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

This information is current as Nanine de Groot, Gaby G. Doxiadis, Natasja G. de Groot, of September 25, 2021. Nel Otting, Corrine Heijmans, Annemiek J. M. Rouweler and Ronald E. Bontrop J Immunol 2004; 172:6152-6157; ; doi: 10.4049/jimmunol.172.10.6152

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Genetic Makeup of the DR Region in Rhesus Macaques: Gene Content, Transcripts, and Pseudogenes1

Nanine de Groot,2 Gaby G. Doxiadis, Natasja G. de Groot, Nel Otting, Corrine Heijmans, Annemiek J. M. Rouweler, and Ronald E. Bontrop

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 ␣1 domain involved in peptide binding, are conserved Downloaded from 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 infor- mation provided in this communication is essential to define restriction elements and to monitor immune responses in SIV/simian human http://www.jimmunol.org/ immunodeficiency virus-infected rhesus macaques. The Journal of Immunology, 2004, 172: 6152Ð6157.

he rhesus monkey provides a valuable model in preclin- The organization of the Mamu-DRB region is complex and it dis- ical studies of infectious and chronic diseases as well as plays variation at the population level with regard to number T for tissue and organ transplantation (1–11). The applica- and/or combination of loci present per configuration (24–28). In tion of macaques in immunological research necessitates an ex- humans, the number of -DRB loci present per configuration differs tensive characterization of the MHC region, because the high de- from one to four, whereas in the rhesus macaque, one to eight loci gree of polymorphism of most of its genes is not only a main can be observed (12, 28). Only five HLA-DRB region configura- by guest on September 25, 2021 characteristic in humans, but also in nonhuman primates (12). Cell tions are known, and all of them display a high degree of poly- surface glycoproteins of the MHC, divided into class I and class II morphism, mainly at the -DRB1 locus (29, 30). The situation in gene products, present peptides to effector T cells, and therefore rhesus macaques is radically different, as Ͼ30 Mamu-DRB region play an important role in adaptive immunology. As one would configurations have been described so far (28). Although the total expect, several susceptibility or resistance traits have been mapped number of apparent Mamu-DRB alleles is comparable to those of to particular Mhc alleles in humans and rhesus macaques (13–22). the HLA-DRB1 locus, the Mamu-DRB region configurations them- The polymorphic MHC class II genes of the rhesus macaque selves show only a limited degree of allelic variation (25, 26). (MhcMamu) map to the DP, DQ, and DR regions. One expects The absence or lack of allelic polymorphism at Mamu-DRB re- that, as is found in humans, the actual polymorphism is mostly gion configurations can be explained in several ways. One inter- confined to exon 2 of the Mamu-DPB1, -DQA1, -DQB1, and -DRB pretation is that these configurations are relatively young and did loci encoding the contact residues of the peptide binding site. As not have time to accumulate variation. Alternatively, it is conceiv- a consequence, Mamu class II sequencing has been mainly focused able that these configurations experience conservative selection on the determination of allelic variation at exon 2 segments. and have been maintained over longer evolutionary time spans. In The Mamu-DRA locus encoding the DR ␣-chain is thought to be both cases, rhesus macaques used a radically different strategy than monomorphic and highly conserved through primate evolution, as humans to initiate Th cell responses to combat infections. While the it shows only limited variation in comparison to HLA-DRA (23). human population invested mainly in generating a high degree of allelic variation at the various DRB loci, the rhesus macaque popula- Department of Comparative Genetics and Refinement, Biomedical Primate Research tion primarily generated a large number of singular combinations of Centre, Rijswijk, The Netherlands DRB loci. We published evidence recently that Mamu-DR/DQ con- Received for publication November 14, 2003. Accepted for publication February figurations appear to be unique for a given population living at distinct 27, 2004. geographic locations (31). This implies that Mamu-DR/DQ region The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance configurations originated after the separation of eastern and western with 18 U.S.C. Section 1734 solely to indicate this fact. rhesus macaque populations, which is thought to have been caused by 1 This study was supported in part by the European Union Project IMGT-QLG2-CT a glacial ice barrier during the Pleistocene era (32). 2000-01287 and the National Institutes of Health Project 1-R24-RR16038-01 (Cata- Most of the Mamu-DRB alleles belong to lineages or loci that log of Federal Domestic Assistance 93.306). are shared between humans and macaques. In addition, present in 2 Address correspondence and reprint requests to Dr. Nanine de Groot, Biomedical Primate Research Centre, Lange Kleiweg 139, 2288 GJ Rijswijk, The Netherlands. the rhesus macaque are loci/lineages for which no human equiv- E-mail address: [email protected] alent is known (-DRBW). Some of the Mamu-DRB loci appear to

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 6153 have been duplicated and can be present twice, or even three times, phocytes or immortalized B cell lines used in this study originate from 14 on the same configuration (26). pedigreed animals in Biomedical Primate Research Center’s self-sustaining One needs to realize that many pseudogenes have been identi- colony (Indian origin). Of these rhesus macaques, five were homozygous and derived from consanguineous origin, two were homozygous, and seven were fied in the various HLA-DR regions. However, little is known heterozygous for their Mhc regions. This panel covers 11 of the most frequent about the expression of the various Mamu-DRB loci, lineages, or Mamu-DRB region configurations present in our colony, as well as some ex- alleles. For example, the Mamu-DRB6 locus, although it may be amples of DRB region configurations displaying allelic polymorphism (12, 26, transcribed (33, 34), does not seem to code for a functional class II 28, 31). gene product, because its exon 2 sequences show various character- RNA extraction, cDNA synthesis, and amplification istics such as inserts, stop codons, and deletions that would render it RNA was extracted from immortalized B cell lines of rhesus macaques a pseudogene (24). Only for some alleles of the Mamu-DRB1*03, using the RNeasy mini kit (Qiagen, Hilden, Germany) according to the -DRB1*10, -DRB1*04,-DRB*W3, DRB*W4, and -DRB*W5 lin- manufacturer’s recommendations. cDNA was then synthesized from eages, immunoprecipitation studies suggested that these particular al- freshly extracted mRNA using the Universal RiboClone cDNA Synthesis leles code for a class II molecule (12, 35). Restriction element studies System (Promega, Madison, WI) according to the manufacturer’s recom- revealed that certain alleles of the -DRB1*03 lineage, as well as mendations. Full-length Mamu-DRA sequences were amplified by PCR from cDNA using primers specific for human DRA 5Ј and 3Ј untranslated -DRB1*0406 and -DRB*W201, encode gene products which are able sequences (1): 5ЈDRA-SalI, 5Ј-TCC CGT CGA CCG CCC AAG AAG ϩ to present peptides to CD4 Th cells (1, 36, 37). Only for these last AAA ATG GCC-3Ј, and 3ЈDRA-BamHI, 5Ј-CAT TGG ATC CGA AGT two alleles, and one particular -DRB1*03 allele, has the complete TTC TTC AGT GAT CTT-3Ј. cDRB sequence been published (23). Mamu-DRB1*0406, and espe- Likewise, Mamu-DRB sequences were amplified by PCR using primers specific for human 5Ј- and 3Ј-untranslated sequences (1): 5ЈDRB-SalI, 5Ј- cially -DRB*W201, seem to be significant restriction elements in cel- GCC CGT CGA CCT GTC CTG TTC TCC AGC ATG-3Ј, and 3ЈDRB- Downloaded from lular response to conserved regions of SIV/simian human immuno- BamHI, 5Ј-GGC GGG ATC CCT TTT CAT CCT GCA AAG CTG-3Ј. deficiency virus (38, 39). Primers were synthesized by Invitrogen (Paisley, U.K.). PCR was performed To assign a full-scale analysis of the Mamu-DR region genetic in a 100 ␮l reaction volume containing5UofTaq polymerase (kindly donated by M. Mo¨rl, Max-Planck-Institut, Saarbru¨cken, Germany) with 0.5 ␮Mof makeup and the transcription of its genes, we analyzed cDRA and ␮ ϫ each primer, 1.5 mM MgCl2, 250 M dNTPs, 1 PCR buffer II (Applied cDRB alleles present on the most prominent -DR region configu- Biosystems, Foster City, CA) , and 10 ␮l of DNA. The cycling parameters rations in our pedigreed colony of rhesus macaques. were a 2 min at 94°C initial denaturation step, followed by 25 cycles ofa2min http://www.jimmunol.org/ at 94°C denaturation step, a 2 min at 60°C annealing step, and a 2 min at 72°C Materials and Methods extension step. A final extension step was performed for 7 min at 72°C. Animals and cells Cloning and sequencing The rhesus macaques (Macaca mulatta) were serologically typed for their PCR products were digested with the restriction enzymes SalI and BamHI MHC class I (Mamu-A and -B) and class II (Mamu-DR) Ags. In the Bio- (Invitrogen). The 5Ј SalI and 3Ј BamHI restriction sites facilitated sticky-ended medical Primate Research Center breeding colony (Rijswijk, The Nether- ligations into the multiple cloning site of the sequencing vector M13mp18 lands), 253 MHC haplotypes have been defined based on the segregation of (Qbiogene, Montreal, Canada). The M13mp18 vector includes a M13 se- 13 Mamu-A, 14 Mamu-B, and 9 Mamu-DR serotypes. Peripheral blood lym- quence before the cloning site, which is used for the sequencing of the product. by guest on September 25, 2021

FIGURE 1. Polymorphic sites of the full-length coding Mamu-DRA nucleotide sequences (A) and deduced amino acid sequences (B) in comparison to HLA-DRA and Patr-DRA. Only polymorphic sites are shown using small and capital letters, which indicates synonymous and nonsynonymous mutations, respectively. Identity to the consensus is illustrated by a dash. The Mamu-DRA*0101 sequence published earlier is boxed (23). The two shadowed boxes represent two distinct lineages. 4 Patr, Pan troglodytes; 5 LP, leader peptide; 6 TD, transmembrane domain; 7 CD, cytoplasmatic domain. 6154 MAMU-DR REGION

sequences have been deposited in the databank (cDRA accession numbers: AJ586874–AJ586884, and cDRB accession numbers: AJ601348–AJ601351, AJ601354–AJ601362, and AJ601364–AJ601372) and are also available via the IMGT/MHC database (www.ebi.ac.uk/ipd/mhc/nhp; European Bioinfor- matics Institute, Cambridge, U.K.). Phylogenetic analysis Phylogenetic analysis of the cDRA and cDRB sequences was performed using PAUP, version 4.0b.10 (40). Pairwise distances were calculated us- ing the Kimura-2 parameter, and the neighbor-joining method was used to create a phylogram. Confidence estimates of the groupings were calculated according to the bootstrap method generated from 1000 replicates. Results and Discussion Mamu-cDRA alleles As in humans, the Mamu-DRA gene encoding the DR ␣-chain is thought to be monomorphic. However, only one study on a full- length cDRA sequence has been published (23) and to our knowl- edge, population analyses have not been conducted. To investigate the existence of Mamu-DRA polymorphism, full-length cDRA al- Downloaded from leles of 17 selected B cell lines have been amplified and se- quenced. This enterprise resulted in the detection of 11 unpub- lished cDRA alleles. Based on deduced amino acid sequences, four distinct Mamu-cDRA transcripts could be distinguished in our FIGURE 2. Phylogenetic tree of HLA-, Patr-, and Mamu-cDRA alleles. panel named Mamu-DRA*0102 to -DRA*0105 (Fig. 1, A and B). As in Fig. 1, the shadowed boxes represent two distinct lineages. The tree The existence of the previously reported Mamu-DRA*0101 allele http://www.jimmunol.org/ is rooted with Patr-DRA as the outgroup. 4 Patr, Pan troglodytes. could not be confirmed in our panel of Indian monkeys, but its sequence is nevertheless included in the alignments. Most of the alleles have point mutations with a synonymous character, as is The purified cDNA was sequenced on the ABI 3100 genetic analyzer (Applied reflected in the names of the alleles: Mamu-DRA*01021 to Biosystems, Foster City, CA) using 0.2 ␮M M13 primer, 1 ␮l of BigDye Terminator (Applied Biosystems), and 2 ␮lof5ϫ dilution buffer (400 mM -DRA*01027 (30). The end of exon 3 of the Mamu-DRA alleles is ␮ characterized by the presence of two different motifs (Fig. 1A). Tris-HCl, 10 mM MgCl2) in a total of 10 l. The resulting sequences were analyzed using the Sequence Navigator program (Applied Biosystems). All Phylogenetic analysis demonstrates that, based on the presence of by guest on September 25, 2021

FIGURE 3. Deduced amino acid sequences of full-length Mamu-cDRB alleles. The HLA-DRB1*0101 sequence is chosen as reference. Identity to the consensus is illustrated by dashes. The three Mamu-DRB alleles published earlier are boxed (23). The underlined and shadowed amino acid sequences of ␤ the 1 domain, represent the motifs differentiating the lineages. CP, Connecting peptide; TD, transmembrane domain; CD, cytoplasmatic domain. The Journal of Immunology 6155

␤ these motifs, the Mamu-DRA alleles cluster into two distinct lin- and cytoplasmatic domain. As expected, the 1 domain encoded eages (Fig. 2). Based on the generally accepted divergence time of by exon 2 represents the most polymorphic part of the -DRB gene. rhesus monkeys and humans of 35 million years, the mean evo- Most of the residues that are known from the HLA-DRB1 molecule lutionary rate of HLA-DRA and Mamu-DRA alleles can be calcu- to contribute to the peptide binding are variable in the rhesus ma- lated to be ϳ0.31 ϫ 10Ϫ9 substitutions per site, per year (41). The caque (45). Phylogenetic analysis shows that alleles of the same divergence time of the two Mamu-DRA lineages is then calculated lineage within one species, for example, members of the Mamu- to be Ͼ10 million years. Thus, the Mamu-DRA lineages seem to be DRB*W6 lineage, cluster tightly together. A similar observation relatively old and, as a consequence, they may also be present in other can be made for rhesus macaque, chimpanzee, and human -DRB5 macaque and Old World monkey species. The Mamu-DRA*01041 lineage (Fig. 4). If sequences fall apart in the phylogenetic anal- and -DRA*01042 alleles code for the same protein (Fig. 1B). How- ysis, an identical or similar motif constituted by aa 9–13, has been ever, they do group into different lineages (Figs. 1A and 2). This the decisive factor for lineage designation. (Figs. 3 and 4). example illustrates that the boxed polymorphic motif probably has been exchanged in a recombination-like event (Fig. 1A). Hence, at this Mamu-DR configurations stage we have decided not to implement a nomenclature protocol that Although Mamu-DRB sequences show a high degree of variability, discriminates between the two Mamu-DRA lineages. the main feature is its unprecedented -DRB region configuration Whereas the synonymous mutations are randomly distributed polymorphism, which is defined by a variable number and content over the entire cDRA sequence, the nonsynonymous mutations ob- of -DRB genes per haplotype (28). Animals selected for this study served in the rhesus macaque panel result in amino acid replace- possess the 11 most prominent Indian DRB region configurations ments restricted to the leader peptide or the cytoplasmic domain in our colony and each configuration harbors 2–4 -DRB genes (28, Downloaded from (Fig. 1, A and B). If one takes the HLA-DR ␣-chain as a reference, 31). In addition, nearly every Mamu-DRB configuration contains the chimpanzee orthologue displays only two polymorphic resi- 1–3 DRB6 genes, most of them characterized by a 62 bp deletion dues, whereas in total, 16 aa replacements have been observed in (24, 26). The choice of animals, which are mainly homozygous for the Mamu-DR ␣-chains (Fig. 1B). Humans, chimpanzees, and rhe- their MHC, allowed us to define the DRA/DRB gene combinations sus macaques shared a common ancestor Ͼ35 million years ago. segregating on one haplotype (Fig. 5). As can be seen, the 11

Because all the anchor residues of the ␣1 domain have been con- Mamu-DRB region configurations analyzed previously could be http://www.jimmunol.org/ served in all three primate species studied (Fig. 1B), this observa- divided into 12 different DRA/DRB combinations. Only one -DRB tion underlines the importance of these specific amino acids for peptide binding. The question left to answer is why do rhesus macaques display polymorphism at the Mamu-DRA gene, and is a similar phenomenon apparently not the case for HLA-DRA gene in the human population? One argument could be that not many hu- man subjects have been studied for HLA-DRA polymorphism. A more plausible explanation takes the differential divergence times of both species into account, which is thought to be 250,000 years by guest on September 25, 2021 for modern humans and ϳ700,000 years for rhesus monkeys (32, 42, 43). In brief, the time period needed to accumulate point mu- tations was approximately three times longer for rhesus macaques. On top of that, and as will be discussed later in detail, the Mamu- DRA alleles appear to be more or less Mamu-DR configuration- specific. This suggests that some of these DR configurations may have been stable entities over a relatively long evolutionary time span. Mamu-cDRB alleles To date, 134 Mamu-DRB exon 2 sequences have been identified (30). Most of these alleles belong to loci/lineages that are shared between humans and rhesus macaques, whereas for the DRB*W alleles, no human equivalents have been described. Little is known about the gene products (35) and only a meager number of full- length cDRB sequences have been published so far (23, 37). To learn more about the peptide-binding profiles of rhesus macaque class II molecules it is, in the first place, essential to know if the class II genes are actually expressed. An example is provided by the Mamu-DRB*W201 molecule, which plays an important role in the peptide binding of conserved epitopes of the simian human immunodeficiency virus (39, 44). To obtain more fundamental insight into the genetics of the Mamu-DR region and the expression status of the various genes, cDRB genes were analyzed in a panel of 15 animals with thor- oughly defined DRB configurations. From these animals, 28 cDRB FIGURE 4. Phylogenetic tree of HLA-, Patr-, and Mamu-cDRB alleles. alleles could be isolated, and the deduced amino acid sequences Some selected HLA-DRB and Patr- alleles, representing different lineages, have been determined (Fig. 3). Moderate heterogeneity is observed have been added to the phylogenetic analysis. The tree was rooted with the ␤ within the 2 domain, whereas relatively little variation is noticed -DRB orthologue, Sanguinus oedipus -DRB*02, of the cotton-top tamarin in the leader peptide, connecting peptide, transmembrane domain, as the outgroup. 9 Saoe, Sanguinus oedipus. 6156 MAMU-DR REGION

FIGURE 5. Overview of the gene content of Mamu-DRA/DRB configu- rations. The different configurations are numbered arbitrarily in Arabic nu- merals; same configurations with al- lelic variations are indicated by A and B, respectively. Expressed loci/alleles are depicted in black, whereas pseudo- genes are boxed. aOnly DRB6 alleles without the 62-bp deletion are listed. bMHC homozygous animals from con- sanguineous matings. cMHC homozy- gous animals. dDRA allele belonging to this confirmation was detected by se- quencing analysis of genomic DNA. Downloaded from eThe serological Mamu-DR nomencla- ture is described by Bontrop et al. (46). http://www.jimmunol.org/ region configuration displayed limited allelic variation at the DRB to be due to a glacial barrier during the Pleistocene era, the prob- loci (Fig. 5; 1a, 1b, 2a, and 2b), which can be separated into two able inactivation of a formerly active gene took place before that DRA/DRB configurations according to different accompanying time point (32). In the case of the MHC, it has been shown that -DRA alleles. The DRB combinations 1a/1b and 2a/2b differ only pseudogenes are maintained over long evolutionary time spans. for one nucleotide in their DRB1*03 or DRB1*10 lineage alleles, This may be due to a “piggy back” effect (one gene is linked to respectively. This is most likely an example of a polymorphism another that is experiencing strong conservative or positive selec- that was generated after the DRB region configurations themselves tion). It has also been hypothesized that pseudogenes are a reser- by guest on September 25, 2021 were established. We have postulated that the differential numbers voir for gene segments that can be exchanged between related of Mamu-DR genes and their order is due to rearrangements by genes by recombination-like processes. In contrast, some pseudo- unequal crossing-over events (12, 26). In this light, the following genes contain premature stop codons and may be partially trans- observation is highly indicative. In one rhesus macaque of Bur- lated into proteins. In such a case, a pseudogene may encode pep- mese origin, three different -DRA alleles have been detected, and tides that are important for thymic education/selection. the presence of two -DRA alleles on one haplotype was proven by In five other -DRA/DRB conformations (Fig. 5; n ϭ 3, 6, 9, 11, segregation analysis (data not shown). This unique configuration is and 12), there is one -DRB allele of which the exon 2 sequence of probably caused by an unequal crossing-over event, once again genomic DNA was sequenced, but a transcript has not been detected. illustrating the great plasticity of the Mamu-DR region. Future The results have been confirmed by analysis of a second animal with plans to initiate the sequencing of the whole genome of the rhesus the same region configuration and whenever necessary, they have macaque will elucidate the order of the genes and shed light on the been repeated (data not shown). These untranscribed alleles belong to recombination hotspots and physical distances between the differ- various loci/lineages that have most likely been inactivated and now ent loci. On average, one can conclude, however, that most of the can be considered as pseudogenes. This phenomenon is known from DRB configurations segregate in combination with a unique the human situation where DRB6 as well as other DRB loci, for ex- Mamu-DRA allele (Fig. 5). ample DRB2, are rendered as pseudogenes. However, in rhesus macaques, a certain -DRB locus or even a DR region: transcripts and pseudogenes lineage may harbor transcribed, as well as untranscribed, alleles Some of the -DRB exon 2 sequences detected by amplifying (pseudogenes); examples are given for -DRB1*03,-DRB3, and genomic DNA could not be recovered at the transcript level. In the Mamu-DRB*W6 (Fig. 5; n ϭ 3, 4, 6, 9, and 11). In relation to HLA, case of the configuration n 4, the Mamu-DRB6*0101 and the Mamu-DRB5 locus appears to be coding, whereas none of the -DRB1*0309 genes are not transcribed in such a way that they -DRB6 alleles was completely transcribed, which is an indication result in a functional ␤-chain (Fig. 5). This result was confirmed by that this locus is a pseudogene in the rhesus macaque as well. The the analysis of cDRB of a Burmese-origin rhesus macaque family, number of Mamu-DRB genes that are transcribed into RNA can in which exon 2 of the DRB1*0309 allele can also be detected on vary from 1 to 3 per haplotype. the genomic, but not on the cDNA level. These results have been The results of this study provide a detailed answer to the question validated by dot-blot experiments (results not shown). In the case of which -DRB alleles are transcribed, and they unravel the complex- of the Mamu-DRB6 gene, this observation is not surprising be- ity caused by the large number of Mamu-DRB region configurations. cause this gene is a pseudogene in humans and chimpanzees (33). In conclusion, the data presented here will be invaluable in preclinical Because the separation of eastern (Burmese and Chinese) and studies in which a detailed knowledge of the rhesus macaque DR western (Pakistan and Indian) macaque populations is speculated region makeup is essential. The Journal of Immunology 6157

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