A Small, Variable, and Irregular Killer Cell Ig-Like Locus Accompanies the Absence of MHC-C and MHC-G in Gibbons

This information is current as Laurent Abi-Rached, Heiner Kuhl, Christian Roos, of September 29, 2021. Boudewijn ten Hallers, Baoli Zhu, Lucia Carbone, Pieter J. de Jong, Alan R. Mootnick, Florian Knaust, Richard Reinhardt, Peter Parham and Lutz Walter J Immunol 2010; 184:1379-1391; Prepublished online 21

December 2009; Downloaded from doi: 10.4049/jimmunol.0903016 http://www.jimmunol.org/content/184/3/1379

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A Small, Variable, and Irregular Killer Cell Ig-Like Receptor Locus Accompanies the Absence of MHC-C and MHC-G in Gibbons

Laurent Abi-Rached,* Heiner Kuhl,† Christian Roos,‡,x Boudewijn ten Hallers,{ Baoli Zhu,{ Lucia Carbone,{ Pieter J. de Jong,{ Alan R. Mootnick,‖ Florian Knaust,† Richard Reinhardt,† Peter Parham,* and Lutz Walter‡,x

The killer cell Ig-like receptors (KIRs) of NK cells recognize MHC class I ligands and function in placental reproduction and im- mune defense against pathogens. During the evolution of monkeys, great apes, and humans, an ancestral KIR3DL expanded to become a diverse and rapidly evolving gene family of four KIR lineages. Characterizing the KIR locus are three framework regions, defining two intervals of variable gene content. By analysis of four KIR haplotypes from two species of gibbon, we find that Downloaded from the smaller apes do not conform to these rules. Although diverse and irregular in structure, the gibbon haplotypes are unusually small, containing only two to five functional . Comparison with the predicted ancestral hominoid KIR haplotype indicates that modern gibbon KIR haplotypes were formed by a series of deletion events, which created new hybrid genes as well as eliminating ancestral genes. Of the three framework regions, only KIR3DL3 (lineage V), defining the 59 end of the KIR locus, is present and intact on all gibbon KIR haplotypes. KIR2DL4 (lineage I) defining the central framework region has been a major target for elimination or inactivation, correlating with the absence of its putative ligand, MHC-G, in gibbons. Similarly, the MHC- http://www.jimmunol.org/ C–driven expansion of lineage III KIR genes in great apes has not occurred in gibbons because they lack MHC-C. Our results indicate that the selective forces shaping the size and organization of the gibbon KIR locus differed from those acting upon the KIR of other hominoid species. The Journal of Immunology, 2010, 184: 1379–1391.

iller cell Ig-like receptors (KIRs) recognize MHC class I bination with HLA class I, have been associated with infectious (3– ligands and are expressed on NK cells and subsets of 7), autoimmune (8), and malignant (9) diseases, as well as the out- T cells. The human KIR locus, in the leukocyte receptor come of transplantation (10) and success in reproduction (11, 12).

K by guest on September 29, 2021 complex on 19, exhibits extensive variation in gene So far, comparison of mammalian species shows that only copy number and in allelic polymorphism of the individual genes (1, catarrhine (Old World monkeys and hominoids) (13) and platyr- 2). Various aspects of KIR gene variation, either alone or in com- rhine primates (New World monkeys) (14), as well as cattle (15), have a variable family of KIR genes. In other species, the KIR locus can consist of a single functional gene (e.g., wild seals) (16), † *Department of Structural Biology, Stanford University, Stanford, CA 94305; Max be completely absent from the genome (e.g., domestic dog) (16), Planck Institute for Molecular Genetics, Berlin-Dahlem; ‡Primate Genetics Labora- x tory and Gene Bank of Primates, German Primate Center, Go¨ttingen, Germany; or have been translocated from the leukocyte receptor complex to {Children’s Hospital Oakland Research Institute, Oakland, CA 94609; and ‖ the X chromosome and not expressed by NK cells (e.g., laboratory Gibbon Conservation Center, Santa Clarita, CA 91380 mice) (17). Certain species, notably mouse, rat, and horse, have Received for publication September 11, 2009. Accepted for publication November evolved by convergence an equally diverse family of structurally 14, 2009. divergent Ly49 receptors with remarkably similar functions to This study was supported by Pakt fu¨r Forschung und Innovation of the Leibniz- Gemeinschaft Biodiversita¨t der Primaten: Erfassung, Ursachen und Erhalt (to C.R. KIRs (18). Ancient duplication of the primordial KIR3D gene and L.W.), by Bundesministerium fu¨r Bildung, Wissenschaft, Forschung und Tech- gave rise to two distinct KIR lineages: KIR3DX and KIR3DL (15). nologie (Germany)–Nationales Genomforschungsnetz Grant 01GR0414 (to R.R.), Modern cattle have an expanded family of KIR3DX genes and and by National Institutes of Health Grant AI024258 (to P.P.). retain KIR3DL as a single-copy gene, whereas modern catarrhine The sequences presented in this article have been submitted to GenBank (www.ncbi. nlm.nih.gov/Genbank/) under accession numbers FP476133 (BAC86P8), FP476132 primates retained KIR3DX as a single-copy gene while expanding (BAC116G5), FP565168 (BAC423M19), GQ398089 (Nole-KIR2DL1), GQ438254 the family of KIR3DL genes. Thus, the complex KIR gene families (Nole-KIR2DH1), and GQ438255 (Nole-KIR3DL1). present in cattle and catarrhine primates are the consequence of Address correspondence and reprint requests to Dr. Peter Parham, 299 Campus Drive independent expansions driven by forces of natural selection on West, Fairchild Building, Stanford University, Stanford, CA 94305. E-mail address: [email protected] different orders of placental mammals. The online version of this article contains supplemental material. Variable KIR gene families have been studied in one species of New World monkey: owl monkey (14); three Old World monkeys: Abbreviations used in this paper: BAC, bacterial artificial chromosome; BLAST, basic local alignment search tool; BP, bootstrap proportion; Csa, green monkey species; rhesus macaque (19, 20), cynomolgus macaque (21), and green CYT, cytoplasmic tail; Hoho, eastern hoolock gibbon primate species; Hosa, Human monkey (22); four great apes: orangutan (23), gorilla (24), bonobo primate species; KIR, killer-cell Ig-like receptor; ML, maximum-likelihood; Mm, rhesus macaque primate species; MP, maximum-parsimony; NJ, neighbor-joining; (25), and chimpanzee (26); as well as the human species (27). The Nole, northern white-cheeked gibbon primate species; Poab, Pongo abelii; Popy, Pon- majority of KIR genes are species-specific, consistent with their go pygmaeus; Pt, chimpanzee primate species; SNP, single nucleotide polymorphism; rapid evolution through extensive gene duplication, deletion, and TM, transmembrane region; UTR, untranslated region. hybridization by nonhomologous recombination (14, 22, 24, 28, Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 29). Phylogenetic analyses show there are four lineages of KIR in www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903016 1380 KIR-MHC COEVOLUTION IN GIBBONS these species (23, 24). Of the known ligand specificities, MHC-G white-cheeked gibbon primate species [Nole]-H1) and that clone 423M19 recognition is associated with lineage I, MHC-A and -B recog- contained much of the second KIR haplotype (Nole-H2) but lacked the 39 nition with lineage II, MHC-B and MHC-C recognition with region. Further sequence analysis of both haplotypes was undertaken. Phylogenetic analysis of gibbon KIR haplotypes was facilitated by the lineage III, and for lineage V, represented by KIR3DL3, neither sequence of a KIR haplotype from a Sumatran orangutan (Pongo abelii). ligand nor function has yet to be identified (1, 30). The BAC clone containing this haplotype (CH276-525G20) was sequenced Whereas lineage II KIR makes up the major lineage in macaques by the Washington University Genome Sequencing Center, as part of the (20, 21), it is represented by only one or two genes in hominids (great orangutan genome project, and is deposited in GenBank (www.ncbi.nlm. nih.gov/Genbank/) under accession number AC200148. apes and humans). Conversely, lineage III KIR is the major hominid lineage but is represented by a single gene in macaques. Coevolution BAC DNA sequencing with rapidly evolving MHC class I ligands is thought to have con- BAC clone DNA was purified by CsCl density centrifugation and then tributed to such KIR diversification, and in great apes, expansion of sheared by sonication. DNA fragments were separated by agarose gel lineage III KIRs coincides with formation of MHC-C (23), whereas electrophoresis, and fragments of 1.5–3.5 kb were cloned into plasmid in Old World monkeys, the expansion of lineage II KIRs correlates pUC19. Clones were sequenced using M13 forward and reverse primers. with the expansion of the MHC-A and MHC-B genes (19–21, 31–33). The sequence data were processed by Phred (www.phrap.org) and assem- bled into a contiguous sequence by Phrap (www.phrap.org). Both the Phred Uniquely missing from our picture of hominoid KIRs are the and Phrap computer programs can be obtained from P. Green (Genome smaller apes, as represented by diverse species of gibbon. There are Sciences Department, University of Washington, Seattle, WA) (47). at least 14 extant species of smaller apes, or hylobatids, who are For the eastern hoolock gibbon, BAC clones CH278-86P8 and CH278- subdivided into four genera: Nomascus, Hylobates, Hoolock 116G5 were selected for sequencing because they cover the same genomic region and contain the Hoho-H1 and Hoho-H2 KIR haplotypes, re- (formerly named Bunopithecus), and Symphalangus (34–37). Downloaded from spectively. Complete sequences for these clones were determined to high- Gibbons are arboreal apes inhabiting Southeast Asia and small quality standard (error rate for each of the consensus # 1in regions of south and east Asia. Although they share habitat and 100,000; Phred score $ 50). White-cheeked gibbon BAC clone CH271- geographical range with orangutans, gibbons are equally related to 423M19, containing the incomplete N. leucogenys haplotype Nole-H2, was all the great apes and humans (38). Compared with great apes and completely sequenced using the same approach, but the resulting sequence did not adequately cover the Nole-KIR2DL1 gene. To determine the se- humans, hylobatids have smaller bodies and are social animals quence of Nole-KIR2DL1 to high accuracy, we first performed a new as- that generally live in monogamous family groups engaging in sembly of the ENCODE project sequence reads (from the National http://www.jimmunol.org/ relatively few contacts between groups (39). The gibbon genome Institutes of Health Intramural Sequencing Center) using the STADEN is characterized by an accelerated chromosomal evolution, re- package (48). This allowed precise identification of the regions of the Nole-KIR2DL1 gene that required further sequence analysis. These regions cently evaluated to be 10–20 times higher than is typical for were amplified by PCR and cloned using the TOPO-TA system (In- mammalian evolution rates (40). A preliminary study of gibbon vitrogen, Carlsbad, CA). Sequencing was performed using the M13 for- MHC class I identified only MHC-A and MHC-B (41), raising the ward and reverse primers, and each nucleotide in the final sequence was possibility that gibbons lack the MHC-C gene present in hominids, minimally covered by three sequences representing at least two different which in humans and chimpanzees is the predominant source of templates and had a quality $ 50 (1 putative mistake/100,000 bases). Unlike the other three BAC clones studied here, BAC clone CH271- KIR ligands (42). If that were the case, then the gibbon KIR 127M10 containing the complete Nole-H1 KIR haplotype was not com- system is predicted to be significantly different from what has pletely sequenced to high precision. Instead, an accurate sequence of the by guest on September 29, 2021 been described for other hominoid species. To investigate this Nole-H1 haplotype was obtained by combining and refining the sequence intriguing possibility, we characterized KIR haplotypes from two data obtained by the ENCODE project and the N. leucogenys whole-genome ∼ Nomascus Hoolock shotgun sequencing project. To do this, a region of 105 kb containing the diverse genera of gibbons ( and ). KIR locus was targeted for analysis. A draft assembly of this region was obtained by assembling the ENCODE project sequence reads (from the Materials and Methods National Institutes of Health Intramural Sequencing Center) using the KIR nomenclature STADEN package (48). This initial assembly was then complemented by sequence reads generated at Washington University Genome Sequencing KIR genes and alleles were named by the KIR Nomenclature Committee Center and Baylor College of Medicine in the course of the ongoing N. (43), formed from the World Health Organization Nomenclature Com- leucogenys whole-genome shotgun project. These sequence reads were mittee for factors of the HLA system and The Organi- obtained by basic local alignment search tool (BLAST) searches, using as sation Genome Nomenclature Committee. A curated database is available probes the genomic segments with low coverage in the initial assembly. at www.ebi.ac.uk/ipd/kir/ (44). Those sequence reads that displayed a well-supported mismatch (i.e., a difference at a position with a good quality in both sequence reads) with Bacterial artificial chromosome library screening sequence reads in the initial assembly likely represented the other KIR haplotype and were discarded. The final assembly had no gaps, and each High-density filters of bacterial artificial chromosome (BAC) libraries base pair of the final sequence had a quality $ 50 (1 putative mistake/ derived from northern white-cheeked gibbon (Nomascus leucogenys leu- 100,000 bases), except for two intronic regions of Nole-KIR3DL3 (∼500 bp cogenys, CHORI-271) and eastern hoolock gibbon (Hoolock hoolock in intron 3 and ∼1 kb in intron 4). leuconedys, CHORI-278) were obtained through the BACPAC resources of Sequencing of the four BAC clones was performed using BigDye ter- the Children’s Hospital Oakland Research Institute (http://bacpac.chori. minator chemistry and either an ABI 3730xl or an ABI 377 sequencer org/home.htm). Both libraries were screened with a rhesus macaque KIR (Applied Biosystems, Foster City, CA). Exons and introns of genes were cDNA probe, and 11 clones were selected for exploratory analysis. Clones either identified manually or by using the BLAST (www.ncbi.nlm.nih.gov/ CH271-127M10, -109N22, -44K12, -49H24, -423M19, and -182G20 are BLAST) and FGENESH-2 (www.softberry.com/all.htm) algorithms. derived from N. leucogenys; clones CH278-86P8, -66L16, -114J2, -141F13, and -116G5 are derived from H. hoolock. Phylogenetic analysis of KIR Preliminary characterization by Southern blotting and end sequencing showed that hoolock gibbon clones 86P8 and 66L16 represent one KIR KIR gene sequences were aligned using MAFFT (49) and manual cor- haplotype (eastern hoolock gibbon primate species [Hoho]-H1), whereas rection of the resulting alignments. The aligned sequences were then divided clones 116G5, 141F13, and 114J2 represent the second KIR haplotype into 12 segments as described previously (23). Each segment was analyzed by (Hoho-H2). Clone CH278-86P8 from Hoho-H1 and clone CH278-116G5 three methods: maximum-likelihood (ML), neighbor-joining (NJ), and max- from Hoho-H2 were selected for complete sequence determination. imum-parsimony (MP). NJ analyses were performed with MEGA4 (50) using As part of the ENCODE project (45), clones CH271-127M10 and the Tamura-Nei method with 500 replicates. PAUPp4.0b10 (51) and the tree CH271-423M19 from white-cheeked gibbon were sequenced to compar- bisection-reconnection branch swapping algorithm were used for MP analy- ative grade standard (46) by the National Institutes of Health Intramural ses with 500 replicates and a heuristic search. ML analyses were performed Sequencing Center (www.nisc.nih.gov). Our analysis of these sequences with RAxML7 (52) under the GTR+CAT model with 500 replicates (rapid showed that clone 127M10 contained a complete KIR haplotype (northern bootstrapping). The results of these analyses are in Supplemental Fig. 1. The The Journal of Immunology 1381 analysis of KIR intergenic regions was similarly performed on an alignment of genera (34, 53, 54). Three BAC clones contained complete gibbon sequences that started ∼600 bp after the end of exon 9 of the first KIR gene and KIR haplotypes: two from H. hoolock (Hoho-H1 and Hoho-H2) ∼ ended 40 bp before exon 1 of the second KIR gene. and one from N. leucogenys (Nole-H1) (Fig. 1A). In these BAC Analysis of the deletions forming gibbon KIR haplotypes clones, the gibbon KIR genes are flanked on the 59 side by the LILR gene cluster and on the 39 side by the FCAR gene, an ar- To identify deletion events leading to modern gibbon KIR haplotypes, we first aligned the intergenic and intragenic segments of the four sequenced KIR rangement identical to that observed in all other primates studied haplotypes based on the results of the phylogenetic analysis; the segments (23) as well as in most other mammalian species (16). used in this analysis are those described above, under “Phylogenetic analysis BAC clone analysis also defined the 59 part of a second KIR of KIR.” The structure of the predicted progenitor for all hominoid KIR haplotype from N. leucogenys (Nole-H2), which extended from the haplotypes was then used to identify deletion and duplication events and the ∼ KIR gene segments lost or gained as a consequence of these events [the flanking LILR gene through 3DL3, 2DL1, and 2DP2 to end 300 bp structure of the progenitor for all hominoid KIR haplotypes used here is before the first exon of 2DL4. To characterize this haplotype further, a refinement of previous evolutionary models (23), as detailed in Results]. we took advantage of the fact that genomic DNA from the same KIR haplotype comparisons in Old World monkey and hominoids pointed to individual gibbon was used to make the BAC library and to conduct the lineage Ia, II, III, and V KIRs as having been present on all ancestral KIR the N. leucogenys whole-genome shotgun-sequencing project. By haplotypes (23), so absence of any of these from gibbon KIR haplotypes was considered a gibbon-specific loss. The lineage Ib KIR2DL5 is typically extracting and assembling all the sequences generated in the latter present on only a fraction of haplotypes in hominoid species, so it is possible project, we showed that the Nole-H2 haplotype contains three ad- that it was not present on all ancestral haplotypes. As a result, absence of ditional KIR linked upstream from 2DP2 in the following order: KIR2DL5 was only considered a gibbon-specific loss when KIR2DL5 seg- 2DL4, 2DH1, and 3DL1, with 3DL1 being the last KIR of the hap- ments could be found on the haplotype; otherwise, the lineage Ib deletion Downloaded from was only considered “possible” and labeled accordingly. lotype (Fig. 1A). Two of these three genes, 2DH1 and 3DL1 repre- sent alleles of Nole-H1 genes and minimally differ by 33 (2DH1) Characterization of MHC class I, Nole-H2 from N. leucogenys and 55 (3DL1 ) nucleotide substitutions distributed along the length To assess the MHC class I gene content of the gibbon genome, we searched the of the gene. Although unambiguous complete sequences were not database from the ongoing N. leucogenys whole-genome shotgun-sequencing obtained (contrasting with the other three haplotypes), there was in project generated by the Washington University Genome Sequencing Center this extensive dataset no evidence for any deletion, insertion, or and the Baylor College of Medicine. First, BLAST searches were performed substitution that would corrupt the functions of 2DH1 and 3DL1. All http://www.jimmunol.org/ using sequences corresponding to the six functional human HLA class I genes: HLA-A, -B, -C, -E, -F,and-G. More than 200 sequences were gathered for the evidence is thus consistent with Nole-H2 having functional each of the six independent BLAST searches. The resulting sequences were 2DH1 and 3DL1 genes. This complementary analysis of shotgun pooled and assembled using the STADEN package (48). The contiguous se- sequences allowed the KIR gene content of the Nole-H2 haplotype to quences thus generated were aligned with human and macaque MHC class I be described completely (Fig. 1A). genes and pseudogenes using MAFFT (49), and the resulting alignment was corrected manually. The alignment was then divided into two halves, and phylogenetic analyses were performed on each half as described above for the KIR genes. The results of these analyses are in Supplemental Fig. 2. Several gene fragments had insufficient sequence for analysis by this approach. For

each of these gene fragments, a customized dataset was made, which was by guest on September 29, 2021 limited to the particular genomic region covered by the gene fragment under test. Phylogenetic analyses were performed using the NJ method, as described above for the KIR. To determine whether the Nole-H2 haplotype contained KIR genes additional to those in BAC423M19, we investigated the N. leucogenys whole-genome shotgun sequences through BLAST searches with KIR gene sequences from the three complete haplotypes as queries. This initial analysis revealed three additional KIR genes related to 2DL4, 2DH1, and 3DL1. Sequence reads for these three genes were then obtained by BLAST searches using the Hoho-2DL4, Nole-2DH1, and Nole-3DL1 gene se- quences as queries. For each of these three genes, the sequence reads gathered were assembled using the STADEN package (48). For Nole-KIR2DL4, the analysis was facilitated by the absence of 2DL4 on Nole-H1, the final assembly containing only one gap of ∼100 bp in intron 6 (assembly represents .99% of the Hoho-2DL4 gene sequence). For KIR2DH1 and 3DL1, the final assemblies had four and five gaps, re- spectively, covering ∼70% of the sequence of their respective equivalent on Nole-H1. This coverage likely represents an underestimation of the coverage from the whole-genome shotgun sequences, because only the sequences that unambiguously represented the Nole-H2 haplotype (i.e., that contained at least one unique polymorphism) were included. The positions of these additional three genes on the Nole-H2 haplotype (following 2DP2) were determined using read-pair information and are as FIGURE 1. Gibbons have small, irregular, and diverse KIR haplotypes. A, follows: 2DL4 - 2DH1 - 3DL1, with 3DL1 being the 39-most KIR of the Schematic representation of the four gibbon KIR haplotypes. Haplotypes Hoho- haplotype, as indicated by the presence of a G3 intergenic segment in its 59 H1 and Hoho-H2 are from Hoolock hoolock leuconedys, and haplotypes Nole-H1 (data not shown). The sequences of these three genes were not included in and Nole-H2 are from Nomascus leucogenys leucogenys. Double arrows indicate the phylogenetic analyses, because they are incomplete and contain seg- genes that are either equivalent within species (alleles) or between species (or- ments of draft quality. thologs). Black boxes represent genes encoding functional KIR, whereas gray boxes represent KIR pseudogenes, and white boxes represent FCAR and LILR Results genes flanking the KIR locus. pActivating KIR. For Nole-H2, KIR3DL3, 2DL1, Gibbons have small KIR haplotypes with diverse gene content and 2DP2 were fully characterized through sequencing of a BAC; 2DL4, 2DH1, and 3DL1 were characterized by analysis of whole-genome shotgun sequences. We investigated KIR haplotype structures in the eastern hoolock B, Shown are the numbers of KIR genes, KIR pseudogenes, and the average (Hoolock hoolock leuconedys) and northern white-cheeked (No- number of KIR genes and pseudogenes; the size of the KIR locus in the KIR mascus leucogenys leucogenys) gibbons, two species that diverged haplotypes of several primate and nonprimate species is also given. For gibbons, ∼11 million years ago and represent the most divergent gibbon the size of each of the four haplotypes is given. 1382 KIR-MHC COEVOLUTION IN GIBBONS

Three of the four gibbon KIR haplotypes are much smaller than results demonstrate that all four KIR lineages present in great apes their counterparts in other hominoids and rhesus macaque, con- and humans are also present in gibbons: Hoho-KIR2DL4 from lin- taining between two to four genes and pseudogenes and being 30– eage I, Hoho- and Nole-KIR3DL1 from lineage II, Nole-KIR2DL1 62 kb in length (Fig. 1B). The largest gibbon haplotype (Nole-H2) from lineage III, and two alleles each of Hoho- and Nole-KIR3DL3 is also smaller than the haplotypes of other hominoids but has the from lineage V (Fig. 3A). The other four gibbon KIRs are either same number of KIR genes as the one rhesus macaque haplotype recombinants, having segments derived from at least two lineages defined (Fig. 1B). The mean number of genes per gibbon KIR (Nole-KIR2DH1 and Hoho-KIR2DL2), or pseudogenes (Nole- haplotype (3.75) is thus lower than that in other hominoids and KIR2DP2 and Hoho-KIR2DP1) lacking the last four exons of rhesus macaque (6–11). Despite their size, the four gibbon KIR complete KIR genes (Fig. 3A–D). Whereas a majority of haplotypes haplotypes all differ in gene content, with a total of six different in other hominoid species contain genes from all four KIR lineages, genes and two pseudogenes being represented. Of these, 2DL4, three of the four gibbon haplotypes do not carry intact genes from all 3DL3, and 3DL1 are common to the two gibbon species and four lineages (Fig. 3D). represent orthologous genes. KIR3DL3 is polymorphic in both gibbon species and the two allotypes differ by either four or five KIR haplotypes lacking the central framework region are amino acid substitutions (Fig. 2). Such allelic variability is small common in gibbons compared with the divergence between the gibbon species, which The basic organization of the gibbon KIR locus is distinguished constitutes 25–27 aa differences for 3DL3. The species divergence from that seen in other species. Characteristic of KIR haplotypes for 3DL1 is even higher, 48 residues (Fig. 2), suggesting 3DL1 has in other hominoids, and the rhesus macaque, are three conserved accumulated substitutions at a higher rate than 3DL3. In addition regions common to all haplotypes (23). These framework regions Downloaded from to their variability in gene content, gibbon KIR haplotypes also make up the 59 part of a lineage V KIR at the 59 end, 2DL4 and the 39 encode a variety of KIR structures: together the four haplotypes part of a pseudogene in the central part, and the 39 part of a lineage encode 10 functional KIRs, 8 inhibitory, and 2 activating (Fig. II KIR at the 39 end. This arrangement is preserved in one haplotype 1A). Six of the eight inhibitory KIRs have three Ig domains, from each gibbon species: Hoho-H1 and Nole-H2, but not in the whereas both activating receptors and the remaining two in- other. Notably, the only genes in Hoho-H1 are the framework hibitory KIRs have two Ig domains (Fig. 2). genes. The Hoho-H2 and Nole-H1 haplotypes both lack the central http://www.jimmunol.org/ framework region. In addition, Hoho-H2 lacks part of the 39 All four hominid KIR lineages are represented in gibbons framework region. The only complete gene common to the four To assess the relationships of gibbon KIR with the four primate KIR haplotypes is lineage V KIR3DL3, which forms the 59 framework lineages (I, II, III, and V) defined in previous analyses (23, 24), we region. Three haplotypes (Hoho-H1, Nole-H1, and Nole-H2) have divided the gene sequences into 12 segments on which phylogenetic a complete lineage II 3DL1 gene as the most 39 gene, whereas analysis was independently performed. The results are summarized Hoho-H2 has a recombinant gene, 2DL2, in which the 39 part de- in Fig. 3A, and two examples of the phylogenetic trees are given in rives from a lineage II gene and the 59 part is from lineage III (Fig. Fig. 3B and 3C. The other trees are in Supplemental Fig. 1. The 3D). The lineage III pseudogene KIR2DP and the lineage I by guest on September 29, 2021

FIGURE 2. Structural diversity of gibbon KIR. Shown is an alignment of the deduced amino acid se- quences of the 10 functional gibbon KIR encoded by genes in the four KIR haplotypes of Fig. 1. The alignment is organized according to the seven functional domains of the : D0, D1, and D2 are the three Ig-like domains, the Stem separates the ligand-binding Ig-like domains from the transmembrane (TM) region and the cytoplasmic tail (CYT). Sequence identity with Nole-KIR3DL1 is indicated by a dot, and an asterisk (p) denotes a stop codon. ITIMs are boxed. Other functionally significant resi- dues are gray shaded. The charac- teristic arginine residue at position 323 in the transmembrane region of activating KIR is also indicated by an arrowhead. Nole-KIR are from Nomascus leucogenys leucogenys; Hoho-KIR are from Hoolock hoo- lock leuconedys. Nole-H2 KIR2DL4, 2DH1, and 3DL1 genes character- ized by analysis of whole-genome shotgun sequences were not in- cluded in this alignment. The Journal of Immunology 1383 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. All four primate KIR lineages are represented in gibbons. To assess the lineage affinities of gibbon KIR with other hominoid KIR, the genes were divided into 12 segments that were independently subjected to phylogenetic analyses. A summarizes the results of the phylogenetic analyses. Lineage assignment for each segment is shown, and the boxes are color-coded according to lineage: Ia (dark purple), Ib ( purple), II (pink), III (green), and V (blue). Segments for which lineage affinity could not be narrowed down to a single lineage are colored in light brown: exon 1 (lineages Ib, II, and III), exon 5 (lineages II and III), and intron 6c to exon 9 (lineages III and V). Light gray boxes indicate gene segments absent in some gibbon KIR. B and C, Representative phylogenetic trees for two of the 12 KIR gene segments are shown in B, for intron 3, and in C, for the segment from intron 6c to exon 9; analyses for the other 10 segments are in Supplemental Fig. 1. Phylogenetic reconstruction was performed using NJ, ML, and MP approaches. The NJ tree is shown (midpoint rooting), with support from all three methods being indicated at the nodes where the bootstrap proportion (BP) $50 for two of the three methods (from top to bottom: NJ, MP, and ML). At the nodes, d denotes strong phylogenetic support (BP $ 80 with the three methods), and gray circles denote moderate support (BP $ 60). pBP , 50. Mm, Macaca mulatta; Poab, Pongo abelii; Popy, Pongo pygmaeus; Pt, Pan troglodytes. D, Relationships between the four gibbon KIR haplotypes. Regions that are equivalent are shaded in gray. Black arrows indicate equivalent genes. A–D, The KIR2DL4, 2DH1, and 3DL1 genes of Nole-H2 were not included in the phylogenetic analysis because they are incomplete and contain segments of draft quality.

KIR2DL4, which provide the central framework region, are present in the same region of exon 6 or in exon 8 (24–26, 55, 56). On the in both Hoho-H1 and in Nole-H2. Both KIR2DP and KIR2DL4 are basis of the characteristics of these sequences and the relationships absent from haplotypes Hoho-H2 and Nole-H1. between them, we estimate at least six independent events have Among humans, great apes, and macaques, KIR2DL4 is the most inactivated ITIMs in hominoid KIR2DL4 allotypes (Fig. 4B). The conserved KIR gene. In addition to the absence of KIR2DL4 from situation in gibbons is extreme, because none of four haplotypes we two of the gibbon KIR haplotypes, the Hoho-2DL4 and Nole-2DL4 characterized has a KIR2DL4 gene encoding a 2DL4 protein with an genes have a single nucleotide insertion near the 39 end of exon 6 that ITIM. The recurrent selection against 2DL4 inhibitory function in causes frameshift and premature termination (Fig. 4A). Conse- hominoids stands in dramatic contrast to Old World monkeys, where quently, these genes encode truncated KIR2DL4 that lack the KIR2DL4 ITIMs are intact (Fig. 4B). ITIMs in the cytoplasmic tail and thereby lack inhibitory signaling The contrasting fate of the KIR2DL4 and KIR3DL3 framework function (Fig. 2). Several KIR2DL4 alleles in other hominoids are genes in gibbons is striking. Whereas all forms of 2DL4 have either similarly affected by single base pair deletions or point substitutions been deleted or lost inhibitory function, 3DL3 is not only fixed in 1384 KIR-MHC COEVOLUTION IN GIBBONS Downloaded from http://www.jimmunol.org/

FIGURE 4. Uniquepatterns of evolution forgibbon KIR. A, Sequencealignmentof exon 7 and the 39 partof exon 6 for KIR2DL4 fromsixprimate species: gibbon (Hole and Nole), human (Hosa), chimpanzee (Pt), orangutan (Popy), and rhesus macaque (Mm). A common dimorphism in KIR2DL4 is the run of either 9 (9A) or 10 (10A) adenosines in exon 6. Positions of frameshift are marked by n. Intronic and exonic nucleotides are indicated by lowercase and uppercase letters, re- spectively. Splice sites are shaded gray. The Nole-2DL4 sequence was obtained by analysis of the N. leucogenys whole-genome shotgun sequences (see Materials and Methods); each base pair of the segment presented here has a quality .80 (1 error/100,000,000 bp). B and C, Schematic representation of the potential for signal transduction by primate KIR2DL4 (B)andKIR3DL3(C): the letter “R” in the transmembrane (TM) domain indicates the presence of a charged residue (arginine). Dots in the cytoplasmic tail (CYT) domain indicate canonical ITIMs. B, The numbers (1–6) in the gray circles denote six independent evolutionary events that affected KIR2DL4 ITIMs. pHuman KIR2DL4 with 9A does not encode a cell surface receptor (71, 72). C,InorangutanKIR3DL3, the exons encoding D2 and Stem

(S) originate from lineage III and are shaded gray. D, Summary of the relationships between the gibbon and hominid (great apes and humans) lineage III KIR by guest on September 29, 2021 sequences. The KIR genes were divided into 11 segments, which are shown to scale. For eachof the fourgibbon KIR with lineage III gene segments, the phylogenetic relationships with hominid lineage III sequences are indicated as follows: I, ingroup; NR, not resolved; O, outgroup (orthologous to all hominid lineage III KIR). For the “outgroup” and “ingroup” categories, the strength of the phylogenetic support is indicated by the shading underlying the segment: black for strong support (BP $ 80 with the three methods used), dark gray for moderate support (BP $ 60 with three methods), and light gray for weak support (BP $ 50 with two or three methods). Csa, green monkey; Hosa, human; MM, rhesus macaque; Poab and Popy, orangutans; Pt, chimpanzee. a form that preserves both ITIMs, but it also displays polymorphism functional. Furthermore, good evidence supporting deletion of the in both gibbon species (with 4–5 aa substitutions), as also observed genomic region carrying MHC-G was the absence from N. leucog- in humans (30, 57–59) (Fig. 2). These properties strongly suggest enys of gibbon counterparts to the human HLA-V, -P, -H, -T,and-K that KIR3DL3 has been of persistent value to gibbons. pseudogenes that flank MHC-G (Fig. 5B), despite the fact that macaques have orthologs for three of these five human pseudogenes Nonfunctional forms of gibbon KIR2DL4 correlate with (MHC-V, -T and -K). Thus, we can conclude with confidence that the genomic deletion of MHC-G genome of the individual gibbon used to make the shotgun library Because HLA-G is the ligand for human KIR2DL4, we examined lacks MHC-G and, using the structure of the HLA region as a model, gibbon MHC class I genes for the presence of MHC-G.Thiswas predict that this deletion in gibbons involves 135–211 kb. We cannot achieved by using HLA-A, -B, -C, -E, -F,and-G sequences in BLAST rule out the possibility that some gibbon MHC haplotypes have searches to extract all MHC class I sequences from the database MHC-G, but the fact that both MHC haplotypes in the animal we generated by the N. leucogenys whole-genome shotgun-sequencing studied lack the gene indicates that MHC haplotypes lacking MHC-G project. Over 300 unique sequence reads were obtained and then are common. In summary, deletion and loss of function of gibbon assembled into 28 gibbon MHC class I contigs. Phylogenetic com- KIR2DL4 correlates with the absence of MHC-G. parison of gibbon, human, and macaque MHC class I sequences (Supplemental Fig. 2) allowed us to identify functional gibbon genes Minimal representation of lineage III KIR in gibbons corresponding to the expressed HLA-A, -B, -E,and-F genes and correlates with absence of MHC-C gibbon pseudogenes corresponding to the HLA-J, -L, -S,-W,and-X Lineage III is the most highly represented KIR lineage in great apes pseudogenes. However, no gibbon counterpart of HLA-G emerged and humans (23). In the latter species, for example, lineage III from this analysis (Fig. 5). Our failure to find gibbon MHC-G was makes up 8 of the 14 KIRs and ∼50% of the genes per KIR unlikely to have been the consequence of insufficient coverage by the haplotype. As a group, the four gibbon haplotypes contain only shotgun sequences, because two alleles were identified for the other one intact lineage III KIR (Nole-KIR2DL1). In addition, two re- MHC class I loci and five for MHC-B (and MHC-S) indicating combinant KIR contain lineage III segments (Nole-KIR2DH1 and presence of at least three MHC-B genes, at least two of which are Hoho-KIR2DL2) (Fig. 3A). Phylogenetic analysis shows that the The Journal of Immunology 1385 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Gibbon MHC haplotypes lack MHC-C and MHC-G. The MHC class I gene sequences generated by the whole-genome shotgun-sequencing project for N. leucogenys were assembled into 28 contiguous sequences (contigs) and compared with human HLA class I by phylogenetic analysis (Supplemental Fig. 2). Although the majority of gibbon MHC class I have orthologs in the HLA class I region, notably absent are gibbon counterparts to HLA-G, and closely linked pseudogenes and HLA-C. A, In this summary diagram, the inferred MHC class I gene content of the gibbon MHC is compared with the established map of the human HLA class I genomic region. Functional genes are indicated by n and pseudogenes by N. The terms centromeric and telomeric refer to the orientation of the HLA complex on the short arm of human chromosome 6. Results from a study that combined paired-end sequence analysis and molecular cytogenetics suggest that in N. leucogenys the MHC is on chromosome 1b, with an opposite centromeric/telomeric orientation comparing to human (73). B, This shows the characteristics of the 28 gibbon MHC class I-containing contigs and their relationships to human HLA class I genes and pseudogenes. The first column on the left lists the six HLA class I genes and the 11 largest HLA class I pseudogenes; the second column indicates presence or absence of orthologs in the gibbon genome. Under “Phylogeny,” gray boxes indicate whether phylogenetic analysis was performed on the 59 untranslated region (UTR)-E4 part of the class I gene and/or the I4-39UTR part, and they also show the part of the MHC-class I gene region covered by each of the 28 gibbon MHC class I contigs. Parentheses indicate segments that were analyzed separately (see Materials and Methods), and “xx” indicates a sequence that cannot be aligned with the other MHC class I sequences, suggestive of a class I gene fragment. Under “Notes,” the sequence motif at positions 76 and 80 is given for MHC-B, because valine 76, asparagine 80 defines the C1 epitope, a ligand for lineage III KIR; also given is the diagnosis of certain sequences as pseudogenes or null genes. To demonstrate the coverage of analysis, the single nucleotide polymorphism (SNP) content of each contig is given, excluding those representing the duplicated MHC-B and MHC-S, for which the allelic relationships are uncertain. gibbon lineage III sequences are orthologous to all the great ape Lineage III KIRs are receptors for MHC-C, and expansion of the and human lineage III KIR but not to any particular KIR (Fig. 4D). lineage III KIR genes is associated with presence of MHC-C in This shows that the difference in lineage III KIR gene content was humans and great apes but not in Old World monkeys (23). A pre- not caused by gene loss in the gibbons, because in that circum- vious study of cDNA characterized expressed MHC-A and -B genes stance the residual gibbon KIR would exhibit greater similarity to in gibbons but failed to detect any MHC-C transcripts (41). Our particular KIR in great apes and humans. Consequently, the dif- analysis of 28 N. leucogenys MHC class I sequences from one in- ference in gene content is due to expansion of the lineage III KIR dividual also failed to identify any gene or pseudogene that was genes in great apes and humans. orthologous to the MHC-C locus shared by orangutan, chimpanzee, 1386 KIR-MHC COEVOLUTION IN GIBBONS bonobo, and human (60). Two copies of a pseudogene with some deletion of KIR haplotypes has been a widespread phenomenon similarities to MHC-B and -C were defined (contig number during the history of gibbons. Whereas partial or complete gene 276081453; Fig. 5B). Thus, the paucity of gibbon lineage III KIR duplications are the main mechanism for diversifying KIR hap- correlates with the absence of a functional MHC-C gene in gibbons. lotypes in great apes, in smaller apes KIR haplotype diversity has In humans and great apes, the specificities of lineage III KIRs for been generated mostly through successive events of deletion. the C1 and C2 epitopes of MHC-C are determined by substitutions Deletion is a mechanism that can create new genes by hybrid- at position 44 in the D1 domain (61). Ancestral reconstructions and izing parts originating from two parental genes. Of the five genomic species comparison have shown that the C1 epitope and its deletions in the gibbon KIR haplotypes, three caused only gene complementary KIR (containing lysine 44) were the first to loss but the other two combined gene loss with the creation of emerge (42). Consistent with this model, Nole-KIR2DL1, Nole- a new gene (Fig. 6C). Assessing the effect of deletion on the KIR2DH1, and Hoho-KIR2DL2 all have lysine 44, which corre- presence and integrity of exons encoding the Ig domains showed sponds to position 139 in the alignment in Fig. 2. The C1 epitope that lineage I (KIR2DL4-5) was a major target, accounting for at is dependent upon valine 76 and asparagine 80 in the a1 domain least four of the seven losses of an Ig domain. Of the other three Ig of MHC-C. Certain MHC-B allotypes also have both valine 76 domain losses, two targeted lineage III KIR and one targeted and asparagine 80 and can function as ligands for C1-specific lineage II KIR. Three genes were more than once a target for KIRs (42). One of the four gibbon MHC-B sequences (number deletion: KIR2DL1, KIR2DL4, and KIR2DL5 (Fig. 6D). 282426381) has both valine 76 and asparagine 80 and represents Among the genomic deletions that created new hybrid KIR genes, a candidate ligand for the gibbon lineage III KIR (Fig. 5B). one produced Hoho-KIR2DL2 in H. hoolock, the other KIR2DH1 in

N. leucogenys (Fig. 6D). Hoho-KIR2DL2 represents a fusion be- Downloaded from The ancestral hominoid haplotype contained five KIR genes tween a lineage III inhibitory KIR and a lineage II inhibitory KIR, and a pseudogene where a fragment of D2, the stem, transmembrane and cytoplasmic As a foundation upon which to investigate how the short gibbon tail of the former were replaced by those of the latter KIR (Fig. 3A). KIR haplotypes arose, we reconstructed ancestral hominoid hap- Because the coding sequences of lineage II and III KIR are closely lotypes using intragenic and intergenic sequence comparisons. To related in the signaling domains (Supplemental Fig. 1) and because

refine previous evolutionary models (23), information from the they share the same ITIM sequences (Fig. 2), the function of the new http://www.jimmunol.org/ four gibbon KIR haplotypes was incorporated, as well as data from hybrid gene is likely to be similar to that of the original 59-donor a KIR haplotype from a Sumatran orangutan (Pongo abelii). This gene (lineage III KIR). In contrast, the gene fusion in N. leucogenys haplotype differs from the previously reported orangutan haplo- had more dramatic effect. Nole-KIR2DH1 combines the Ig domains type (23) by presence of KIR2DL5 and an activating lineage II of an inhibitory lineage III KIR with a KIR2DL4 transmembrane and KIR3DS and absence of activating lineage III KIR2DS (Fig. 6A). cytoplasmic tail, which lacks an ITIM and has only activating po- Paralleling the four human and great ape KIR lineages defined by tential (Figs. 2, 3A). As an activating KIR with lineage III Ig domains, intragenic sequences (23), the intergenic sequences also fall into four Nole-KIR2DH1 represents a functional, and independently evolved, groups (G1, G2a, G2b, and G3). Each of these groups is represented in equivalent to the great ape activating KIR2DS of lineage III. Nole- gibbons (Fig. 6B). For three groups, the genes flanking the intergenic KIR2DH1 is located between 2DL4 and 3DL1 in the Nole-H2 hap- by guest on September 29, 2021 sequence are similar in all hominoids: these are composed of G3, lotype, representing the first example of a nonhuman primate gene in between the most 39 KIR and FCAR; G2b, between KIR2DL4 and the 39 part of the KIR locus that encodes lineage III Ig domains. alineageIIKIR; and G2a, between one lineage III KIR and either In summary, lineage I and III KIRs have been the main targets of KIR2DL5 or a second lineage III KIR (one exception is the orangutan the deletions diversifying gibbon KIR haplotypes. For lineage I, KIR2DL5-3DS segment). The fourth group of intergenic sequences these events caused loss of Ig domain function, whereas for lin- (G1) contains the two gibbon sequences located 59 of hybrid genes eage III KIR, two events caused loss of Ig domain function, and whose 59-most segments are related to KIR2DL5 (Fig. 6B). The two events involved swapping of the signaling domains with either segment 59 of orangutan P. abelii-KIR2DL5 also belongs to the G1 preservation of function (Hoho-KIR2DL2) or functional shift from group, whereas the intergenic sequences 59 of human KIR2DL5 be- an inhibitory to an activating receptor (Nole-KIR2DH1). These long to the G2a group (Fig. 6B). Discovery of a complete KIR2DL5 deletions are correlated with absence of MHC-C and MHC-G from gene in orangutan (Fig. 6A)andofKIR2DL5 segments in gibbon some, if not all, gibbon MHC haplotypes. shows KIR2DL5 was a component of early hominoid haplotypes. Our analysis also indicates that KIR2DL5 in early hominoids was 39 of Acquisition of novel signaling function accompanied lineage a G1 intergenic region, later to be replaced by the G2a segment III KIR expansion present in modern humans. Taken together with phylogenetic analysis In humans and great apes, the activating KIR of lineages II and III of the intragenic sequences, these observations allowed us to refine have a lysine residue in the transmembrane region that mediates previous models of KIR haplotype evolution. Ancestral hominoids are interaction with the DAP12 signaling adaptor (62). In contrast, the now seen to have had KIR haplotypes with five complete genes, each gibbon-activating KIR2DH1 has an arginine residue in the trans- corresponding to one of the five human KIR lineages, and a lineage III membrane region that is shared with Mm-KIR3DH, a macaque- pseudogene (Fig. 6A). activating KIR of lineage II. KIRs having arginine in the trans- membrane region are predicted to interact with the FcRg signaling The irregular structures of gibbon KIR haplotypes were shaped adaptor and not with DAP12 (63). Because KIR signaling domains by successive deletions with lysine evolved from inhibitory lineage III KIR (28) and To discern how modern gibbon KIR haplotypes evolved from their gibbon lineage III KIR are equally related to all great ape and common hominoid ancestor, we aligned their intergenic and in- human lineage III KIR (Fig. 4D), activating KIRs with lysine- tragenic segments on the basis of the results of phylogenetic analysis mediated signaling via DAP12 are seen to be specific to great apes (Fig. 6A). This comparison showed that a minimum of five deletions and humans. Thus, the emergence of MHC-C and lineage III KIR and one duplication were necessary to evolve the four gibbon hap- specific for MHC-C in a great ape ancestor was also accompanied lotypes from the ancestral KIR haplotype. That three deletions are by acquisition of a lysine-containing transmembrane by the KIR specific for N. leucogenys and two for H. hoolock suggests partial and recruitment of DAP12. The Journal of Immunology 1387 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. Successive deletions diversified gibbon KIR haplotypes. A, Relationships between the genes and intergenic segments of the four gibbon KIR haplotypes. Vertical black arrows indicate equivalent genes or gene segments. Red arrows indicate deletion (Del) or duplication (Dup) events. Dotted arrows denote hybrid genes with segments originating from different ancestral genes. The KIR intergenic regions are color-coded based on the phylogenetic analysis presented in B. At the top is shown the predicted ancestral hominoid KIR haplotype, with framework genes in bold. At the bottom are shown two orangutan KIR haplotypes. KIR genes are color-coded according to lineage: Ia (dark purple), Ib (light purple), II (pink), III (green), and V (blue). B, Phylogenetic analysis of KIR intergenic segments. Analysis and display are as described for Fig. 3B and 3C. Sequences characterized in this study are colored yellow (gibbon) or pink (orangutan). C and D, Summary of the functional effects of the five deletions shown in A. All five deletions led to the loss of Ig-like domains; two of them also formed new hybrid genes (C). Both species of gibbon experienced deletions that caused loss of Ig-like domains and formation of new genes (D). Because KIR2DL5 is not a framework gene, its Ig domains were only considered “lost” when a KIR2DL5 remnant was present on the haplotype; other potential KIR2DL5 “losses” were are indicated by parentheses. Deletions were numbered as in A. ppTwo independent events. A–D, The KIR2DL4, 2DH1, and 3DL1 genes of Nole-H2 were not included in the analysis because they are incomplete and contain segments of draft quality. 1388 KIR-MHC COEVOLUTION IN GIBBONS

Discussion ∼50% of orangutan MHC haplotypes and the MHC-C allotypes The expansion of the KIR locus, which now constitutes a large, only carry one (C1) of the two epitopes (C1 and C2) elaborated in plastic gene family in humans, occurred in primates during the human and chimpanzee (55, 60, 66). In this context, the structures past ∼85 million years (64). Four ancient KIR lineages predate the of the gibbon haplotypes are both striking and informative. Three of separation of hominoids from Old World monkeys 24–34 million the haplotypes have no intact lineage III KIR gene, and the fourth years ago (65), and they have been retained to the present day. (Nole-H2) has only one (KIR2DL1). Thus, we have no evidence for Additional species-specific gene expansions have, however, led to expansion of lineage III KIR genes in gibbons, but in fact the exact markedly different repertoires of KIRs in modern primate species. opposite. For most KIR haplotypes, lineage III has been extinguished In great apes and humans, the lineage III KIRs form the largest as a distinct entity (although segments can be preserved in recom- and most diverse group of KIR (23), whereas this role has been binant forms). This feature of gibbon KIR and its contrast with the taken by the lineage II KIR in Old World monkeys (macaques and situation in the orangutan (23, 55) suggest that absence of MHC-C is green monkeys) (19–22). Functional interaction with rapidly likely to be a general property of gibbon MHC haplotypes (Fig. 7). evolving and highly polymorphic MHC class I is considered to Before this study, the lineage I KIR2DL4 in the central framework drive the dynamic evolution of KIRs. For example, in great apes, region was seen as the most conserved and phylogenetically stable the expansion of lineage III KIR correlates with emergence of KIR gene (23). But in the gibbon, KIR2DL4 has been eliminated from MHC-C (23). Notably absent from the current picture of hominoid two of the haplotypes and the 2DL4 genes of the other two haplotypes KIR evolution have been the smaller apes, the gibbons, equally encode proteins that lack the ITIMs necessary for inhibitory function. close relatives of great apes and humans. Filling this gap are the Whereas 2DL4 in other species has the potential for both activating results from this study of gibbon KIR and MHC class I. and inhibitory function, gibbon 2DL4 has only the potential for ac- Downloaded from Previously, gibbon cDNA sequences corresponding to poly- tivation. The defined ligand for human KIR2DL4 is HLA-G, which is morphic MHC-A and MHC-B genes were described, but no cDNA physiologically expressed during pregnancy by fetal extravillous corresponding to MHC-C (41). From a systematic search through trophoblast (67). Interactions between trophoblast HLA-G and the genome of an individual gibbon, we identified 28 MHC class I KIR2DL4 expressed by maternal uterine NK cells are implicated in sequences, including two alleles for MHC-A, -E, and -F genes and the invasive remodeling of maternal blood vessels that accompanies

MHC-J, -L, -W, and -X pseudogenes that are orthologous to the implantation (68). The observed deletion of gibbon MHC-G,com- http://www.jimmunol.org/ corresponding HLA class I. Contrasting with such similarities was bined with deletion of 2DL4 from some KIR haplotypes and func- the presence of three MHC-B like genes in the gibbon and the tional incapacitation on others, suggests that cognate interactions absence of MHC-C and MHC-G. Also absent from the gibbon between MHC-G and KIR2DL4 have been selected against in gib- were several MHC-G–linked pseudogenes conserved by human bons. Trophoblast also expresses HLA-C, but not HLA-A and -B (67), and rhesus macaque, consistent with deletion of an extended ge- and polymorphic interactions between trophoblast HLA-C and cog- nomic segment containing MHC-G from the gibbon genome. nate lineage III KIR on uterine NK cells are associated with human Multiple MHC-B and absence of MHC-C are features the gibbon pregnancy syndromes (11, 12). Our results suggest that gibbons lack shares with rhesus macaque and other Old World monkeys. Ab- MHC-C and have few MHC-C–specific KIRs, implying that neither sence of MHC-C could reflect the fact that gibbons, like Old of the two types of HLA-KIR interaction that are important for human by guest on September 29, 2021 World monkeys, diverged from great apes and humans before reproduction contribute to gibbon reproduction. Whereas human MHC-C arose. Alternatively, gibbon ancestors may have had placentation is associated with a deep trophoblast invasion of the MHC-C, but it was subsequently lost, as clearly happened for uterine wall, pregnancy in gibbons involves a shallow invasion gibbon MHC-G. A third possibility is that MHC-C is not fixed in comparable to that seen for Old World monkeys (69), species that also gibbon genomes, as is the case for the orangutan MHC-C (66), and lack MHC-C and functional MHC-G (33). by chance the individuals studied here and in the previous in- KIR3DL3, the 59 framework gene, stands out as the only gene in vestigation (41) had two MHC-C negative MHC haplotypes. the gibbon KIR locus that has withstood the effects of widespread Further perspective on the presence or absence of gibbon MHC-C deletion and has done so for ∼11 million years, since gibbon came from the study of gibbon KIRs. Comparison of four gibbon genera diverged (53). KIR3DL3 is the only gene common to the KIR haplotypes showed that they have fewer genes on average than four haplotypes. All four KIR3DL3 variants have two intact KIR haplotypes in other hominoids and rhesus macaque. They are ITIMs in the cytoplasmic tail, and they appear fully functional. also highly variable, each haplotype having a different KIR gene These properties point to KIR3DL3 having been of persistent content and irregular organization. This state of affairs arose by value for gibbons. Human KIR3DL3 is a highly polymorphic gene a series of independent deletions that targeted different parts of (30, 57–59) that has a distinctive promoter (70). Transcription a common ancestral haplotypes and differentially affected in- appears not to occur in mature CD56dim NK cells, but can be bright dividual KIR genes. As a consequence of these deletions, the gen- detected in the CD56 NK cells that are a minority population erally conserved organization of the KIR locus into two variable in peripheral blood, but constitute the majority of uterine NK cells regions bounded by three constant framework regions (23) has been (30). Although in vitro transfection of KIR3DL3 into a cell line eroded in gibbons. That these unusual features are common to gives rise to surface expression of KIR3DL3 protein, no expres- species representing the most divergent genera of gibbon (separated sion of KIR3DL3 by either blood or uterine NK cells has been by ∼11 million years of evolution) (34, 53, 54) points to their detected using specific mAb. No ligand for KIR3DL3 has been generality in this taxonomic group. identified, and the functions of this receptor are unknown. A po- All known MHC-C–specific KIRs are lineage III KIR. In hu- tentially important difference between human and gibbon is that mans and chimpanzees, these make up the most numerous KIRs, human KIR3DL3 has only one ITIM, compared with two in and several lineage III KIR genes are represented on each haplo- gibbons, because the protein terminates at a position within the type (2, 19). In contrast, the rhesus macaque, which lacks MHC-C, second ITIM present in other inhibitory KIR. Loss of functional has a single lineage III gene (19). Expansion of lineage III KIR ITIMs is a common feature of great ape and human KIR3DL3 has been correlated with the presence of MHC-C and is clearly (Fig. 4C), suggesting that 3DL3 in these species has acquired evident in the orangutan, even though MHC-C is present only on a function that distinguished them from gibbon 3DL3. The Journal of Immunology 1389

FIGURE 7. Model for the di- versification of KIR in hominoids and macaques. KIR genes are color-coded according to lineage: Ia (dark pur- ple), Ib (light purple), II (pink), III (green), and V (blue). In great apes and humans, the lineage III genomic regions are highlighted by green boxes; in macaques, the lineage II region is highlighted by a pink box. To simplify the display, KIR pseu- dogenes were not included in the Downloaded from model, except for gibbons. http://www.jimmunol.org/

From our analysis, it is clear that the KIR locus in gibbons does not Disclosures conform to the principles previously established from comparison The authors have no financial conflicts of interest. of an Old World monkey (rhesus macaque), two great apes (orangutan and chimpanzee), and the human species. In these spe- cies, one can see a progression of increasing complexity, which References by guest on September 29, 2021 roughly correlates with the emergence, fixation, and diversification 1. Vilches, C., and P. Parham. 2002. KIR: diverse, rapidly evolving receptors of of MHC-C (23). During gibbon evolution, we see that three of the innate and adaptive immunity. Annu. Rev. Immunol. 20: 217–251. 2. Bashirova, A. A., M. P. Martin, D. W. McVicar, and M. Carrington. 2006. 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