Characterization of the major histocompatibility complex class II DOB, DPB1, and DQB1 alleles in cynomolgus macaques of Vietnamese origin
Major histocompatibility complex (MHC) molecules play an important role in the susceptibility and/or resistance to many diseases. To gain an insight into the MHC background and to facilitate the experimental use of cynomolgus macaques, the second exon of the MhcMafa-DOB, -DPB1, and -DQB1 genes from 143 cynomolgus macaques were characterized by cloning to sequencing. A total of 16 Mafa-DOB, 16 Mafa-DPB1, and 34 Mafa-DQB1 alleles were identified, which revealed limited, moderate, and marked allelic polymorphism at DOB, DPB1, and DQB1, respectively, in a cohort of cynomolgus macaques of Vietnamese origin. In addition, 16 Mafa-DOB, 5 Mafa-DPB1, and 8 Mafa-DQB1 alleles represented novel sequences that had not been reported in earlier studies. Almost of the sequences detected at the DOB and DQB1 locus in the present study belonged to DOB*01 (100%) and DQB1*06 (62%) lineages, respectively. Interestingly, four, three, and one high-frequency alleles were detected at Mafa-DOB, -DPB1, and -DQB1, respectively, in this monkeys. The alleles with the highest frequency among these monkeys were Mafa-DOB*010102, Mafa-DPB1*13, and Mafa-DQB1*0616, and these were found in 33 (25.6%) of 129 monkeys, 32 (31.37%) of 102 monkeys, and 30 (31%) of 143 monkeys, respectively. The high-frequency alleles may represent high priority targets for additional characterization of immune function. We also carried out evolutionary and population analyses using these sequences to reveal population-specific alleles. This information will not only promote the understanding of MHC diversity and polymorphism in the cynomolgus macaque but will also increase the value of this species as a model for biomedical research.
KeywordsHigh frequency Major histocompatibility complex class II Macaca fascicularis
Rhesus macaques have been used as animal models for various human diseases for a long time. With the 1978 ban on exportation of Rhesus macaques from India, researchers have become increasingly interested in an alternative macaque, the cynomolgus macaque, which has a shorter breeding cycle, a docile personality, and requires lower dosages of drugs. The cynomolgus macaque (Macaca fascicularis), also known as the crab-eating monkey or long-tailed macaque, is used mainly in animal models of diabetes, renal transplantation, virological research, SARS, tuberculosis, studies of the pathogenesis of simian immunodeficiency virus (SIV), and pharmacodynamic evaluation (O'Sullivan et al. 1997; Menninger et al. 2002; McAuliffe et al. 2004; Reed et al. 2009). Owing to the need for reliable data on experimental drug reactions provided by animal models, researchers have focused on genes of the immune system of cynomolgus monkeys, in particular, the genes of the major histocompatibility complex (MHC). Molecules of MHC class I and II play an important role in immune regulatory processes by presenting peptides of intracellular or extracellular origin to CD8+ or CD4+ T cells, respectively.
The classical MHC genes of cynomolgus macaques can be divided into MHC-I and MHC-II genes. The MHC-I genes include mainly -A and -B alleles, and MHC-II genes include mainly -DM, -DO, -DP, -DQ, and -DR alleles. DO is a non-classical class II heterodimer that consists of α and β chains, which are encoded by the DOA and DOB genes located in the MHC class II region. The function of MHC-DO is poorly understood; it may act as a negative regulator by binding to HLA-DM and inhibiting the exchange reaction of class II-associated invariant chain peptides for antigenic peptides (Fernandez-Donoso et al. 1970; O'Sullivan et al. 1997). HLA-DPB1 alleles have been demonstrated to be involved in corneal and renal transplantation, myasthenia gravis, multiple sclerosis, Hodgkin’s disease, Beryllium disease, and sarcoidosis. In addition, an MHC-DPB1 allele was found to be involved in the susceptibility to experimental autoimmune encephalomyelitis in rhesus macaques (Slierendregt et al. 1995a, b). Thus, the primate MHC-DPB1 plays a fundamental and important role in the peptide-binding selectivity of the DP antigen (Uda et al. 2005) and is a significant factor in the humoral response (Krebs et al. 2005). It has been reported that HLA-DQB1 alleles have been associated as an increased risk of developing type 1 diabetes (Todd 1990; Todd 1997; Redondo et al. 2001), celiac disease (Murray et al. 2007), multiple sclerosis (Dyment et al. 1997; Schmidt et al. 2007, and narcolepsy (Kadotani et al. 1998).
It is not easy to elucidate the mechanism of naturally occurring immune protection in human immunodeficiency virus (HIV), and most direct supporting data are available from animal models in non-human primates. The SIVmac virus, which was isolated originally from cynomolgus monkeys, is now used frequently for research on vaccines against acquired immune deficiency syndrome (AIDS; Hu 2005). Many reports show that polymorphism of MHC genes in the cynomolgus monkey affects the results obtained with drugs significantly (Walsh et al. 1996; Mothe et al. 2003; Hao and Nei 2005) and is associated with control of viral diseases (Florese et al. 2008). Cynomolgus macaques from Mauritius may be particularly valuable because more than half of these animals share the MHC class I allele combination Mafa-B*430101, Mafa-B*440101, and Mafa-B*460101. The increased sharing of MHC-I allele in cynomolgus macaques of Mauritian origin may reduce the overall number of animals needed to study cellular immune responses in non-human primates dramatically, while simultaneously reducing the confounding effects of genetic heterogeneity in HIV/AIDS research (Krebs et al. 2005).
The MHC class II molecules of Rhesus macaques (Macaca mulatta) have been studied methodically, especially in animals of Indian origin. In contrast to Rhesus macaques, although alleles of the Mafa II class molecules, including Mafa-DPB1 and Mafa-DQB1, have been identified by several groups (Otting et al. 2002; Doxiadis et al. 2006; Blancher et al. 2006; O'Connor et al. 2007), knowledge is limited of the MHC class II genes of the cynomolgus monkey and their degree of polymorphism. In addition, a more detailed study of the Mafa-DPB1 sequence in three different geographical variants of cynomolgus monkeys from south-east Asia demonstrated that polymorphism of MHC-II genes is influenced by species and geography (Sano et al. 2006). It is very rare that cynomolgus macaques from different regions share MHC-II genes (Krebs et al. 2005).
Haplotype screening, which employs multiple markers rather than single genes, would be meaningful in MHC disease association studies because it is well-known that most of the MHC loci are tightly linked and exhibit very little recombination (Dukkipati et al. 2006). The high-frequency alleles may represent high priority targets for additional characterization of immune function (Wiseman et al. 2009). Therefore, to examine whether the MHC class II genes found in a cohort of Vietnamese cynomolgus macaques are common to for other geographical populations of cynomolgus macaques, the Mafa class II region was characterized in the present study by sequencing of the polymorphic exon 2 of the -DOB, -DPB1, and -DQB1 genes.
Materials and methods
Whole blood samples from 150 unrelated cynomolgus macaques, originally from Vietnam, were provided generously by South China Primates Research Central. Whole blood samples (3–5 ml) withdrawn from each monkey were collected into EDTA vacuum tubes. All the monkeys were clinically normal with no known diseases.
DNA isolation and sequencing of exon 2 of Mafa-DOB, -DPB1, and -DQB1
Primers used to amplify MHC class II alleles
Sequence (5′ to 3′)
The sequences of the exon 2 regions of Mafa-DOB, -DPB1, and -DQB1 obtained in the present study were aligned, and the phylogenetic tree was generated, which was done using the neighbor-joining method (Saitou and Nei 1987) and Mega 4.0 software (Tamura et al. 2007). The bootstrap consensus tree inferred from 1,000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein 1985). Branches corresponding to partitions reproduced in fewer than 50% of bootstrap replicates are collapsed. The evolutionary distances were computed using the Kimura 2-parameter method (Kimura 1980) and are in the units of the number of base substitutions per site. The rate of variation among sites was modeled with a gamma distribution (shape parameter = 1). New alleles were confirmed by three repeats sequencing. The names of new sequences were derived according to the published guidelines, and the Immuno Polymorphism Database of Major Histocompatibility Complex for Non-Human Primates was searched to avoid the same name(s) being assigned to different alleles (Klein et al. 1990; Robinson et al. 2005).
Results and discussion
Allele frequencies of Mafa-DOB, -DPB1, and -DQB1 in Vietnamese cynomolgus macaques
Limited polymorphism at the MHC-DOB locus and extensive polymorphism at the MHC-DPB1 and -DQB1 loci have been reported in the primates tested until now, and most of the variability is confined to exon 2, which encodes a major part of the peptide-binding site.
MhcMafa-DOB alleles identified in 129 randomly sampled cynomolgus macaques
No. of haplotypes
MhcMafa-DPB alleles identified in 102 randomly sampled cynomolgus macaques
No. of haplotypes
It has been shown that the most frequent alleles in Vietnam cynomolgus macaques are Mafa-DPB1*13 and -DPB1*35 (Sano et al. 2006), which supports our results above. In contrast to the result from Sano et al. (Sano et al. 2006), a high frequency of the Mafa-DPB1*21 allele was detected in our study. The Mafa-DPB1*21 allele was also observed in Mauritian cynomolgus macaques, but was not at a high frequency (O'Connor et al. 2007). Like HLA-DPB1, the Mafa-DPB1 gene of the cynomolgus macaque also displays moderate polymorphism, and more than 50 alleles have been documented to date (Slierendregt et al. 1995a, b; Otting et al. 1998; Marsh et al. 2005; O'Connor et al. 2007). In cynomolgus macaques, point mutations might play crucial role in generating DPB1 polymorphism, whereas in humans, much of the variability has been produced by frequent exchange of polymorphic sequence motifs (Zangenberg et al. 1995; Bontrop et al. 1999; Doxiadis et al. 2001).
MhcMafa-DQB alleles identified in 143 randomly sampled cynomolgus macaques
No. of haplotypes
Although the MhcMamu-DQB1*06111 (equivalent to the -DQB1*061101) allele was the most frequent (13%) in 105 randomly sampled Chinese rhesus macaques (Qiu et al. 2008), the allele, which corresponds to MhcMafa-DQB1*0613 in the present study, was at a low frequency (2%) in the 143 monkeys tested. The Mafa-DQB1 polymorphism has been studied earlier (Otting et al. 2002), and only eight of the 34 alleles detected in this study have not been reported previously. Given that the number of different -DQ alleles observed is nearly as high as the number of animals tested, it is likely that cynomolgus macaques display abundant Mafa-DQB1 polymorphism and, when other populations are tested, the levels may reach or even exceed those reported for rhesus macaques (Robinson et al. 2003; Doxiadis et al. 2006).
Amino acid sequences encoded by the MhcMafa-DOB, -DPB1, and -DQB1 genes
Eighty-seven amino acid residues of DPB1 exon 2, which encodes the DP antigen β1 domain, were aligned using 52 Mafa-DPB1, 33 Mamu-DPB1, and 18 HLA-DPB1 sequences (Fig. 2). All 103 MHC-DPB1 alleles had different amino acid sequences. Although all the Mafa-DPB1 alleles were conserved at two cysteine sites (amino acid positions 10 and 72) and did not contain a terminator codon, an amino acid insertion or deletion was seen in nine Mafa-DPB1 alleles (Sano et al. 2006). Mafa-DPB1*16 and Mafa-DPB1*49 contained a methionine residue inserted between amino acid positions 43 and 44, whereas Mafa-DPB1*35, which was the most frequent in the Vietnamese population (Table 2), had a single amino acid residue deleted from position 58 (Fig. 2). Of the 87 residue positions, 54 residues were polymorphic among the Mafa-DPB1 alleles, in contrast to 33 residues among the Mamu-DPB1 alleles (Sano et al. 2006). In addition, 62 species-specific amino acid residues, located at 43 positions, were identified from the comparison between the Mafa- and Mamu-DPB1 alleles, and of these, 59 were specific to Mafa-DPB1 (Fig. 2).
Phylogenetic tree of the MhcMafa-DOB, -DPB1, and -DQB1 genes
In conclusion, the Mafa-DOB, -DPB1, and -DQB1 alleles detected in this manuscript are mostly specific for a given geographic area, and only a small number of alleles appears to be shared with other populations, providing an important addition to the limited immunogenetic information available for Vietnamese cynomolgus macaques. This suggests the fast evolution of Mafa-DOB, -DPB1, and -DQB1 alleles due to adaptation to new environments. The high-frequency alleles among Vietnamese population, Mafa-DOB*010102, Mafa-DPB1*13, and Mafa-DQB1*0616, may represent high priority targets for additional characterization of immune function. Characterization of shared and unique MHC class II DNA sequences may be vital for disease research and may help better elucidate the biogeography of non-human primates.
We thank the Primate Research Center of South China for providing blood samples from animals. We thank Dr. Natasja de Groot, Dr. Nel Otting, and IMGT Non-human Primate Nomenclature Committee for naming the Mafa-DOB, -DPB1, and -DQB1 sequences. We wish to thank Dr. Xiao-hui Liu and David Cushley for editing the manuscript. This project was supported by the National Basic Research Program of China (2006CB701500 and 2007CB512402), the Fundamental Research Funds for the Central Universities of South China University of Technology (2009ZM0105) and Science and Technology Planning Project of Guangdong Province, China (2010B060200007).
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