Class I MHC Expression in the Yellow David A. Sidebottom, Ronald Kennedy and William H. Hildebrand This information is current as of October 2, 2021. J Immunol 2001; 166:3983-3993; ; doi: 10.4049/jimmunol.166.6.3983 http://www.jimmunol.org/content/166/6/3983 Downloaded from References This article 41 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/166/6/3983.full#ref-list-1

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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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Class I MHC Expression in the Yellow Baboon1,2

David A. Sidebottom, Ronald Kennedy, and William H. Hildebrand3

MHC class I molecules play a crucial role in the immune response to pathogens and vaccines and in self/non-self recognition. Therefore, characterization of MHC class I gene expression of Papio subspecies is a prerequisite for studies of immunology and transplantation in the baboon (Papio hamadryas). To elucidate MHC class I expression and variation within Papio subspecies and to further investigate the evolution of A and B loci in Old World , we have characterized the expressed class I repertoire of the yellow baboon (Papio hamadryas cynocephalus) by cDNA library screening. A total of nine distinct MHC class I cDNAs were isolated from a spleen cDNA library. The four A alleles and four B alleles obtained represent four distinct loci indicating that a duplication of the A and B loci has taken place in the lineage leading to these Old World primates. No HLA-C homologue/ orthologue was found. In addition a single, nonclassical homologue of HLA-E was characterized. Examination of nucleotide and extrapolated protein sequences indicates that alleles at the two B loci are much more diversified than the alleles at the A loci. One of the A loci in particular appears to display very limited polymorphism in both Papio hamadryas cynocephalus and Papio hamadryas anubis subspecies. The failure to detect a homologue of HLA–C in the baboon provides additional evidence for the more Downloaded from recent origin of this locus in the Pongidae and Hominidae. Further comparative analysis with MHC sequences among the species reveals specific patterns of divergence and conservation within class I molecules of the yellow baboon. The Journal of Immunology, 2001, 166: 3983–3993.

lassical MHC class I glycoproteins are highly polymor- by the association of particular class I molecules with disease re- phic gene products that are expressed in all nucleated sistance and susceptibility (5). Extensive class I diversification is http://www.jimmunol.org/ C cells and play a central role in immune recognition and not a random or neutral evolutionary event. regulation. In primates and other , the classical MHC The number of class I genes in the MHC varies between species class I genes encode related families of cell surface molecules. (6), as does the extent of their degree of polymorphism (7). Class These class I proteins are characterized by a peptide binding region I cDNAs have been isolated from several species of great apes and responsible for the presentation of processed peptide fragments, Old World monkeys, including (8–12), pygmy chim- primarily generated by cytosolic protein degradation, to the recep- panzees (bonobos) (13, 14), gorillas (9), orangutans (15, 16), gib- tors of CD8ϩ T cells (1). In addition, MHC class I molecules bons (15), and rhesus (17, 18). Orthologues of HLA-A regulate the activity of NK cells via interaction with both inhibi- and HLA-B have been identified in every species of ape and Old by guest on October 2, 2021 tory and activating receptors. Thus, MHC class I molecules are World monkey examined, while orthologues of HLA-C have been inextricably linked in a coevolutionary scheme with different im- reported in the Hominidae and Pongidae (great apes), mune effector cells that interact with these MHC molecules. (8, 10), pygmy chimpanzee (bonobo) (13, 14), gorilla (9), and A functional hallmark of MHC class I proteins is sequence vari- orangutan (Pongidae) (16). To date the occurrence of an HLA-C- ability focused on the ␣1 and ␣2 heavy chain domains (2). Poly- like locus has not been determined in the Hylobatae (lesser apes). morphism in these two domains results in different class I mole- The failure to detect a homologue of HLA-C in Old World mon- cules binding different subsets of peptides for presentation on the keys, Cercopithecoids (17, 18), suggests differing pathways of cell surface. The nonrandom nature of ␣1/␣2 diversity is reflected class I evolution between Old World monkeys and the great apes in the prevalence of nonsynonymous over synonymous substitu- and humans following divergence of their common ancestor. tions at nucleotides encoding amino acid residues within this pep- Although the immune systems of and humans share tide binding region (3). Indeed, a predominance of nonsynony- substantial similarities, results in other primate species indicate mous substitutions has been cited as support for overdominant that the rapidly evolving MHC molecules will have diverged after selection (heterozygote advantage) at MHC class I loci (4). The 35 million yr of phylogenetic disparity (19). We initially used an functional significance of class I polymorphism is further revealed RT-PCR methodology using oligonucleotide primers specific for human class I molecules to sample class I gene expression in the (Papio hamadryas anubis) (20). Our preliminary data Department of Microbiology and Immunology, University of Oklahoma Health Sci- found three HLA-A homologues, two HLA-B homologues, and no ences Center, Oklahoma City, OK 73190 homologue of human HLA-C. By comparison, an accumulation of Received for publication August 30, 2000. Accepted for publication January 12, 2001. data in the rhesus monkey indicates that macaques have multiple The costs of publication of this article were defrayed in part by the payment of page HLA-B like loci and that macaques might vary the number of func- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tional class I loci from to animal. Although no HLA-C-like 1 This work was supported by a grant from the National Institutes of Health Baboon molecule has been reported in rhesus macaques, an HLA-C ortho- Research Resource Program (Grant RR12317-03/P40 to R.K.). logue has recently been found in ape species previously thought to 2 The nucleotide sequence data reported in this paper have been submitted to the predate the formation of HLA-C (16). Therefore, reported data dif- GenBank nucleotide sequence database and have been assigned accession numbers fer from animal to animal within the nonhuman primates sampled. AF288698 to AF288706. A picture of the nature and number of functional class I loci in 3 Address correspondence and reprint requests to Dr. William H. Hildebrand, Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, P.O. the nonhuman primates, including the baboon, is still emerging. Box 26901, Oklahoma City, OK 73190. E-mail address: [email protected] Although our previous characterization of the baboon class I MHC

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 3984 BABOON CLASS I MHC

molecules provided insight pertaining to the nature of polymor- primary library size) was amplified in a 50-␮l reaction mixture comprising phism in this species, the reliance on human PCR primers may 1ϫ Pfx PCR buffer (Life Technologies, Gaithersburg, MD), 1.0 mM MgCl ,50␮M of each dNTP (final concentrations) together with 20 pmol have biased our sampling of class I expression in the baboon. Full 2 of each required primer, and 1.25 U of Platinum Pfx DNA polymerase characterization of baboon MHC is a prerequisite for addressing (Life Technologies). The PCR profile used was an initial denaturation at questions on immunologic mechanisms influencing cross-species 95°C for 2 min, 35 cycles of 15 s at 94°C, 30 s at 55°C, and 68°C for 1 min MHC restriction, and knowledge of interspecies variation in pep- and 20 s in a Perkin-Elmer 9700 thermocycler (Foster City, CA). tide pocket architecture is essential for extrapolation of primate- derived vaccine test data to human systems. Here, we performed Cloning and sequencing screening of a yellow baboon (Papio hamadryas cynocephalus) PCR products were cloned into vector pCR-Blunt II (Invitrogen, San Di- spleen cDNA library to facilitate full characterization of the ex- ego, CA). Plasmids were isolated from individual bacterial clones using the pressed class I repertoire. The resulting MHC class I data in the Wizard SV mini-preps DNA purification system (Promega, Madison, WI). baboon are discussed in terms of current knowledge in humans and other nonhuman primate species. Sequence analysis Resulting sequences were assembled with GCG fragment assembly soft- Materials and Methods ware (Wisconsin Package version 10.0, Genetics Computer Group, Mad- Isolation and characterization of full-length Pacy MHC class I ison, WI). Characterized alleles were named according to the criteria sug- alleles: cDNA library screening gested by Klein et al. (23). A baboon spleen ␭Zap II cDNA library constructed with mRNA isolated from an 18-yr-old Papio hamadryas cynocephalus male (Stratagene, La Phylogenetic/evolutionary genetic analysis ϫ 5 Downloaded from Jolla, CA) was screened for full-length MHC class I cDNAs (6 10 Phylogenetic trees were constructed based on individual exons and full- ␭ plaques). The library had been through one round of amplification. Zap II length coding sequence for comparison using the program PAUP (phylo- phage were plated and plaque lifts were performed according to standard genetic analysis using parsimony and other methods) 4.0b4a (David Swof- procedures (21). Duralon-UV filters (Stratagene) were hybridized with a ford, University of Illinois, Urbana, IL) using the neighbor-joining tree previously characterized, fluorescein-labeled, full-length Papio hamadryas construction method (24). Genetic distances were estimated using the anubis baboon class I cDNA isolate (GenBank accession no. U35624). Jukes-Cantor distance measure (25). Bootstrap analysis was performed Homologous plaques were identified by screening hybridized filters on a (10,000 replicates) to assign confidence to tree branch nodes (26). Only Storm 860 fluorescent imaging system (Amersham Pharmacia Biotech, Pis- nodes supported by 50% bootstrap support or greater were included. Rates http://www.jimmunol.org/ cataway, NJ) according to the manufacturer’s protocol. Picked plaques of synonymous and nonsynonymous substitution and associated variance were resuspended in SM buffer, and size screened for insert by PCR with were calculated according to the methods of Nei and Gojobori (27) and Nei Ϫ ␮ M13 universal ( 20) and reverse primers. A total of 5.0 l of PCR product and Jin (28), respectively, using MEGA (molecular evolutionary genetic from positive reactions was incubated for 15 min at 37°C with2Uof analysis) software (University of Pennsylvania, Philadelphia, PA) (29). shrimp alkaline phosphatase and 10 U of exonuclease I to remove unin- Jukes-Cantor distances were calculated from aligned sequences (GCG) for corporated dNTPs and residual single-stranded PCR primers, respectively all pairwise comparisons of human A, B, and C locus alleles for which (U.S. Biochemical, Cleveland, OH). The resulting mixture was subse- complete exon 2 and 3 sequence data were available (IMGT/HLA data- quently diluted 10-fold with H2O, and the PCR products were cycle se- base) (30), and Pacy/Paan (baboon) class I alleles using PAUP 4.0b4a quenced bidirectionally, using nested, vector-specific Cy5-labeled SK and (31). The numbers of nucleotide substitutions per site (Jukes-Cantor dis- KS sequencing primers with a Thermo Sequenase-labeled primer cycle ␣ tance) in exons encoding the 2 domain were plotted as a function of the by guest on October 2, 2021 sequencing kit (Amersham Pharmacia Biotech) according to the manufac- corresponding nucleotide substitution rate per site within ␣1 to facilitate turer’s instructions. Resulting fragments were loaded and electrophoresed comparison of the range of intra- and interlocus polymorphism together on an ALFexpress automated sequencer (Amersham Pharmacia Biotech). with relative substitution rates within ␣1 and ␣2. Scatterplots were con- On selected plaque isolates demonstrated to contain Pacy MHC class I structed using Statistica version 5.5 (Statsoft, Tulsa, OK). inserts, in vivo excision of pBluescript SKϪ phagemid from ␭Zap II vector was performed to generate a plasmid stock, using ExAssist interference- resistant helper phage (Stratagene) with Escherichia coli SOLR host strain GeneBank accession numbers (Stratagene). Confirmatory sequencing on excised clones was performed using vector-specific (pBluescript) SK and KS Cy5-labeled sequencing Names for alleles characterized in nonhuman primates are assigned ac- primers in combination with Cy5-labeled HLA class I internal sequencing cording to the convention recommended by Klein et al. (23), with the first two letters of the genus name being combined with the first two letters of primers 4N and 3S (22) to generate full-length bidirectional sequence on ϭ each class I clone. the species or subspecies name as appropriate (i.e., Hyla Hylobates la; Mafa ϭ Macaca fascularis; Mamu ϭ Macaca mulatta; Paan ϭ Papio Confirmatory PCR amplification of isolated Pacy class I alleles hamadryas anubis; Pacy ϭ Papio hamadryas cynocephalus, Patr ϭ Pan troglodytes; Papa ϭ Pan paniscus; Popy ϭ Pongo pygmeaus; Gogo ϭ To control for reverse transcriptase errors and cloning artifacts, confirma- Gorilla gorilla). Sequences reported in the text have been submitted to tory amplification and sequencing of isolated Pacy MHC class I alleles GenBank and have been assigned the following accession numbers: Pacy- were performed. Full-length baboon MHC class I molecules were ampli- A*01, AF288698; Pacy-A*02, AF288699; Pacy-A*03, AF288700; Pacy- fied from the ␭Zap II cDNA library, using locus-specific primer pairs (Ta- A*04, AF288701; Pacy-B*01, AF288702; Pacy-B*02, AF288703; ble I) specifically designed to anneal to sequences located within the 5Ј- Pacy-B*03, AF288704; Pacy-B*04, AF288705; and Pacy-E*01, and 3Ј-untranslated regions of previously isolated and characterized Pacy AF288706. Human HLA sequences described on phylogenetic trees were class I alleles, thereby facilitating isolation of the full-length coding se- downloaded from the IMGT/HLA database (30). Accession numbers for quence. DNA template corresponding to 2.0 ϫ 107 PFU (10-fold above other sequences used in the construction of phylogenetic trees are as follows:

Table I. Locus-specific primer pairs used in confirmatory PCR amplification of isolated alleles

Locus Primer Pair Specificity Sequence (5Ј-3Ј)

OWP5ЈUTA A locus GCG CGT CGA CCC CAG ACG CCG AGG ATG GC OWP3ЈUTA AG GCG TGA ACA AAT CTT GCC TCG OWP5ЈUTB B locus GGA GAG TCT CCT CAG ACG CCG A OWP3ЈUTB GC CAG AGG CTC TTG AAG TCA CAA OWP5ЈUTE E locus AGT CAG GGG CTC GCA CGA AGC OWP3ЈUTE AAG AAA TCC TGC ATC TCA GTC G The Journal of Immunology 3985

Hyla-A*01, U50089; Hyla-B*01, U50091; Gogo-A*03, X54375; Gogo- B*0101, X60255; Gogo-B*0205, X60253; Gogo-C*0105, X60252; Gogo- C*0205, AF118898; Mamu-A*01, U50836; Mamu-A*02, U50837; Mamu- A*03, U41379; Mamu-A*04, U41380; Mamu-A*05, U41831; Mamu-A*06, U41834; Mamu-A*07, U41832; Mamu-B*01, U42837; Mamu-B*02, U41833; Mamu-B*03, U41825; Mamu-B*04, U41826; Mamu-B*05, U41827; Mamu- B*06, U41828; Mamu-B*07, U41829; Mamu-B*08, U41830; Mamu-I*01051, AF161865; Mamu-I*02012, AF161869; Mamu-I*03051, AF161870; Mamu- I*10, AF161878; Mamu-E*05, U41837; Mafa-E*01, U02976; Mafa-E*01, U02977; Mamu-F, Z21819; Mamu-AG*0202, U84786; Mamu-AG*03011, U84787; Paan-A*01, U35624; Paan-A*02, U35625; Paan-A*03, U35626; Paan-B*01, U35627; Paan-B*02, U35628; Paan-AG*01, AF059191; Paan- AG*02, AF059192; Papa-A*01, L39093; Papa-B*03, U05575; Papa-B*04, U05577; Patr-A*14, U10544; Patr-B*02, X13116; Patr-B*10, U05582; Patr- H*01, AF157393; Popy-A*01, M30680; Popy-B*01, U50086; Popy-B*0701, AF118895; and Popy-C*0205, AF118897.

Results Isolation of nine baboon (Papio hamadryas cynocephalus) MHC class I cDNAs from a spleen cDNA library A total of nine distinct MHC class I molecules were identified in the course of cDNA library screening (four A, four B, and one E Downloaded from locus allele). To confirm the fidelity of the clones, we designed Pacy A, B, and E locus-specific oligonucleotide primers based on 5Ј/3Ј-untranslated region consensus sequences within each Pacy A, B, and E clone identified during the screening of the cDNA library. These oligonucleotide primers were used to amplify, clone, and sequence the respective class I cDNAs from the cDNA library. http://www.jimmunol.org/ Every class I molecule identified through screening of the cDNA library was confirmed with the PCR amplification. No additional FIGURE 1. Alignment of predicted amino acid sequences of Papio class I molecules were identified during the course of confirmatory hamadryas cynocephalus, Papio hamadryas anubis, Macaca mulatta, and PCR screening. The overall sequence alignments of the expressed selected human class I alleles. A dash denotes positional identity with Pacy class I alleles isolated are shown in Fig. 1 and include MHC consensus; a dot signifies gaps introduced for optimal alignment (truncated class I alleles isolated from the rhesus (Macaca mulatta; leader sequence/cytoplasmic domain; B loci); shaded areas denote locus- animal 88090) (17, 18) and selected human MHC class I alleles for specific deletions, group A1 alleles and E locus. comparison. The predicted protein products of the characterized by guest on October 2, 2021 nucleotide sequences range in length from 359 to 365 aa in length. Alleles Pacy-A*03 and -A*04 encode 365-aa heavy chains, while E*01, a characteristic of numerous human B locus alleles not all other Pacy class I alleles encode shorter heavy chains. Leader previously reported in other primate E locus alleles (33). sequence deletions result in Pacy-E*01 being 359 residues in As with other human/nonhuman primate class I MHC mole- length, and a two-codon deletion in the transmembrane regions of cules, a putative glycosylation site is located at residue 86. In ad- Pacy-A*01/A*02 translates into a 363-aa heavy chain. As in hu- dition, conserved cysteine residues occur at positions 101 and 164 mans, all Pacy-B alleles characterized have a shortened cytoplas- in ␣2 and at positions 203 and 259 in ␣3. Other areas of similarity mic tail compared with A and E loci. The Pacy-B loci molecules with human class I molecules include a region of variability at lack exon 8 due to the occurrence of a stop codon just before the residues 77–83 near the C terminus of the ␣1 ␣ helix, analogous end of exon 7, a characteristic of human B loci, resulting in Pacy- to the site of the Bw4/Bw6 motif in humans. Positions of novel B*01 and -B*02 being 362 aa in length. Combined leader and Pacy amino acid polymorphism in the animal studied, otherwise cytoplasmic tail region deletions result in Pacy-B*03/B*04 being conserved in orthologous human alleles, occur at residue positions 359 residues in length. Deletions and insertions located at three Ϫ13, Ϫ9, 1, 6, 23, 34, 45, 50, 121, 157, 165, 178, 197, 206, 231, positions within leader, transmembrane, and cytoplasmic regions 267, 268, 291, 296, 303, and 315 within Pacy-A loci and at posi- therefore distinguish the four pairs of baboon A and B molecules. tions Ϫ19, Ϫ5, 5, 6, 10, 18, 21, 34, 48, 50, 75, 79, 98, 102, 105, Pacy-A*01 and Pacy-A*04 were identical with alleles Paan-A*03 107, 111, 121, 128, 135, 141, 142, 146, 150, 151, 155, 174, 182, (accession no. U35626) and Paan-A*02 (accession no. U35625), 191, 196, 214, 220, 223, 236, 263, 264, 283, 294, 308, 315, 320, respectively, previously characterized in the olive baboon (Papio 326, 329, and 335 within Pacy-B loci. hamadryas anubis) (20). The reading frame of the single Pacy-E molecule isolated starts Comparative phylogenetic analysis of isolated MHC class I at the second methionine in the overall alignment. Such a nested alleles of the yellow baboon start codon is identical with those seen in Pacy-B*03/B*04 alleles Phylogenetic and sequence comparisons were performed to re- and in human and macaque E locus leader sequences. The Pacy- late class I molecules in the yellow baboon with previously E*01 allele characterized also contains an additional 9-nucleotide/ characterized Hominoid and Cercopithecoid class I molecules. 3-aa deletion (Ϫ8toϪ10) identical with that seen in macaque E Numerous analyses including phylogenetic (24), distance mea- locus alleles, not seen in humans. Pacy-E*01 differs from HLA- sure (25), and BLAST (34) unanimously grouped the Pacy-A E*01 by 16 residues within ␣1 and ␣2 domains (Fig. 2), but only and Pacy-B alleles into A and B lineages together with other a single predicted peptide pocket change of threonine for isoleu- orthologous nonhuman primate class I MHC sequences (Fig. 3 cine occurs at position 73 (32). Of additional interest is the occur- and data not shown). We first focused our phylogenetic com- rence of a cysteine residue within the cytoplasmic domain of Pacy- parisons upon class I MHC molecules for which a full-length 3986 BABOON CLASS I MHC Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. Continued. sequence had been reported. The phylogenetic neighbor-joining (Papio hamadryas anubis)orMamu (Macaca mulatta) alleles. tree (24) depicted in Fig. 3 demonstrates that Pacy-A*01/Paan- The Pacy-B*01, -B*02, -B*03, and -B*04 group with macaque A*03 and Pacy-A*02 group together on their own branch, B locus molecules on a branch just below the ape and human B whereas Pacy-A*03 and Pacy-A*04 group with existing Paan locus allele clusters. Pacy-B*02 and Pacy-B*03 group together The Journal of Immunology 3987 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. Continued. with Paan-B*01 and Paan-B*02 with strong bootstrap support, gether with Mamu-I variants. Mamu-I is a novel macaque MHC in contrast to Pacy-B*01 and -B*04, which reside with Mamu- class I B-related locus that exhibits unusually low variability B*02 and -B*04. None of the Pacy-B locus alleles groups to- (18). Sequence homology analysis of Papio class I sequences 3988 BABOON CLASS I MHC

92.4%; mean, 89.9%; SD, 2.0%). In contrast, Pacy-E*01 exhibits a GC value of 83.0%, substantiating the nonclassical nature of Pacy-E*01.

Specific Pacy classical class I loci exhibit a high degree of intralocus variability Comparisons of intra-allelic variability reveal a high degree of divergence between specific Pacy class I alleles even within as- signed allelic groups (Table II). Pairwise differences between full- length Pacy group A2, B1, and B2 alleles reveal large intra-allelic differences: A*03/A*04, 65 nucleotides; B*01/B*02, 70 nucleo- tides; and B*03/B*04, 98 nucleotides. Scatterplots further reflect the high level of intralocus diversity of Pacy alleles within ␣1 and ␣2 domains (Fig. 4). The number of nucleotide substitutions per site (Jukes-Cantor distance) (25) in the exons encoding the ␣2 domain (y-axis) are plotted as a function of ␣ FIGURE 2. Ribbon diagram of characterized Pacy-E*01 allele, high- substitutions in the 1 domain (x-axis). The described prediction lighting foci of polymorphism and position residue divergence from human interval ellipse for human HLA-A, -B, and -C loci depicts the area

E*0101 within ␣-1/␣-2 domains. for each locus in which a single new observation can be expected Downloaded from to fall with 95% probability and illustrates the extent of variation apparent at each classical human class I locus. All pairwise in- with human A, B, C, and E locus consensus sequences and in- tralocus comparisons among human A, B, and C loci provide for terlocus comparisons of Pacy vs human exon 3 Jukes-Cantor comparison to the limited numbers of alleles at Pacy-A loci and distance measures additionally confirmed the Pacy class I locus Pacy-B loci. Fig. 4 first demonstrates that Pacy-A locus intra/in- designations (data not shown). terlocus allelic variation lies at the upper range of human intralo- http://www.jimmunol.org/ ␣ ␣ Exons 4–8 of HLA class I genes generally delineate loci and cus variability. Furthermore, differences within 1 and 2 domains lineages in contrast to exons 1–3, which reflect alleles and allele on pairwise comparison of the Pacy-B loci alleles lie beyond the ␣ families. Fifty percent bootstrap consensus tree construction ex- normal human range, particularly within 2 (combined intra/inter- cluding exons 2 and 3 resulted in a tree with little substructure locus comparisons within orthologous group). For all comparisons ␣ resembling a “lawn” rather than a tree, as did the exon 3 only tree. within Pacy-A and -B loci, differences within 2 exceed those ␣ The corresponding exon 2 tree demonstrated increased substruc- within 1. ture compared with the exon 3 tree or with trees excluding exon 2 Fig. 4 (ii, iii, and iv) further demonstrate that Pacy-A and -B loci ␣ and 3, but produced a topology analogous to the overall tree, with share greater homology with HLA-A within their 1 domains and ␣ by guest on October 2, 2021 reduced discrimination. Pacy groupings observed on the full- greater homology with HLA-B and HLA-C within their 2 do- length tree were mirrored on the additional bootstrap trees con- mains. We first observed this trend for closer homology with hu- ␣ ␣ structed with no new allelic groupings with strong bootstrap sup- man B and C loci within 2 than 1 domains when phylogenetic port apparent. trees were constructed with individual exons (data not shown). In the majority of comparisons, Pacy class I alleles share greater ho- The isolated classical Pacy class I alleles reflect four mology with human A locus alleles within ␣1. In corresponding functional loci scatterplots of ␣2 Jukes-Cantor distance plotted as a function of ␣3 Jukes-Cantor distance, the range of ␣3 variation observed on Pacy On the basis of characteristic sequence deletions, the isolated al- intra/interlocus comparison lies within the range or variation ob- leles may be paired into allelic groups A1 (Pacy-A*01/Pacy-A*02), served in corresponding comparisons at human classical class I A2 (Pacy-A*03/Pacy-A*04), B1 (Pacy-B*01/Pacy-B*02), and B2 loci (data not shown). Therefore, Pacy class I alleles tend to be (Pacy-B*03/Pacy-B*04). Assigning alleles to loci on the basis of HLA-A-like in ␣-1, HLA-B/C-like in ␣-2, and like their ortholo- sequence homology, transmembrane deletion pattern, and differ- gous allelic HLA class I counterparts throughout the remainder of ences observed in pairwise nucleotide comparison (Table II) their coding sequence. places Pacy alleles A*01/A*02 and Pacy-A*03/A*04 at separate loci. Assignment of B locus alleles to individual loci on the basis of deletion pattern and homology designates B*03 and B*04 at one The high degree of intralocus variability is reflected in the loci and B*01 and B*02 at another. However, overall phylogenetic peptide pocket architecture analysis (Fig. 3) and differences observed in pairwise nucleotide The overall high degree of intra-allelic group variation (A1, A2, comparison (Table II) indicate that alleles B*03 and B*04 at the B2 B1, and B2) observed when comparing full-length molecules is locus more closely resemble alleles B*02 and B*01 at the B1 locus, reflected in the ␣1 and ␣2 domain peptide binding pockets. Of the respectively, than each other. Segregation analysis is clearly re- six specificity pockets, A–F (36–38), that comprise the class I quired to substantiate allele assignment on the basis of the signal binding groove, the high degree of overall variability between al- peptide deletion profile. leles within the designated Pacy allelic groups is reflected in res- It has been demonstrated (35) that nonclassical HLA class I idue positions that constitute specificity pockets. In overall com- genes exhibit a reduced percent GC content at third codon posi- parisons of all Pacy classical class I molecules, 8 of 12 residues tions within the ␣1/␣2 domains. The percent GC compositions at that interact with P1 are polymorphic, and polymorphism occurs at third codon positions within the ␣1 and ␣2 domains of Pacy-A*01 other specificity pockets influencing: P2, 12/18 residues; P3, 11/13 and -A*02 alleles are 91.2 and 89.6%, respectively. This GC con- residues; P4, 7/8 residues; P5, 10/11 residues; P6, 14/18 residues; tent lies within the range of variation of the other isolated classical P7, 9/12 residues; P8, 5/8 residues; and P9, 3/19 residues. There- Pacy alleles A*03, A*04, B*01, B*02, B*03, and B*04 (87.9– fore, Pacy polymorphism is positioned to modify ligand binding. The Journal of Immunology 3989 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. Phylogenetic unrooted bootstrap tree of classical (A– C) and nonclassical (E–G) MHC class I genes from various Old World primate species (Catarrhines). Full-length nucleotide sequences from primate MHC class I alleles (leader/␣-1/␣-2/␣-3/trans-membrane/cytoplasmic domain) were used for tree construction. Corresponding segments were aligned using the default options of the program PILEUP (Wisconsin Package version 10.0, Genetics Computer Group, Madison, WI) and the phylogenetic tree constructed using PAUP 4.0b4a with the neighbor-joining method (Jukes-Cantor distance). The final 50% bootstrap consensus tree was displayed with TreeView version 1.6.1. Confidence values resulting from 10,000 replicates are denoted on the nodes. Only nodes supported by 50% bootstrap support or greater are included. The baboon alleles characterized in this study are highlighted in reverse text. Nonhuman primate alleles are named according to the accepted nomenclature recommended by Klein et al. (23), i.e., Papio hamadryas anubis ϭ Paan.

Discussion and evolutionary studies. The MHC of Papio species has not pre- Precise knowledge of the range and nature of the class I MHC viously been investigated by cDNA library screening. Therefore, molecules expressed in baboons is a prerequisite for medical and we screened a yellow baboon (Papio hamadryas cynocephalus) immunological applications in vaccine study, xenotransplantation, spleen cDNA library to identify the expressed class I repertoire 3990 BABOON CLASS I MHC

Table II. Pairwise differences in nucleotide sequence for characterized Pacy allelesa

Allelic Group (length/b.p.) Allele Pacy-A*01 Pacy-A*02 Pacy-A*03 Pacy-A*04 Pacy-B*01 Pacy-B*02 Pacy-B*03 Pacy-B*04 Pacy-E*01

A1 (n ϭ 1092) Pacy-A*01 0 Pacy-A*02 1 0 A2 (n ϭ 1098) Pacy-A*03 53 54 0 Pacy-A*04 59 60 65 0 B1 (n ϭ 1089) Pacy-B*01 119 120 116 124 0 Pacy-B*02 112 113 113 116 70 0 B2 (n ϭ 1080) Pacy-B*03 119 120 129 121 96 77 0 Pacy-B*04 125 126 131 130 79 93 98 0 E(n ϭ 1080) Pacy-E*01 143 144 151 165 150 156 165 152 0

a Intra-allelic group comparisons are in bold. and to understand the total number of expressed loci within this purifying selection (39) or because this locus arose from a recent animal. duplication (18). To date, substantial variability has been observed in the num- In contrast to Pacy group A1 alleles, the Pacy group A2 alleles ber of reported loci expressed in primates, especially class I loci (Pacy-A*03/04) appear more polymorphic, differing from each in the rhesus macaque in which A and B locus duplications are other by 65 nucleotides. Such disparity bears the hallmark of Ag- Downloaded from evident (7, 17, 18). Variability in expressed alleles and func- driven selection and meets or exceeds human HLA-A variation. tional loci has also been reported in other species of Old World Numerous polymorphic differences are evident within the ␣1 and primate, with multiple B locus duplications observed in the or- ␣2 ribbons; these differences are at the extremes of human ␣1/␣2 angutan (15, 16). Five functional class I loci are apparent in the variation evident from scatterplots (Fig. 4). Examination of the Papio subspecies studied, two functional A loci, two functional number of nucleotide substitutions and the frequency of synony- B loci, and one functional E locus. The A and B loci were mous/nonsynonymous substitution in the Ag recognition site fur- http://www.jimmunol.org/ heterozygous, while a single E locus molecule was identified. ther indicates that Ag-driven selection is a factor at this locus. These data are consistent with previous observations in another Although Pacy group A1 and A2 alleles both exhibit A locus phy- baboon using a PCR-based strategy. Therefore, a picture is logenetic characteristics, we anticipate that the A2 loci will be emerging whereby the baboon has four functional classical polymorphic as is the human HLA-A locus, while the A1 loci will class I loci and one nonclassical HLA-E equivalent. be monomorphic/oligomorphic. Several parameters led us to assign A locus, B locus, and E locus The Pacy-B alleles cluster with macaque B alleles upon phylo- designations to the baboon class I molecules we found. The first genetic analysis, and Pacy B locus alleles display evidence of long- parameter was phylogenetic. Comparing an assortment of classical term accumulation of polymorphism. Like the Pacy-A2 locus, the by guest on October 2, 2021 class I molecules from several species demonstrates that four of substantial number of nucleotide/amino acid differences between the Pacy class I molecules clearly group with B locus molecules the Pacy B alleles within the ␣1/␣2 domains (Fig. 5) indicates that from other species. Likewise, four of the Pacy class I molecules the baboon will be extremely polymorphic at the B loci. Compar- clearly group with A locus molecules, and the remaining Pacy isons of B*01/B*02, and B*03/B*04 alleles within ␣1 and ␣2 do- class I molecule clearly groups with E locus homologues. Assign- mains demonstrates that Pacy-B intralocus/intra-allelic group vari- ing these four A and four B alleles to their individual loci was then ation tends to exceed intralocus disparity at human A, B, and C loci achieved through comparisons of the predicted primary amino acid (40). Although specific deletions in the B leader sequence were sequences of these molecules. We assigned Pacy-A alleles to par- used to designate B loci, no clear assignment of alleles at the ticular loci on the basis of a 2-aa deletion in the transmembrane Pacy-B locus can be made on the basis of sequence homology. domain, a deletion that also distinguishes the A loci previously Sequence comparison of Pacy-B alleles demonstrates greater ho- reported in Papio hamadryas anubis. In a similar fashion, the four mology between B alleles assigned to different loci than between Pacy-B molecules can be separated into two loci on the basis of B alleles at a locus. Such sharing of residues at polymorphic sites deletions in the leader sequence. Again, B locus alleles in Papio between alleles at different loci may result from direct descent hamadryas anubis are likewise distinguished. Such data indicate (41), convergent evolution (42, 43), or interlocus gene conversion duplication of the A and B loci in the baboon. Because the rhesus (44). Regardless, the Pacy-B alleles appear highly polymorphic, macaque has also been reported to express class I from multiple A possibly having borne the brunt of MHC class I-selective and/or B loci, we propose that the A and B loci duplicated before pressures. divergence of the Cercopithecoid species (19). The single nonclassical class I allele isolated was a baboon Extension of the phylogenetic analysis to examine the extent of HLA-E locus allele that displays a high degree of homology and a polymorphism and its location reveals unique properties of the third codon percent GC content similar to that of other primate Pacy-A alleles. Most striking is that alleles A*01 and A*02 differ HLA-E genes. It resides with previously characterized E locus al- by a single nonsynonymous nucleotide substitution. This result leles on phylogenetic analysis and reflects functional conservation could be interpreted to mean that this Pacy-A locus, A1, is rela- of key peptide pocket residues. Pacy-E and HLA-E molecules tively monomorphic. Alternatively, perhaps this animal simply is probably share ligand binding profiles, and the Pacy and Mamu heterozygous for two closely related alleles. The fact that our pre- leaders appear identical, both being shortened with respect to the vious examination of an unrelated baboon found a single analo- human HLA-E leader (it has been suggested the leader deletion gous A locus allele at this locus, and that this single allele is iden- prevents the E locus from presenting its own leader). These at- tical with the one found in this study argues that little tributes prompt assignment of the nonclassical name Pacy-E.It polymorphism will be found at the Pacy-A1 locus. We suspect that must be noted here that other than showing limited diversity, the the Pacy-A1 locus alleles display limited polymorphism due to Pacy A1 locus does not display characteristics of a nonclassical The Journal of Immunology 3991 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. Comparison of range of intra- and interlocus variation between expressed Pacy and human classical class I loci. The number of nucleotide substitutions per site in exon 3/␣-2 (Jukes-Cantor distance) is plotted as a function of the number of nucleotide substitutions in exon 2/␣-1 (Jukes-Cantor distance): i, pairwise intralocus comparisons at human A, B, and C loci and Pacy intra/interlocus comparisons at A1/A2 and B1/B2 loci; ii, iii, and iv, interspecies/interlocus comparisons between Pacy and human classical class I alleles. molecule, including a percent GC representative of classical class No HLA-C homologue has been found in the rhesus macaque, I alleles. The similarity of the Pacy-E allele with macaque and and to date the occurrence of an HLA-C orthologue has not been human E molecules indicates a conservation of structure and, reported outside the Hominidae or Pongidae (16). We anticipate therefore, function at this locus. that ancestral human and baboon lineages diverged before the 3992 BABOON CLASS I MHC Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 5. Ribbon diagrams of characterized Pacy group A1, A2, B1, and B2 alleles depicting foci of polymorphism and positions of ␣1/␣2 poly- morphism/residue substitution observed in intra-allelic group comparisons. event that gave rise to HLA-C. In addition, the Pacy-A,-B, and -E Acknowledgments alleles reported here are clustered with respective macaque and We thank Dr. G. White, Dr. M. Carey, and members of the University of human counterparts, indicating that no equivalent of Mamu-I is Oklahoma Health Sciences Center Animal Resources Facility for helpful present in the yellow baboon. It is unclear what the functional discussions and support. impact of not expressing HLA-C or Mamu-I equivalents will be, but the expression of eight classical class I molecules would seem References to provide the baboon with a full complement of MHC class I. 1. Parham, P., C. E. Lomen, D. A. Lawlor, J. P. Ways, N. Holmes, H. L. Coppin, In summary, the MHC of Papio genera has not previously been R. D. Salter, A. M. Wan, and P. Ennis. 1988. The nature of polymorphism in HLA-A -B -C molecules. Proc. Natl. Acad. Sci. USA 85:4005. investigated by cDNA library screening. Nine alleles were char- 2. Pamer, E., and P. Cresswell. 1998. Mechanisms of MHC class I-restricted antigen acterized, presumably reflecting five functional class I loci in the processing. Annu. Rev. Immunol. 16:323. 3. Klein, J., Y. Satta, C. O’Huigin, and N. Takahata. 1993. The molecular descent baboon. Although one A locus was monomorphic/oligomorphic, of the major histocompatibility complex. Annu. Rev. Immunol. 11:269. the other A and two B loci appear to encode vastly diverse alleles. 4. Hughes, A. L., and M. Nei. 1988. Pattern of nucleotide substitution at major The single E locus allele isolated possesses a high degree of ho- histocompatibility complex class I loci reveals overdominant selection. Nature 335:167. mology to previously reported E locus alleles. Overall, the level of 5. Lechler, R., and A. Warrens. 2000. HLA in Health and Disease. Academic Press, nucleotide sequence diversity at three of the four baboon classical San Diego. class I loci exceeds that observed in humans. Inclusion of addi- 6. Watkins, D. I. 1995. The evolution of major histocompatibility class I genes in primates. Crit. Rev. Immunol. 15:1. tional from other Papio subspecies will clarify the degree 7. Vogel, T. U., D. T. Evans, J. A. Urvater, D. H. O’Connor, of polymorphism at the various loci, the apparent lack of an A. L. Hughes and D. I. Watkins. 1999. Major histocompatibility complex class I genes in primates: co-evolution with pathogens. Immunol. Rev. 167:327. HLA-C counterpart, and possible interlocus exchange mechanisms 8. Mayer, W. E., M. Jonker, D. Klein, P. Ivanyi, G. van Seventer, and J. Klein. 1988. that might be at play at the B loci. Knowledge of the range and Nucleotide sequences of chimpanzee MHC class I alleles: evidence for trans- nature of the class I MHC molecules in baboons will serve vaccine species mode of evolution. EMBO J. 7:2765. 9. Lawlor, D. A., E. Warren, P. Taylor and P. Parham. 1991. Gorilla class I major studies, evaluation of xenotransplantation, and evolutionary stud- histocompatibility complex alleles: comparison to human and chimpanzee class ies of the MHC. I. J. Exp. Med. 174:1491. The Journal of Immunology 3993

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