an in-vitro-model of the graft versus host reaction (GVHR), to analyze the expression of MHC, natural killer complex (NKC), and other genes in cutaneous GVHR. We observed a statistically significant and strong up or down regulation of 11 MHC, 6 NKC, and 168 genes encoded in other genomic regions, i.e. 4.9%, 14.0%, and 2.6% of the tested genes respectively. The regulation of 7 selected MHC and 3 NKC genes was confirmed by quantitative real-time PCR and in independent skin explant assays. In addition, similar regulations of most of the selected genes were observed in GVHD-affected skin lesions of transplanted rats and in human skin explant assays.
Conclusions/Significance
We identified rat and human MHC and NKC genes that are regulated during GVHR in skin explant assays and could therefore serve as biomarkers for GVHD. Several of the respective human genes, including HLA-DMB, C2, AIF1, SPR1, UBD, and OLR1, are polymorphic. These candidates may therefore contribute to the genetic risk of GVHD in patients.
doi:10.1371/journal.pone.0016582
PMCID: PMC3030590  PMID: 21305040
20.  Activity-dependent regulation of MHC class I expression in the developing primary visual cortex of the common marmoset monkey 
Background
Several recent studies have highlighted the important role of immunity-related molecules in synaptic plasticity processes in the developing and adult mammalian brains. It has been suggested that neuronal MHCI (major histocompatibility complex class I) genes play a role in the refinement and pruning of synapses in the developing visual system. As a fast evolutionary rate may generate distinct properties of molecules in different mammalian species, we studied the expression of MHCI molecules in a nonhuman primate, the common marmoset monkey (Callithrix jacchus).
Methods and results
Analysis of expression levels of MHCI molecules in the developing visual cortex of the common marmoset monkeys revealed a distinct spatio-temporal pattern. High levels of expression were detected very early in postnatal development, at a stage when synaptogenesis takes place and ocular dominance columns are formed. To determine whether the expression of MHCI molecules is regulated by retinal activity, animals were subjected to monocular enucleation. Levels of MHCI heavy chain subunit transcripts in the visual cortex were found to be elevated in response to monocular enucleation. Furthermore, MHCI heavy chain immunoreactivity revealed a banded pattern in layer IV of the visual cortex in enucleated animals, which was not observed in control animals. This pattern of immunoreactivity indicated that higher expression levels were associated with retinal activity coming from the intact eye.
Conclusions
These data demonstrate that, in the nonhuman primate brain, expression of MHCI molecules is regulated by neuronal activity. Moreover, this study extends previous findings by suggesting a role for neuronal MHCI molecules during synaptogenesis in the visual cortex.
doi:10.1186/1744-9081-7-1
PMCID: PMC3023691  PMID: 21205317
21.  Human box C/D snoRNAs with miRNA like functions: expanding the range of regulatory RNAs 
Nucleic Acids Research  2010;39(2):675-686.
Small nucleolar RNAs (snoRNAs) and microRNAs are two classes of non-protein-coding RNAs with distinct functions in RNA modification or post-transcriptional gene silencing. In this study, we introduce novel insights to RNA-induced gene activity adjustments in human cells by identifying numerous snoRNA-derived molecules with miRNA-like function, including H/ACA box snoRNAs and C/D box snoRNAs. In particular, we demonstrate that several C/D box snoRNAs give rise to gene regulatory RNAs, named sno-miRNAs here. Our data are complementing the increasing number of studies in the field of small RNAs with regulatory functions. In massively deep sequencing of small RNA fractions we identified high copy numbers of sub-sequences from >30 snoRNAs with lengths of ≥18 nt. RNA secondary structure prediction indicated for a majority of candidates a location in predicted stem regions. Experimental analysis revealed efficient gene silencing for 11 box C/D sno-miRNAs, indicating cytoplasmic processing and recruitment to the RNA silencing machinery. Assays in four different human cell lines indicated variations in both the snoRNA levels and their processing to active sno-miRNAs. In addition we show that box D elements are predominantly flanking at least one of the sno-miRNA strands, while the box C element locates within the sequence of the sno-miRNA guide strand.
doi:10.1093/nar/gkq776
PMCID: PMC3025573  PMID: 20846955
22.  Erratum to: Rhesus macaque MHC class I molecules show differential subcellular localizations 
Immunogenetics  2010;62(6):409-418.
The MHC class I gene family of rhesus macaques is characterised by considerable gene duplications. While a HLA-C-orthologous gene is absent, the Mamu-A and in particular the Mamu-B genes have expanded, giving rise to plastic haplotypes with differential gene content. Although some of the rhesus macaque MHC class I genes are known to be associated with susceptibility/resistance to infectious diseases, the functional significance of duplicated Mamu-A and Mamu-B genes and the expression pattern of their encoded proteins are largely unknown. Here, we present data of the subcellular localization of AcGFP-tagged Mamu-A and Mamu-B molecules. We found strong cell surface and low intracellular expression for Mamu-A1, Mamu-A2 and Mamu-A3-encoded molecules as well as for Mamu-B*01704, Mamu-B*02101, Mamu-B*04801, Mamu-B*06002 and Mamu-B*13401. In contrast, weak cell surface and strong intracellular expression was seen for Mamu-A4*1403, Mamu-B*01202, Mamu-B*02804, Mamu-B*03002, Mamu-B*05704, Mamu-I*010201 and Mamu-I*0121. The different expression patterns were assigned to the antigen-binding α1 and α2 domains, suggesting failure of peptide binding is responsible for retaining ‘intracellular’ Mamu class I molecules in the endoplasmic reticulum. These findings indicate a diverse functional role of the duplicated rhesus macaque MHC class I genes.
doi:10.1007/s00251-010-0447-y
PMCID: PMC3128699  PMID: 20445972
MHC class I; Mamu-A; Mamu-B; Subcellular localization; Macaca mulatta
23.  The endogenous danger signals HSP70 and MICA cooperate in the activation of cytotoxic effector functions of NK cells 
Abstract
Although natural killer (NK) cells are often described as first line defence against infected or malignant cells which act without the need of prior activation, it is known now that the NK cell activity is tightly regulated by other cells and soluble factors. We show here that the stress-inducible heat shock protein (HSP) 70 activates human NK cells to kill target cells expressing major histocompatibility complex class I chain-related molecule A (MICA) in a natural killer group 2 member D (NKG2D-) dependent manner. The HSP70-derived peptide TKD (TKDNNLLGRFELSG) was able to replace the full-length HSP70 and to exert the same function. Interestingly, the expression of the cytotoxic effector protease granzyme B in NK cells was increased after TKD stimulation. When MICA and MICB expression was induced in human tumour cells by a histone deacetylase inhibitor and NK cells were activated by HSP70 or TKD, both treatments jointly improved the killing of the tumour cells. Thus, the synergistic activity of two stress-inducible immunological danger signals, HSP70 and MICA/B, leads to activation and enhanced cytotoxicity of human NK cells against tumour cells.
doi:10.1111/j.1582-4934.2008.00677.x
PMCID: PMC3823130  PMID: 20569278
heat shock protein 70; NKG2D ligands; natural killer cells; cellular cytotoxicity; cancer; immunotherapy
24.  Neuronal MHC Class I Molecules are Involved in Excitatory Synaptic Transmission at the Hippocampal Mossy Fiber Synapses of Marmoset Monkeys 
Several recent studies suggested a role for neuronal major histocompatibility complex class I (MHCI) molecules in certain forms of synaptic plasticity in the hippocampus of rodents. Here, we report for the first time on the expression pattern and functional properties of MHCI molecules in the hippocampus of a nonhuman primate, the common marmoset monkey (Callithrix jacchus). We detected a presynaptic, mossy fiber-specific localization of MHCI proteins within the marmoset hippocampus. MHCI molecules were present in the large, VGlut1-positive, mossy fiber terminals, which provide input to CA3 pyramidal neurons. Furthermore, whole-cell recordings of CA3 pyramidal neurons in acute hippocampal slices of the common marmoset demonstrated that application of antibodies which specifically block MHCI proteins caused a significant decrease in the frequency, and a transient increase in the amplitude, of spontaneous excitatory postsynaptic currents (sEPSCs) in CA3 pyramidal neurons. These findings add to previous studies on neuronal MHCI molecules by describing their expression and localization in the primate hippocampus and by implicating them in plasticity-related processes at the mossy fiber–CA3 synapses. In addition, our results suggest significant interspecies differences in the localization of neuronal MHCI molecules in the hippocampus of mice and marmosets, as well as in their potential function in these species.
Electronic supplementary material
The online version of this article (doi:10.1007/s10571-010-9510-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s10571-010-9510-3
PMCID: PMC2912721  PMID: 20232136
Calbindin; CA3; GFAP; Piccolo; Neuronal plasticity; PSD95; sEPSC; VGAT; VGlut1; VGlut 2
25.  Mitochondrial evidence for multiple radiations in the evolutionary history of small apes 
Background
Gibbons or small apes inhabit tropical and subtropical rain forests in Southeast Asia and adjacent regions, and are, next to great apes, our closest living relatives. With up to 16 species, gibbons form the most diverse group of living hominoids, but the number of taxa, their phylogenetic relationships and their phylogeography is controversial. To further the discussion of these issues we analyzed the complete mitochondrial cytochrome b gene from 85 individuals representing all gibbon species, including most subspecies.
Results
Based on phylogenetic tree reconstructions, several monophyletic clades were detected, corresponding to genera, species and subspecies. A significantly supported branching pattern was obtained for members of the genus Nomascus but not for the genus Hylobates. The phylogenetic relationships among the four genera were also not well resolved. Nevertheless, the new data permitted the estimation of divergence ages for all taxa for the first time and showed that most lineages emerged during four short time periods. In the first, between ~6.7 and ~8.3 mya, the four gibbon genera diverged from each other. In the second (~3.0 - ~3.9 mya) and in the third period (~1.3 - ~1.8 mya), Hylobates and Hoolock differentiated. Finally, between ~0.5 and ~1.1 mya, Hylobates lar diverged into subspecies. In contrast, differentiation of Nomascus into species and subspecies was a continuous and prolonged process lasting from ~4.2 until ~0.4 mya.
Conclusions
Although relationships among gibbon taxa on various levels remain unresolved, the present study provides a more complete view of the evolutionary and biogeographic history of the hylobatid family, and a more solid genetic basis for the taxonomic classification of the surviving taxa. We also show that mtDNA constitutes a useful marker for the accurate identification of individual gibbons, a tool which is urgently required to locate hunting hotspots and select individuals for captive breeding programs. Further studies including nuclear sequence data are necessary to completely understand the phylogeny and phylogeography of gibbons.
doi:10.1186/1471-2148-10-74
PMCID: PMC2841658  PMID: 20226039

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