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 http://www.jimmunol.org/content/172/10/6152 Downloaded from References This article cites 45 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/172/10/6152.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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: 6152–6157. 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).