Gaby G. Doxiadis

Genetic Makeup of the DR Region in Rhesus Macaques: Gene Content, Transcripts, and Pseudogenes

In the human population, five major HLA-DRB haplotypes have been identified, whereas the situation in rhesus macaques (Macaca mulatta) is radically different. At least 30 Mamu-DRB region configurations, displaying polymorphism with regard to number and combination of DRB loci present per haplotype, have been characterized. Until now, Mamu-DRB region genes have been studied mainly by genomic sequencing of polymorphic exon 2 segments. However, relatively little is known about the expression status of these genes. To understand which exon 2 segments may represent functional genes, full-length cDNA analyses of -DRA and -DRB were initiated. In the course of the study, 11 cDRA alleles were identified, representing four distinct gene products. Amino acid replacements are confined to the leader peptide and cytoplasmatic tail, whereas residues of the α1 domain involved in peptide binding, are conserved between humans, chimpanzees, and rhesus macaques. Furthermore, from the 11 Mamu-DRB region configurations present in this panel, 28 cDRB alleles were isolated, constituting 12 distinct cDRA/cDRB configurations. Evidence is presented that a single configuration expresses maximally up to three -DRB genes. For some exon 2 DRB sequences, the corresponding transcripts could not be detected, rendering such alleles as probable pseudogenes. The full-length cDRA and cDRB sequences are necessary to construct Mhc class II tetramers, as well as transfectant cell lines. As the rhesus macaque is an important animal model in AIDS vaccine studies, the information provided in this communication is essential to define restriction elements and to monitor immune responses in SIV/simian human immunodeficiency virus-infected rhesus macaques.

Genomic plasticity of the immune-related Mhc class I B region in macaque species

BackgroundIn sharp contrast to humans and great apes, the expanded Mhc-B region of rhesus and cynomolgus macaques is characterized by the presence of differential numbers and unique combinations of polymorphic class I B genes per haplotype. The MIB microsatellite is closely linked to the single class I B gene in human and in some great apes studied. The physical map of the Mhc of a heterozygous rhesus monkey provides unique material to analyze MIB and Mamu-B copy number variation and then allows one to decipher the compound evolutionary history of this region in primate species.ResultsIn silico research pinpointed 12 MIB copies (duplicons), most of which are associated with expressed B-genes that cluster in a separate clade in the phylogenetic tree. Generic primers tested on homozygous rhesus and pedigreed cynomolgus macaques allowed the identification of eight to eleven MIB copies per individual. The number of MIB copies present per haplotype varies from a minimum of three to six in cynomolgus macaques and from five to eight copies in rhesus macaques. Phylogenetic analyses highlight a strong transpecific sharing of MIB duplicons. Using the physical map, we observed that, similar to MIB duplicons, highly divergent Mamu-B genes can be present on the same haplotype. Haplotype variation as reflected by the copy number variation of class I B loci is best explained by recombination events, which are found to occur between MIBs and Mamu-B.ConclusionThe data suggest the existence of highly divergent MIB and Mamu-B lineages on a given haplotype, as well as variable MIB and B copy numbers and configurations, at least in rhesus macaque. Recombination seems to occur between MIB and Mamu-B loci, and the resulting haplotypic plasticity at the individual level may be a strategy to better cope with pathogens. Therefore, evolutionary inferences based on the multiplicated MIB loci but also other markers close to B-genes appear to be promising for the study of B-region organization and evolution in primates.

Gluten-sensitive enteropathy coincides with decreased capability of intestinal T cells to secrete IL-17 and IL-22 in a macaque model for celiac disease

Celiac disease (CD) is an autoimmune disorder caused by intolerance to dietary gluten. The interleukin (IL)-17 and IL-22 function as innate regulators of mucosal integrity. Impaired but not well-understood kinetics of the IL-17/22 secretion was described in celiac patients. Here, the IL-17 and IL-22-producing intestinal cells were studied upon their in vitro stimulation with mitogens in class II major histocompatibility complex-defined, gluten-sensitive rhesus macaques. Pediatric biopsies were collected from distal duodenum during the stages of disease remission and relapse. Regardless of dietary gluten content, IL-17 and IL-22-producing cells consisted of CD4+ and CD8+ T lymphocytes as well as of lineage-negative (Lin-) cells. Upon introduction of dietary gluten, capability of intestinal T cells to secrete IL-17/22 started to decline (p<0.05), which was paralleled with gradual disruption of epithelial integrity. These data indicate that IL-17/22-producing cells play an important role in maintenance of intestinal mucosa in gluten-sensitive primates.

Improved xenobiotic metabolism and reduced susceptibility to cancer in gluten-sensitive macaques upon introduction of a gluten-free diet.

Background A non-human primate (NHP) model of gluten sensitivity was employed to study the gene perturbations associated with dietary gluten changes in small intestinal tissues from gluten-sensitive rhesus macaques (Macaca mulatta). Methodology Stages of remission and relapse were accomplished in gluten-sensitive animals by administration of gluten-free (GFD) and gluten-containing (GD) diets, as described previously. Pin-head-sized biopsies, obtained non-invasively by pediatric endoscope from duodenum while on GFD or GD, were used for preparation of total RNA and gene profiling, using the commercial Rhesus Macaque Microarray (Agilent Technologies),targeting expression of over 20,000 genes. Principal Findings When compared with normal healthy control, gluten-sensitive macaques showed differential gene expressions induced by GD. While observed gene perturbations were classified into one of 12 overlapping categories - cancer, metabolism, digestive tract function, immune response, cell growth, signal transduction, autoimmunity, detoxification of xenobiotics, apoptosis, actin-collagen deposition, neuronal and unknown function - this study focused on cancer-related gene networks such as cytochrome P450 family (detoxification function) and actin-collagen-matrix metalloproteinases (MMP) genes. Conclusions/Significance A loss of detoxification function paralleled with necessity to metabolize carcinogens was revealed in gluten-sensitive animals while on GD. An increase in cancer-promoting factors and a simultaneous decrease in cancer-preventing factors associated with altered expression of actin-collagen-MMP gene network were noted. In addition, gluten-sensitive macaques showed reduced number of differentially expressed genes including the cancer-associated ones upon withdrawal of dietary gluten. Taken together, these findings indicate potentially expanded utility of gluten-sensitive rhesus macaques in cancer research.

High resolution definition of HLA‐DRB haplotypes by a simplified microsatellite typing technique

In humans, the region configurations DR1, DR8, DR51, DR52 and DR53 are known to display copy number as well as allelic variation, rendering high resolution typing of HLA-DRB haplotypes cumbersome. Advantage was taken of microsatellite D6S2878, present in all DRB genes/pseudogenes with an intact exon 2-intron 2 segment. This DRB-STR is highly polymorphic in composition and length. Recently, it was proven that all exon 2 sequences could be linked to a certain DRB-STR that segregates with the respective DRB allele. Because haplotypes show differential copy numbers and compositions of exon 2-positive DRB genes/pseudogenes, unique DRB-STR patterns could be described that appear to be specific for a particular DRB haplotype. The aim of this workshop project was to approve and to qualify this simple typing protocol in a larger panel covering different European populations. All participants succeeded in correctly defining the DRB-STR amplicons varying from 135 to 222 base pair (bp) lengths. The panel of 101 samples covered 50 DRB alleles distributed over 37 different haplotypes as defined by exon 2 sequence-based typing. These haplotypes could be refined into 105 haplotypes by DRB-STR typing. Thus, discrimination of exon 2-identical DRB alleles was feasible, as well as the exact description of three different crossing-over events that resulted in the generation of hybrid DR region configurations. This typing procedure appears to be a quick and highly robust technique that can easily be performed by different laboratories, even without experience in microsatellite typing; thus, it is suitable for a variety of researchers in diverse research areas.

Fifty-one full-length major histocompatibility complex class II alleles in the olive baboon (Papio anubis)

Here we report 51 novel major histocompatibility complex (MHC) class II alleles in a group of related olive baboons.

Human and Rhesus Macaque KIR Haplotypes Defined by Their Transcriptomes

The killer-cell Ig-like receptors (KIRs) play a central role in the immune recognition in infection, pregnancy, and transplantation through their interactions with MHC class I molecules. KIR genes display abundant copy number variation as well as high levels of polymorphism. As a result, it is challenging to characterize this structurally dynamic region. KIR haplotypes have been analyzed in different species using conventional characterization methods, such as Sanger sequencing and Roche/454 pyrosequencing. However, these methods are time-consuming and often failed to define complete haplotypes, or do not reach allele-level resolution. In addition, most analyses were performed on genomic DNA, and thus were lacking substantial information about transcription and its corresponding modifications. In this paper, we present a single-molecule real-time sequencing approach, using Pacific Biosciences Sequel platform to characterize the KIR transcriptomes in human and rhesus macaque (Macaca mulatta) families. This high-resolution approach allowed the identification of novel Mamu-KIR alleles, the extension of reported allele sequences, and the determination of human and macaque KIR haplotypes. In addition, multiple recombinant KIR genes were discovered, all located on contracted haplotypes, which were likely the result of chromosomal rearrangements. The relatively high number of contracted haplotypes discovered might be indicative of selection on small KIR repertoires and/or novel fusion gene products. This next-generation method provides an improved high-resolution characterization of the KIR cluster in humans and macaques, which eventually may aid in a better understanding and interpretation of KIR allele–associated diseases, as well as the immune response in transplantation and reproduction.

No postcopulatory selection against MHC-homozygous offspring : Evidence from a pedigreed captive rhesus macaque colony

The heterozygosity status of polymorphic elements of the immune system, such as the major histocompatibility complex (MHC), is known to increase the potential to cope with a wider variety of pathogens. Pre‐ and postcopulatory processes may regulate MHC heterozygosity. In a population where mating occurs among individuals that share identical MHC haplotypes, postcopulatory selection may disfavour homozygous offspring or ones with two MHC haplotypes identical to its mother. We tested these ideas by determining the incidence of MHC‐heterozygous and MHC‐homozygous individuals in a pedigreed, partially consanguineous captive rhesus monkey colony. Bayesian statistics showed that when parents share MHC haplotypes, the distribution of MHC‐heterozygous and MHC‐homozygous individuals significantly fitted the expected Mendelian distribution, both for the complete MHC haplotypes, and for MHC class I or II genes separately. Altogether, we found in this captive colony no evidence for postcopulatory selection against MHC‐homozygous individuals. However, the distribution of paternally and maternally inherited MHC haplotypes tended to differ significantly from expected. Individuals with two MHC haplotypes identical to their mother were underrepresented and offspring with MHC haplotypes identical to their father tended to be overrepresented. This suggests that postcopulatory processes affect MHC haplotype combination in offspring, but do not prevent low MHC heterozygosity.

Novel DRA alleles extracted from seven macaque cohorts

In this document, we report the detection of 37 DRA alleles in macaque cohorts.