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Molecular Immunology 46 (2009) 457–472

Contents lists available at ScienceDirect

Molecular Immunology

journa l homepage: www.e lsev ier .com/ locate /mol imm

he B7 family of immunoregulatory receptors: A comparativend evolutionary perspective

ohn D. Hansena,∗, Louis Du Pasquierb, Marie-Paule Lefrancc, Virginie Lopezd,bdenour Benmansoure, Pierre Boudinote

US Geological Survey—Western Fisheries Research Center, Seattle, WA 98115, USAUniversity of Basel, Institute of Zoology and Evolutionary Biology, Vesalgasse 1, CH-4051 Basel, SwitzerlandInstitut Universitaire de France, Laboratoire d’ImmunoGénétique Moléculaire, Université Montpellier II, UPR CNRS 1142, FranceUMR 6632, Équipe Évolution biologique et Modélisation, Université de Aix Marseille/CNRS, case 19, 3, place Victor-Hugo, 13331 Marseille Cedex 03 FranceInstitut National de la Recherche Agronomique, Unité de Virologie et Immunologie Moléculaires, 78352 Jouy-en-Josas Cedex, France

r t i c l e i n f o

rticle history:eceived 8 October 2008ccepted 9 October 2008vailable online 9 December 2008

eywords:7D80D867-H17-DC

a b s t r a c t

In mammals, T cell activation requires specific recognition of the peptide–MHC complex by the TcR andco-stimulatory signals. Important co-stimulatory receptors expressed by T cells are the molecules of theCD28 family, that regulate T cell activation, proliferation and tolerance. These receptors recognize B7s andB7-homologous (B7H) molecules that are typically expressed by the antigen presenting cells. In teleostfish, typical T cell responses have been described and the TcR, MHC and CD28/CTLA4 genes have beencharacterized. In contrast, the members of the B7 gene family have only been described in mammalsand birds and have yet to be addressed in lower vertebrates. To learn more about the evolution of com-ponents guiding T cell activation in vertebrates, we performed a systematic genomic survey for the B7co-stimulatory and co-inhibitory IgSF receptors in lower vertebrates with an emphasis on teleost fish. Oursearch identified fish sequences that are orthologous to B7, B7-H1/B7-DC, B7-H3 and B7-H4 as defined by

7-H37-H4o-stimulation

sequence identity, phylogeny and combinations of short or long-range syntenic relationships. However,we were unable to identify clear orthologs for B7-H2 (CD275, ICOS ligand) in bony fish, which correlateswith our prior inability to find ICOS in fish. Interestingly, our results indicate that teleost fish possess asingle B7.1/B7.2 (CD80/86) molecule that likely interacts with CD28/CTLA4 as the ligand-binding regionsseem to be conserved in both partners. Overall, our analyses implies that gene duplication (and loss)have shaped a molecular repertoire of B7-like molecules that was recruited for the refinement of T cell

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. Introduction

The activation of T lymphocytes is finely tuned by a combinationf signals delivered through the TcR–CD3 complex and accessoryignals that can be either stimulatory or inhibitory. The initial sig-al through the MHC/peptide/TcR complex termed “signal 1” isot sufficient to induce full activation of naïve T lymphocytes andhus requires an additional co-stimulatory signal, which is antigen-ndependent (signal 2). This co-stimulatory signal is provided by

nteractions between B7.1 (CD80) and B7.2 (CD86) ligands on APCsnd CD28 expressed on T cells (Freeman et al., 1993). Once receivinghis confirmatory signal from the APC (signal 2), the armed T cellill only require signal 1 for future activation and effector function-

∗ Corresponding author.E-mail address: jhansen@usgs.gov (J.D. Hansen).

cam2aIch

161-5890/$ – see front matter. Published by Elsevier Ltd.oi:10.1016/j.molimm.2008.10.007

of the vertebrates.Published by Elsevier Ltd.

lity against non-self. In mammals, both B7.1 and B7.2 are requiredor full complete activation of the naïve T cell by providing a balancef activating and inhibitory signals. However, these two receptorsisplay distinct expression patterns: B7.1 is inducible, while B7.2

s constitutively expressed on APCs, up-regulated upon activationn APCs (Larsen et al., 1994) and is required for the generation of

ature DC repertoires. In contrast, the ligation of B7.1 and B7.2ith CTLA4 exerts an inhibitory effect on T cell activation, blocking

h2 responses and maintaining peripheral tolerance. Accordingly,omparison of B7.1-KO, B7.2-KO and B7.1/.2-KO mice and blockingntibodies suggests a complex role for these receptors in autoim-unity (Larsen et al., 1994; Lenschow et al., 1995; Poussin et al.,

003; Salomon and Bluestone, 2001). Mammalian B7.1 and B7.2re membrane bound receptors containing one IgSF V domain, onegSF C domain, a transmembrane region and rather divergent intra-ytoplasmic regions. Additionally, a splicing variant lacking the TMas also been described for B7-2, that is expressed by non-activated

4 Immunology 46 (2009) 457–472

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onocytes. The secretion of B7.2deltaTM induces proliferation andytokine production by both naive and memory T cells (Greenfieldt al., 1998), providing an example of additional structural com-lexities of B7-mediated co-stimulation. B7.1 and B7.2 genes haveeen found in many mammalian species and are tightly linked onuman chromosome 3 and mouse chromosome 16 (Table 1).

Aside from B7.1 and B7.2, five additional receptors have beenharacterized and named “B7-homologs” (B7-H), owing to sharedtructural features with the primary B7 molecules. Three of these7-H receptors bind members of the CD28 family, providing

unctional support to the notion that the B7 family mirrors theiversity of the CD28-related receptors for evoking T-cell stimu-

atory/inhibitory pathways in the immune system: B7-H1 (PDL-1)nd B7-DC (PDL-2) bind the programmed cell death (PD-1) receptorKeir et al., 2008), and B7-H2 (CD275) is the ligand of ICOS (inducibleo-stimulator) (Wang et al., 2000).

B7-H1 and B7-DC deliver negative co-stimulatory signalshrough PD1. B7-H1 is constitutively expressed on multiple cellypes in mice including activated B, T, myeloid and DC and alson endothelial cells. In contrast, B7-DC is restricted to DC andacrophages and is induced by IL4 while B7-H1 is primarily regu-

ated by IFN�. Both genes are up-regulated during T cell activationnd B7-DC has a higher affinity for PD-1 than B7-H1. Surprisingly,xpression cloning revealed that B7-H1 not only interacts with PD-but also with B7.1 (but not with B7.2). The exact function of B7-H1xpressed by T cells is still unknown, but the large distribution ofhese receptors has to be compared to the broad expression patternor their main ligand, PD-1. Overall, the PD1:PD-ligand pathway isritical for peripheral tolerance (Fife et al., 2006; Keir et al., 2008)nd elicits an essential role in chronic viral infections by balancingmmune responses to pathogens and subsequent CMI mediated tis-ue damage (Barber et al., 2006; Petrovas et al., 2006). The deliveryf an inhibitory signal through B7-H1 and B7-DC has been well doc-mented in the context of anti-tumor immunity since many tumorsxpress B7-H1 and thus down-regulate specific T cell responses.

In contrast, B7-H2 delivers positive co-stimulatory signalshrough ICOS, a member of the CD28 family. It is constitutivelyxpressed on the surface of a broad range of cell types including Bells, macrophages, DC, some T-cell subsets and on certain epithe-ial and endothelial cells. ICOS is induced on CD4+ and CD8+ T cellsuring T cell activation and the ICOS/B7-H2 pathway is critical forhe delivery of T-cell help to B cells to promote humoral immunity.he B7-H2 knockout mice demonstrated that B7-H2 is required forhelper cell activation, differentiation, and the expression of effec-

or cytokines as well as for the development of NKT cells (Chung etl., 2008; Nurieva et al., 2003).

The ligands of the last two members of the B7 family (B7-H3nd B7-H4) have yet to be identified, but their domain composi-ion, sequence similarities and functional properties group themith the B7/B7H receptors. Mammalian B7-H3 has been detected

n the surface of T cells, B cells, DC, macrophages and on specificarcinoma cells. B7-H3 transcripts are up-regulated upon in vitroFN� stimulation (in Th1 responses), but are down-regulated dur-ng Th2 responses (Suh et al., 2003). The functional properties of the7-H3 receptor seem to be rather complex. A B7-H3Ig fusion pro-ein binding to activated T cells provided a positive co-stimulatoryesponse leading to T-cell proliferation, cytotoxicity and IFN� pro-uction, suggesting a positive co-stimulatory function (Chapovalt al., 2001; Sun et al., 2002). Soluble versions of B7-H3 are alsoeleased from monocytes, DC and activated T cells via proteolytic

rocessing resulting in the binding and activation of T cells by theoluble receptor (Zhang et al., 2008). However, B7-H3 KO mice (Suht al., 2003) showed increased T cell responses, accelerated EAEnd severe hyperinflammatory response, supporting an inhibitoryunction. The last member of the B7 family, B7-H4, has the same Ta

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verall domain composition as the other members of the B7 familyut in contrast, B7-H4 is bound to the APC via a GPI-linked (glycosylhosphatidylinositol) anchor. Human and mouse B7-H4 expressionan be induced on freshly isolated T cells, B cells, DC, and mono-ytes and B7-H4 mRNA is broadly expressed in many lymphoid andon-lymphoid tissues, including tumors. Overall, B7H4 is a nega-ive regulator of T cell responses (Prasad et al., 2003; Sica et al.,003; Zang et al., 2003), while the expression of B7-H4 on tumorsuggests that B7-H4 is involved in evasion from anti-tumor immu-ity (Choi et al., 2003). Surprisingly, B7-H4 KO mice develop normalytotoxic T-lymphocyte reactions against viral infection (Suh et al.,006), and thus the precise role of this receptor has yet to be fullynderstood.

The B7 family members are therefore engaged in multi-le stimulatory and/or inhibitory pathways, being expressed onntigen-presenting cells and for the most part, binding a ligand oncells. They represent a dedicated subset of a large extended fam-

ly of B7-related proteins including MOG and butyrophilins (Henryt al., 1999), which includes molecules involved in innate immu-ity. While B7s and B7H molecules share structural features andequence similarity, it is not clear yet whether they all bind one oreveral members of the CD28 family and to which extent they alsoecognize other members of the B7 family. The complexity of theirxpression patterns reflects the complexity of their functional con-ributions to the regulation of immune responses. A comparativenalysis with their homologs involved in the less complex stim-lation pathways of the lower vertebrates may therefore providelues about their functional relevance. Teleost CD28 and CTLA4rthologs which possess conserved B7 ligand-binding sites haveeen recently described suggesting that they likely engage a B7ounterpart (Bernard et al., 2007, 2006). We therefore made a sur-ey of the available fish genomic and transcript databases to searchor B7 family members and we identified several sequences show-ng most of the hallmarks of their mammalian counterparts. Here

e provide a phylogeny-based classification of these molecules andcomparative analysis that is intended to shed light on the originsf T-cell co-stimulation.

. Materials and methods

.1. Identification and sequence characterization of B7 familyembers

An orderly approach utilizing current genome drafts andST indices was taken to identify B7 and B7-H genes from thearious vertebrates. In short, the majority of searches involvedsing mammalian, avian and teleost sequences to search thearious databases using TBLASTN. For EST searches, sequencesere used as queries for TBLASTN analysis of the EST indices

n Genbank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and at thenstitute for Genomic Research (http://compbio.dfci.harvard.edu/gi/tgipage.html). ESTs representing partial sequences were usedo search EST indices using BLASTn and overlapping sequencesere assembled using the Assembler function of MacVector.

ndividual genomes were searched using TBLASTN at ENSEMBLwww.ensembl.org/index.html) or BLAT (http://genome.ucsc.du/cgi-bin/hgBlat). In addition, TBLASTN searches were conductedor Tetraodon (www.genoscope.cns.fr/externe/tetranew/), medakattp://dolphin.lab.nig.ac.pj/medaka/) and the elephant shark (Cal-

orhinchus milii, http://esharkgenome.imcb.a-star.edu.sg/) at the

pecified URLs. Upon assembled consensus sequences, sequencesere used for BLASTP analysis of the NR database at Genbank.mino acid sequences of the B7 members were then scanned

or domain architecture using CDART (http://www.ncbi.nlm.ih.gov/Structure/lexington/lexington.cgi?cmd=rps) and SMART

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nology 46 (2009) 457–472 459

http://smart.embl-heidelberg.de/). Amino acid alignments andMGT numbering were performed using Clustal W and tools foundt IMGT (IMGT®, the International ImMunoGeneTics informationystem, http://imgt.cines.fr/ (Lefranc et al., 2008). Secondarytructures and IMGT Collier de Perles were based upon the IMGTnique number system IgSF V and V-like domains and for C and-like domains using IMGT tools (Lefranc et al., 2005). Signal pep-ides, TM domains and putative N-linked glycosylation sites weredentified with using SignalP (www.cbs.dtu.dk/services/SignalP/),Mpred (www.ch.embnet.org/software/TMPRED form.html),MHMM (www.cbs.dtu.dk/services/TMHMM/) and NetNGlyc1.0 (www.cbs.dtu.dk/services/NetNGlyc/), respectively. GPI pre-ictions for B7-H4 were made using the “Big PI” predictor atttp://mendel.imp.ac.at/gpi/cgi-bin/gpi pred.cgi. Finally, phyloge-etic comparisons were performed using MEGA 3.1 (Kumar et al.,004). Briefly ClustalW alignments of the IgSF domains (single orandem) were used for the generation of Neighbor-Joining treesith Poisson correction, deletion of gaps and bootstrap analysis

1000 replicants).

.2. Synteny analysis using C.A.S.S.I.O.P.E

The syntenic relationships of the B7 family were initially con-ucted using BLAT (http://genome.ucsc.edu/cgi-bin/hgBlat) andnsembl and then more refined analysis of synteny was per-ormed using C.A.S.S.I.O.P.E (Clever Agent System for Syntenynheritance and Other Phenomena in Evolution). Searching foriologically relevant conserved genomic regions requires both phy-

ogenetic orthology assessment and statistical testing for the genesf the relevant regions in as many genomes as possible. The pro-ess developed in C.A.S.S.I.O.P.E. integrates these two importantteps in a single automated process: (1) the phylogeny: orthol-gous/paralogous genes are determined by the aggregation ofhree phylogenetic methods using the Figenix plateform (Gouret etl., 2005), in contrast to over-simplistic BLAST approaches. Addi-ionally, phylogenetic information allows reconstruction of thevolutionary history and thereby more accurate ancestral genomeeconstruction (2) a statistical test: CASSIOPE therefore utilizes apecific statistical test (Danchin and Pontarotti, 2004) to assess theignificance of the predicted, conserved gene clusters.

. Results

.1. B7.1 and B7.2 family members

In mammals, B7.1 and B7.2 are responsible for signal 2 that isbsolutely required for the activation of naïve T cells. Sequencesimilar to B7.1 and B7.2 were easily identified in various mam-alian databases and in the avian databases using TBLASTN with

uman or murine B7.1 or B7.2 as queries. However, the identifica-ion of teleost B7 genes was more elusive. The fish B7 homologswhich we have named B7R, for B7-related) were in fact retrievedsing a fish sequence that was initially identified as an Ig domain-ontaining molecule. Sequences showing significant identity withither B7.1 or B7.2 were identified from zebrafish, fugu, Atlanticalmon, stickleback, fat-head minnow and a cichlid species. Onlyne sequence could be identified per fish species even when theomplete genome sequence was available, suggesting that there issingle, common B7 receptor in teleosts. The human B7.1 and B7.2

mino acid sequences are ∼30% identical to each other, thus the7R sequences display roughly equivalent levels of identity to B7.1nd B7.2 (∼25%).

A partial B7R EST was also identified from the spiny dogfishSqualus acanthias), an elasmobranch, indicating the presence of

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60 J.D. Hansen et al. / Molecular

7 genes in the earliest jawed vertebrates. Interestingly the spinyogfish sequence identified B7-H2 (ICOSL) as the top 4 matches

n BLASTP analyses (29% identity over 212 AA) whereas the fish7R sequences all retrieved either B7.1 or B7.2 sequences as theop matches (avg. 25% identity over 240 AA) when used in BLASTPueries of the NR.

The B7R sequences displayed the characteristic structure of the7 family including a prototypical V-like domain, a C-like domain, aransmembrane hydrophobic stretch and a short intra-cytoplasmicegion. An alignment of the human, murine, avian and fish B7equences was then performed using ClustalW. Additionally, IMGTumbering and modeling was applied to the Ig V and C domainso produce an optimized alignment (Fig. 1). Both V and C domainsisplayed conserved 23C and 104C residues that are required for the

g fold, but all fish B7R molecules lacked the canonical 41W in the Vomain. Instead, 41W is replaced with other amino acids (I/F/L/V)hich possess non-polar side chains like tryptophan. Overall, theammalian B71/B72 and fish B7R sequences are not highly sim-

lar in amino acid identity but, a few common structural featuresppear from the collier de perle representation of the V domainssee Supplementary Fig. I). In particular, the short C → C′ and D → Eoops appear to be a typical feature of the B7 molecules.

Interestingly, the elasmobranch B7-H2-like sequence maintains1W in the presumptive V domain. The C-like domain was slightlyegenerated and was not easily recognized using the conservedomain database (CDD) at NCBI or via SMART analysis.

Although the fish B7R and tetrapod B7 sequences do not showigh sequence identity (avg. 25–30% AA identity) to each other,he multiple alignments highlight important conserved features.reviously we reported that the CDR3 loop of fish CD28 and CTLA4eceptors possess the conserved (L/F/M)(Y/F)PPP(I/L/F) motif thatnteracts with the B7.1 and B7.2 receptors (Bernard et al., 2007,006). This motif was absolutely conserved in all CD28 and CTLA4equences from fish and tetrapods, suggesting that fish shouldossess B7 orthologs with a conserved CD28/CTLA4 binding site.uman CTLA4 contacts residues within the B7.1 or B7.2 IgSF Vomain and this interaction involves the PPP-containing loop athe top surface of the CTLA-4V domain and at the side of the B7Vomain. The positions of the B7V domain involved in interactionith CTLA4 are located in the C, C′, C′ ′, F and G strands and they

re slightly different in B7.1 and B7.2 (Fig. 1). The alignmentuggests that the biochemical properties of the CD28/CTLA4nteracting residues are rather well conserved at most of the keyositions between fish B7R and mammalian B7.1 and/or B7.2. Theotential contact residues at positions 40, 42, 52 and 55 that areritical in the B7.1V domain for CD28/CTLA4 association are wellonserved among the teleost sequences (40Y, 42Q, 51F and 54G).nterestingly, the conserved positions among the fish sequencesmplies that the overall contact interface is more similar to thoseescribed for human B7.1 than B7.2. Site-directed mutagenesisf several conserved residues in the ABED �-sheet of the B7.1Comain completely abrogated the binding to CTLA4, implyinghat the C domain plays an important, although indirect role inhe interaction between CTLA4 and B7. Most of these positionsre well conserved in the fish B7R sequences including positions(F/Y), 4 (P) and 6 (I/L/V/F), that constitute critical residues forinding CD28/CTLA4. Taken together, these observations supportur hypothesis that the fish B7R receptors bind CD28 and CTLA4ia the P-rich loops and that the geometry of the interaction haseen conserved among gnathostomes.

The alignment of the B7R constant domains also reveals con-erved cysteine residues (positions 10 and 19 in strands A and B)hat are limited to the teleost sequences. This is reminiscent of theignature for the cortical thymocyte marker in Xenopus (CTX) con-tant domain, which may reflect special constraints on the structure

a1BPe

nology 46 (2009) 457–472

f the domain. The transmembrane regions did not display obvi-us similarity. Finally, the intra-cytoplasmic regions do not containyrosine-based signaling motifs such as ITAMs or ITIMs. In addition,he fish and avian sequences lack the RRNE motif found in micehat is required for co-stimulation and capping (Doty and Clark,998). However, the same study indicated that a serine residue nearhe RRNE motif was also required for co-stimulation; all of the B7nd B7R intra-cytoplasmic sequences contain serine and prolineesidues, the role of which remains to be determined.

.2. B7-H1 and B7-DC sequences in fish and other vertebrates

B7-H1 and B7-DC are the natural ligands for PD-1, a CD28 familyember, and together they are partially responsible for periph-

ral tolerance of T cells via inhibitory signals. Chicken and murine7-H1 and DC were used as queries in TBLASTN searches to iden-ify orthologous sequences in fish and other tetrapods. Singleequences displaying roughly equivalent identity to chicken andammalian B7-H1 and DC were identified for salmonids, 2 species

f pufferfish and representative cyprinids as well as distinct B7-1 and DC genes in amphibians. Thus for all bony fish speciesxamined, we were only able to define a single B7-H1/DC geneer genome compared to the two genes encoding B7-H1 and DC

n tetrapods. Similar to other B7 family members, all of the B7-H1nd DC sequences identified within this study are composed of aignal sequence, extracellular IgSF V and C domains followed bytransmembrane region and a relatively short intra-cytoplasmic

omain. As shown in Fig. 2, all B7-H1 and DC molecules displayonservation of the canonical cysteines in the IgSF V-like domain23C and 104C) and C-like (23C and 104C) domains that are requiredor the Ig fold. In addition, all of the sequences except human and

ouse (V domain) encode canonical 41W residues in the C strandsor both the V and C-like domains. The V domain of human B7-H1,7-DC and fish B7-H1 also shared a motif GXE in the C′–C′ ′ loop,nd the same length of the D → E loop, as evidenced by the col-ier de perle representation (Supplementary Fig. I). Moreover, allequences contain 3-4 N-linked glycosylation sites (NXS/T) withinhe extracellular domains including the presence of a conserved-linked site in or near the EF loop of the C domain.

Overall, the teleost B7-H1/DC sequences displayed slightlyigher levels of identity (avg. 28–34% ID over 215 AA) to the tetra-od B7-H1 sequences, followed directly by the B7-DC genes in aituation similar to that found for the B7R and the B7.1 and B7.2enes. We have tentatively named the teleost sequences as B7-1 based upon the relative conservation of residues implicated inD-1 association for B7-H1. Similar to CD28 and CTLA4, PD-1 inter-cts with the B7-H1/DC ligands through interaction of the PD-1Vomain with the B7-H1/DC V domains. One major difference is thathe PD-1 CDR3 loop lacks the conserved proline rich motif foundn CD28 and CTLA4. Butte et al. (2007) then identified 10 residuesn murine B7-H1 via site directed mutagenesis that are involvedn PD-1 recognition. In particular, 7 residues impact PD-1 binding

hile three additional residues resulted in increased affinities forD-1 and were considered as stabilizing residues (Fig. 2). Thoseesidues in the IgSF V domain of murine B7-H1 and DC, which areritical for PD-1 association, are well conserved between chickensnd mammals and to some degree between Xenopus and the otheretrapods. Several of these residues are also moderately conservedithin the gnathostomes. Aside from interacting with PD-1, it was

ecently shown that mammalian B7-H1 can also interact with B7.1

nd that the region of interaction overlaps with the for B7.1/PD-. Based upon this, it has been suggested that CD28, CTLA4 and7-H1 could all compete for B7.1 binding and similarly, B7.1 andD-1 may compete for binding to B7-H1 (Butte et al., 2007). Inter-stingly, chemically cross-linked lysine residues that mapped the

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Fig. 1. Multiple sequence alignment of B7 and B7R genes from fish and tetrapods. Sequences are numbered according to the unique IMGT numbering system for V-like and C-like domains and includes IMGT region delimitations(Lefranc et al., 2005, 2008). Signal sequences were removed. Conserved cysteine residues involved in the IgSF fold are highlighted in red on yellow background. V-domain: positions involved in the human B7 dimer interfaceare in bold black lettering with green background. Amino acids directly involved in the B7.1 or .2/hCTLA4 interface that are conserved are indicated with an @ symbol. Positions that are found in the human B7.1/B7.2/CTLA4interfaces that are strictly conserved are in black lettering with red background (i.e. 40Y) and those with common biochemical properties are in red. In addition, amino acid residues involved in the human B7.1/CTLA4 andB7.2/CTLA4 interface that are conserved in fish are shown in black lettering with blue background while those residues possessing similar biochemical properties are in blue lettering. C-domain: positions found in the C domainthat contribute to B7/CTLA4 binding are indicated with a # symbol. The connecting peptide (CP), transmembrane region (TM) and cytoplasmic domain are also shown. Proline residues in the IC are shown in red with bluebackground. Accession numbers or genome positions are as follows: Homo sapiens B7.1: AAA58390, Mus musculus B7.1: X60958, Gallus gallus B7.1: AJ851659, Homo sapiens B7.2: AAA58389, Mus musculus B7.2: AAH13807, Gallusgallus B7.2: CAJ18297, Danio rerio B7R: CN017839, Pimephales promelas B7R: DT293786, Fugu rubripes B7R-scafold 172 (chrUn231812808), Gasterosteus aculeatus B7R: DN656970, Salmo salar B7R: DW580717, Lipochromis. spB7R: DB860025 and Squalus acanthias B7R/B7-H2: ES651617. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

462 J.D. Hansen et al. / Molecular Immunology 46 (2009) 457–472

Fig. 2. Multiple sequence alignment of B7-H1 and DC related sequences from teleost fish and tetrapods. Sequences are numbered according to the unique IGMT numberingsystem for V-like and C-like domains and includes IMGT region delimitations (Lefranc et al., 2005, 2008). Signal sequences were removed. Conserved cysteine residuesinvolved in the IgSF fold as well as those located in TM are highlighted in red with yellow background. Positions with white lettering on red background correspond to aminoacids that interact with PD1 and are relatively conserved in the alignment. Residues in black lettering/red background indicate mutations that increase affinity for PD1. Murinelysine residues with grey background demarcate the boundaries of the B7/B7-H1 interaction site (note K119 and K124 mark remaining boundary). Putative ITM motifs in theIC region are in red on yellow background. Finally, potential N-linked glycosylation sites (N-X-S/T) are underlined. Accession numbers or genome positions are as follows:Homo sapiens B7H1: AAF25807, Mus musculus B7H1: AAG18509, Gallus gallus B7H1: AI980757–BU254037–XM 424811, Xenopus tropicalis B7H1-Scaf 86 (2029395-2024440),Oncorhynchus mykiss B7H1R: CA366631, Danio rerio B7H1R: DN833503–EV760159, Gasterosteus aculeatus B7H1R: DT956717-chrXIV (7425302.27368), Pimephales promelasB7H1R: DT159564–DT173258, Homo sapiens B7DC: BO331153, Mus musculus B7DC: AAD33892, Gallus gallus B7DC: CF251191 and chrZ (28228704–31137), Xenopus tropicalisB7DC-Scaf 86 (1998704.1996153). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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7-H1/B7.1 interaction boundaries via surface plasmon resonancenalysis for B7-H1 are well conserved within the vertebrate B7-H1equences, implying that teleost B7-H1R could interact with B7.1Butte et al., 2008).

.3. B7-H3 family members

By far, the most conserved B7 family member found in thistudy is B7-H3 as sequences were readily retrieved from represen-ative elasmobranch, teleost and tetrapod species using standardBLASTN analysis and conserved synteny examination. Overall, theature proteins (minus leader segment) display >50% amino acid

dentity between the various vertebrates. Higher levels of identityere maintained within individual vertebrate classes as exempli-ed by the teleosts, which had an average amino acid identity of2% across the extracellular portion of the predicted protein.

A multiple sequence alignment (Fig. 3) was generated for theature proteins to exemplify the conserved nature of B7-H3. The

arious vertebrate sequences display absolute conservation withinhe IgSF V-like and C-like domains of cysteine residues requiredor the Ig fold. B7-H3, like other B7 family members is a glyco-rotein. Analysis of potential N-linked glycosylation sites revealedbsolute conservation of two N-linked sites within E strand of thedomain and the D strand within the C-like domain. Strong con-

ervation of additional glycosylation sites were also observed athe C and D strand intersection and within the F strand of the C-ike domain and within the V domain B strand and CDR1 of theeleost sequences suggesting that these sites are likely importantor B7-H3 function. Following the IgSF extracellular domains, theres a short connecting peptide region containing a conserved prolineesidue that is immediately followed by a highly conserved trans-embrane domain (68% average AA identity, 86% between trout

nd zebrafish) which includes a conserved cysteine residue but noharged residues. The cytoplasmic region contains two conservedresidues, which may be important for biological activity. The first

onserved C residue is found at the transmembrane/cytoplasmicunction. The second is located 8 positions downstream in theytoplasmic region and is immediately followed by a series of con-erved acidic and basic residues suggestive that this region interactsith additional proteins based upon charge–charge interactions. In

ilico post-translational analysis did not reveal any conserved phos-horylation motifs. The genomic structure is identical between 2eleosts (zebrafish and tetraodon) and tetrapod B7-H3 (data nothown).

.4. B7-H4 family members

Homologs of the human B7-H4 sequences were detected usingBLASTN in different fish genomes including zebrafish, tetraodon,nd fugu. Additional sequences similar to B7-H4 were found inST databases from rainbow trout, Atlantic salmon, minnow andounder. Overall, the teleost amino acid sequences were 30–35%

dentical across the extracellular region respective to their mam-alian counterparts. B7-H4 homologs were also identified from

he genome assembly and EST indices of chicken and Xenopus.ig. 4 illustrates that these proteins all contain a typical IgSF V-likeomain and a degenerated IgSF C-like domain that is missing oner both of the canonical 23C and 104C residues that maintain the Igold. In addition, the G strand of the V domain contains a diglycineulge (GxG motif) for some species, which is reminiscent of a J seg-

ent. However, this feature is not strictly conserved and one of

he G residues forming the putative GxG bulge is missing in threef the teleosts. The carboxy-terminal portion of these proteins lackecognizable TM domains but they all contain predicted GPI linkageites, both of which are characteristic of B7-H4. The classification of

eofdv

nology 46 (2009) 457–472 463

sh B7-H4 is hindered by the low similarity with their mammalianounterparts and by the absence of obvious motifs conserved in theolecule. However, all sequences maintain a conserved N-linked

lycosylation site in the V domain (E strand). We also examinedhe genomic structure of zebrafish and tetraodon B7-H4. They pos-ess the same exon/intron structure (splice sites and phase) as thatound in mammals: the first exon encodes the signal sequence, fol-owed by exons 2 and 3 which code for the V and C domains andxon 4 which encodes the carboxy-terminus.

.5. Phylogenetic analysis of the B7 family members

All sequences of the B7 family members were added to a sin-le multiple alignment to assess their phylogenetic relationship(s)sing either single IgSF domains (V or C) or both IgSF domainsogether. Both analyses produced nearly identical trees for thehylogeny of the B7 family. In addition, trees produced by eithereighbor-joining, maximum evolution and maximum parsimonyethods produced nearly identical branching orders (data not

hown) thus lending additional support for the designation of the7 and B7H clades.

In general, the newly identified teleost B7R sequences formed aistinct sister-clade from the main branch that separates the B7 and7H sequences. Overall, the B7 (B7.1/.2 and B7R) sequences do not

orm a monophyletic clade, likely due to the highly divergent naturef these genes (Fig. 5). The B7R clade includes a recently depositedequence for trout B7R (Zhang et al., accession ACH58052), whichrouped tightly with the Atlantic salmon sequence (91% AA identifycross the V and C domains). The branch grouping all fish B7R is sup-orted by a fair bootstrap value (>70%) but the group B71/B7R/B72

s not, although a similar consensus tree is observed with all dif-erent phylogeny algorithms. Interestingly, grouping of chicken B71nd B72 with their respective counterparts is not supported by highoostrap values either.

In contrast, each group of the fish B7H-related sequenceslustered in well-defined B7H sister clades (i.e. B7-H3) withinhe overall B7H monophyletic clade which is supported by goodoostrap scores. The fish sequences reproducibly cluster withheir respective mammalian counterparts, although with moder-te bootstrap scores. Within the overall B7-H1/DC clade, there wasrelatively clear demarcation of both B7-H1 and DC within the

etrapods and a single sister clade corresponding to teleost B7-H1Rmplying that all of these genes derived from a common B7-H1

olecule. Finally, the phylogenetic analysis also suggests that the7-H3 and B7-H4 sequences form distinct sister clades within theverall B7H branch (Fig. 5). It should be noted that the “fish” B7-3 group includes two elasmobranch sequences (L. enicacea—a

kate and C. milii—a chimaera) further strengthening the phylo-enic groupings of the B7-H3 clade and that the origins of this groupredate the emergence of the gnathostomes.

Additional sequences displaying similarity with the B7 genesere also retrieved from medaka and rainbow trout EST databases.owever, these last sequences were more similar to mammalianutyrophilins than to the B7.1 and B7.2 genes. Therefore we didot consider these butyrophilin-like molecules as B7 counterpartss they did not cluster with the other fish B7R and B7 moleculesn the phylogenetic analysis (data not shown) nor with the B7Hequences.

Although our phylogenetic analysis was not always supported byigh bootstrap scores (i.e. >80%), the analysis supports our hypoth-

sis that the fish B7R, B7H1, B7H3 and B7H4 are likely orthologsf the mammalian genes and are therefore expected to be derivedrom common ancestral genes. It also suggests a clear and ancientifference between the B7 and the B7H clades. The low bootstrapalues may simply reflect the high evolutionary pressures exerted

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Fig. 3. Multiple sequence alignment of B7-H3 sequences (lacking leader sequences) from teleost fish and tetrapods. Sequences are numbered according to the unique IMGT numbering system for V-like and C-like domainsand includes IMGT region delimitations (Lefranc et al., 2005, 2008). Conserved cysteine residues involved in the IgSF Ig fold are highlighted in red with yellow background. Additionally conserved cysteines located in theTM and cytoplasmic domain are also highlighted in yellow with red background. Potential N-linked glycosylation sites (N-X-S/T) are underlined. Finally, acidic residues found in the cytoplasmic domain are indicated inbold and blue. Accession numbers or genome positions are as follows: Homo sapiens: AAK15438, Mus musculus: AAH19436, Gallus gallus: BM488497–BU249770, Xenopus laevis: CD301231–BJ067228, Oncorhynchus mykiss:CA346702–CA352727, Danio rerio: CD759065–EE719264, Callorhinchus milii-Scaf AAVX01064573 and Leucoraja erinacea: DT378804. (For interpretation of the references to color in this figure legend, the reader is referred tothe web version of the article.)

J.D. Hansen et al. / Molecular Immunology 46 (2009) 457–472 465

Fig. 4. Multiple sequence alignment of mature B7-H4 sequences from teleost fish and tetrapods. Sequences are numbered according to the unique IMGT numbering system forV and C domains and includes IMGT region delimitations (Lefranc et al., 2005, 2008). Conserved cysteine residues involved in the IgSF Ig fold are highlighted in red on yellowbackground. Potential N-linked glycosylation sites (N-X-S/T) are underlined. SMART analysis did not reveal typical TM regions; potential GPI linkage sites are indicated inmagenta background (IMGT prediction) or bold lettering with red background (prediction http://mendel.imp.ac.at/sat/gpi/gpi server.html). Putative C-terminal hydrophobicpeptide removed at the addition of the GPI anchor are underlined. Accession numbers or genome positions are as follows: Homo sapiens: AAP37283, Mus musculus: AAP37284,Gallus gallus: chr1 (82647831.82658137), Xenopus tropicalis: CR439118, Danio rerio-chr9 (13865193.13868734), Tetraodon nigroviridis-chr2 (17459002.17460514), Fugu rubripes-chrUn (318872930.318873762), Oncorhynchus mykiss:BX911540, Salmo salar: DY726411 and Pimephales promelas: DT117185. (For interpretation of the references to color inthis figure legend, the reader is referred to the web version of the article.)

466 J.D. Hansen et al. / Molecular Immunology 46 (2009) 457–472

Fig. 5. Phylogenetic analysis of the B7 family in vertebrates. The tree was constructed from CLUSTAL generated amino acid alignments for the V and C domains combined(∼180 amino acids as deduced by SMART analysis) using the neighbor-joining method. Tree topography was evaluated by bootstrapping 1000 times with percentages shownat nodes. Brackets denote fish (F) and tetrapod (T) groupings. Accession numbers and gene locations in addition to those found in Figs. 1–4 are as follows: Mesocricetusauratus-B7.1: BAC24767, Rattus norvegicus-B7.1: EDM11215. Oryctolagus cuniculus B7.1.: BAA08643, Canis familiaris-B7.1: AAF17293, Sus scrofa B7.1: AAL58443, Felis catus B7.1:AAB53575, Canis familiaris-B7.2: AAF17297, Felis catus B7.2 BAB11688, Sus scrofa B7.2: AAV74621, Oncorhynchus mykiss-B7R: EU927452, Oryzias latipes B7R: AM306315, Salmosalar B7-H1R: DW562815 and DY708145, Tetraodon nigroviridis B7-H1R: CAF93166, Fugu rubripes B7H1R chrUn (268841252.268843284), Rattus norvegicus B7-H1: EDM13096,O 5102( eus acA 918, H

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rnithorhynchus anatinus B7-H1: XP 001506123, Ambystoma tigrinum B7-H1: CN06316485061.31648775), Oryzias latipes B7-H3: DK244157 and DK216942, GasterostAH29227, Rattus norvegicus B7-H2: XP 001079346, Canis familiaris B7-H2: XP 544

n these receptors for example by pathogen subversion. The pres-nce of B7/butyrophilin related sequences in the genomes of fishridoviruses (YP 164128; gb|AAV91039; gb|AAV91038) adds sup-ort to this interpretation.

.6. Identification of conserved syntenies involving B7 familyembers

As our phylogenetic analysis of the B7 family was inconclusiveor the origin(s) of the B7 and B7H genes, we searched for con-erved syntenies involving B7 related genes and other genes inhe B7 genomic neighborhood. For this purpose, we used a dedi-ated program (C.A.S.S.I.O.P.E) that has been developed to comparehylogenetic trees of all genes in a genomic region, leading to thealidation of true ortholog gene sets. The C.A.S.S.I.O.P.E method-

logy establishes that synteny groups correspond to sets of realrthologs by congruent phylogenetic analyses and statistical vali-ation, which avoids the many pitfalls of the classical reverse-blastnalysis. When this approach provides a clear demonstration thateveral markers are true orthologs in two species, it firmly estab-

o

5tr

, Tetraodon nigroviridis B7-H3: chr5 (1823258.1824878), Fugu repices B7-H3 chrUnuleatus B7-H3: DW637643, Gallus gallus B7-H2: CF257773, Mus musculus B7-H2:omo sapiens B7-H2: AAG01176.

ishes that the B7 related sequences flanked by these markersave also been generated by speciation (i.e. true orthologs). Takenogether, this is critical evidence that sequence similarity is dueo inheritance and not convergence, and therefore builds a solidramework for further analysis of the evolution of functional prop-rties governing T cell activation.

Conserved genes in the vicinity of the B7R genes were identi-ed in different fish species. Several genomic markers located onach side of B7R were identified in humans, mice and chickenshich defined two groups of conserved synteny that were located

n the same region in humans and chickens, and in two clustersn different chromosomes in mice. Unfortunately, the B7s genesere located in a totally different region on another chromosome

n tetrapods (Fig. 6) suggesting that genomic translocations affect-ng this region during vertebrate evolution preclude the detection

f a direct conserved synteny involving B7R and B7.1/.2.

In contrast, a conserved synteny involving the B7-H1 genes andmarkers (CDC37, AK3, RCL1, JAK2 and a putative ORF) was iden-

ified in Stickleback, Tetraodon and mammals (Fig. 7). Syntenicelationships (Fig. 7) were also established for regions flanking

J.D. Hansen et al. / Molecular Immunology 46 (2009) 457–472 467

F teny inr corre

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ig. 6. Genes flanking B7R but not B7R/B7 itself display conserved short-range synelationships of genes in the vicinity of fish B7R. The location of each marker on the

he B7-H3 sequences including 6 markers in zebrafish, tetraodon,uman, mouse and chicken: neuroplastin (NPTN), hyperpolariza-ion activated cyclic nucleotide-gated potassium channel 4 (HCN4),ysyl oxidase-like 1 (LOXL1), secretory carrier membrane protein 5SCAMP5), poly (ADP-ribose) polymerase family member 6 (PARP6)nd the pyruvate kinase-muscle gene (PKM2). These genetic mark-rs were also linked to the Xenopus B7-H3 sequence on Scaf 103

data not shown) thereby further supporting the orthologous rela-ionships of the B7-H3 genes. Sun et al. (2002) previously reportedhat the presence of 2 differentially spliced versions of B7-H3 con-aining either 2 or 4 IgSF domains, which was likely due to tandemuplication of the V and C domain within the lineage leading to all

tcfst

fish and tetrapods. The C.A.S.S.I.O.P.E program was used to establish the syntenicsponding chromosome is indicated in the table.

rimates. We analyzed the various genome drafts and found thatuplication event which generated the 4Ig B7-H3 molecule is prob-bly confined to primates as examination of rodent, avian, amphib-an and teleost genomes by BLAT analyses were negative for theresence of duplicated VC domains for B7-H3. Finally, microsyn-enic clustering involving four markers around B7-H4 in zebrafish,ufferfish, mouse and human confirmed the identification of the

eleost sequences as true B7-H4 orthologs (Fig. 7). This conservedluster involved the genes encoding the transcription terminationactor 2 (TTF2), a dsDNA-dependent ATPase which acts as a tran-cription termination factor, mannosidase alpha 1A2 (MAN1a2) andhe “family with sequence similarity 46” (FAM46C) gene.

468 J.D. Hansen et al. / Molecular Immunology 46 (2009) 457–472

F rograml

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ig. 7. Syntenic relationships of the B7H genes in vertebrates. The C.A.S.S.I.O.P.E pocations and identifiers in the various genomes (Ensembl) are shown.

.7. Linkage groups and long-distance conserved syntenies:racking the likely origin of the B7 family

As noted earlier (Fig. 6), the establishment of syntenic relation-hips for the primordial B7 genes in the vertebrates was not as

sffel

was used to establish syntenic relationships for B7-H1, B7-H3 and B7-H4. Gene

traightforward as that for the B7H genes. In addition, searchingor classical synteny groupings to track the origin of the B7/B7Hamily appeared inadequate due to long-term sequence drift andxtensive recombination within the genomes. However, trackingong-range syntenies of well-defined molecules can be used to

J.D. Hansen et al. / Molecular Immunology 46 (2009) 457–472 469

F on oria ns weo

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ig. 8. Long-range synteny analysis of genes flanking B7R suggests an ancient commnd locations in the genomes of medaka, stickleback and humans are shown. Positiof B7R (CD80 homologue) in the appropriate genomes.

efine weakly linked, ancient gene groups that were duplicatednto large paralogous sets (also known as syntenic blocks). This is

ade possible by the fact that intra-chromosomal recombinationvents are more frequent than inter-chromosomal recombinationvents (O’Brien et al., 1999; Richard et al., 2003). Therefore, track-

ng the evolution of gene sets corresponding to old and long-rangeynteny blocks provides a synthetic view of paralogs in a genomicontext, which could help for future nomenclature revisions basedn evolutionary origin. Thus, such an approach revealed the exis-ence of an ancestral cluster of IgSF genes defined by four sets of

ot(c(

gin of B7R and 4 paralogous regions in mammals that includes B7.1. Gene identifiersre determined using Ensembl and BLAST/BLAT analysis. Circles define the location

aralogs located in human chromosomes 1p, 3q, 11, 21q (and 19q),hat contain many genes coding for membrane receptors involvedn APC/lymphocyte interactions or the development of the nervousystem (Daeron et al., 2008; Du Pasquier et al., 2004). These partic-lar paralogous regions (1p, 3q, 11, 21q and 19q) track the common

rigin of many well-known regulatory and signaling molecules ofhe immune system including genes of the B7 family: CD2, CD3�, �, � and �), BTLA (CD272), members of the leukocyte receptoromplex (LRC), and B7.1 (3q), B7.2 (3q, close to B7.1) and B7-H221q). We therefore looked for the fish homologs of the mark-

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70 J.D. Hansen et al. / Molecular

rs located in the mammalian paralogous regions (1p, 19q, 11pnd 21q) which led to the identification of two linkage groupsn both stickleback and medaka (Fig. 8). In such an approach, theurrent nomenclature may be misleading since it has been builtainly from sequence similarity rather than solid phylogenetic

ssignment. Often a name is assigned rather than another basedn minute sequence differences. Moreover, the phylogenetic anal-sis is sometimes difficult due to gene loss and different rates ofenetic drift or selection, and the functional equivalence of theseorces is not a reliable criterion for a common origin. However, thispproach showed that these two teleost genomic regions representn obvious reshuffling of the genes encountered in the 4 paralo-ous regions identified in human, including the B7R. Surprisingly,ven markers from the human 19q region which contains the LRCnd is thought to be a fragment of chromosome 21 are retrievedDaeron et al., 2008), supporting the hypothesis that human regions1q21 and 19q13 constitute two parts of the same ancestral com-lex. Interestingly, the homologs of three markers belonging to theq linkage group containing B7.1 and B7.2 – JAM3, LSAMP and CD96were encoded on the same chomosome as the B7R gene (Fig. 8).

n addition, the markers JAM3, LSAMP, CD47 and CD166 all haveomologs on stickleback LGVII. These observations suggest that theeleost B7R gene belonged to the same overall linkage group thatncludes B7.1/B7.2. In contrast, we did not find any B7H moleculesn these long-range synteny blocks or any obvious syntenic rela-ionships of the fish B7 genes with the major histocompatibilityegions.

. Discussion

In this article, we identified several new sequences of B7 fam-ly members in teleost fish. The fish B7R genes appear to be likelyrthologs of B7.1 and B7.2 based upon several criteria includingest Blast scores (% identity), phylogenetic analysis of Ig domainsnd the presence of conserved contact residues for binding thePP-loop of CD28 and CTLA4. The fish counterparts of B7H1, B7H3nd B7H4 were also retrieved and constitute the likely orthologsf these B7H receptors. Our conclusions are also based upon ourbility to establish syntenic relationships, both long and short-ange, for the B7 and B7H genes. These observations indicate thatrototypical B7 and B7H molecules were present in the commonncestor of teleosts and mammals, defining ancient clades of stim-latory/inhibitory receptors. This is also supported by the findingf elasmobranch sequences related to B7-H2 and B7-H3, indicatinghat processes governing T-cell co-stimulation and inhibition wereresent at the origin of the gnathostomes.

Previously we demonstrated that teleost CD28 and CTLA4 pos-ess canonical B7 ligand-binding motifs, implying that they shouldnteract with a B7 molecule in a classical fashion (Bernard et al.,007, 2006). This observation therefore prompted our search foronserved motifs potentially involved in receptor/ligand interac-ions in the fish B7R sequences. The conserved positions in the fish7R alignment matched well with key positions involved in theinding of B7.1/B7.2 to CD28 and CTLA4 thereby further supportinghat the B7/CD28 pathway is a very ancient regulatory pathway forymphocytes. Moreover, the teleost CD28 molecules are authentico-stimulatory receptors as they are capable of transducing strongctivation signals in a mammalian context (Bernard et al., 2006).he function of fish CTLA4 is not as well defined as we were pre-iously unable to establish any signaling capacity for trout CTLA4.

owever, the significant conservation of the B7-binding region in

he CTLA-4V domain and the presence of the GxG motif responsibleor high affinity interactions between B7/CTLA4 suggests that fishTLA4 most likely binds B7R and may act as an inhibitory receptory competition with CD28.

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nology 46 (2009) 457–472

The discovery of B7-H1 orthologs in fish was slightly unexpecteds we could not identify a fish PD-1 homolog. It is difficult to excluden the basis of unproductive Blast and synteny searches that a PD-1omolog is not present in the genomes of fish. However, we scannedll available databases for sequences similar to PD-1 and foundnly counterparts of other members of the immunoglobulin superene family. In retrospect, two very intriguing reports have recentlyhown a clear interaction between B7.1 and B7-H1 in mammalsuggesting that maybe this interaction was the primordial one for7-H1. Therefore, the fish B7-H1 may have originally bound B7R

n the absence of PD-1 (Butte et al., 2007, 2008). The B7-H1 recep-or would then have acquired the capacity to bind PD-1 after itsppearance in tetrapods. Interestingly, the binding interfaces areell conserved for both B7/CD28 and B7H1/PD-1, implying that

he fish B7-H1 could bind B7R. The presumptive roles of B7-H3 and7-H4 in primitive vertebrates is more elusive since their ligand(s)ave yet to be defined in mammals. However, it is striking that the7-H3 sequences are the most conserved among the whole family,uggesting that the modalities of ligand recognition/interaction for7-H3 have been maintained during vertebrate evolution to fulfilln important functional role.

Aside from the structural signatures that imply functionalonservation, the genomic and phylogenetic analyses provided per-inent information about the origins of the B7 family members.lthough the accumulation of genomic rearrangements makes thelassical short-range synteny approach nearly impossible for track-ng the history of B7, we could assign it to one of the ancient setsf paralogous regions that has been maintained from tunicates toammals which contains many genes involved in APC/lymphocyte

nteractions (Du Pasquier et al., 2004). The functional and evolu-ionary significance of these particular syntenies awaits furthernvestigation in other genomes such as those for elasmobranchs,ut our observations reinforce the hypothesis that B7/B7R geneselong to a primordial set of immune receptors. These observa-ions prompted us to investigate more ancient species such as theamprey, an agnathan. Unfortunately, we could not find any B7-likeenes in the lamprey genomic contigs, but sequences that harbor allhe hallmarks of regulatory receptors with a V domain, a C domain,TM and an intra-cytoplasmic region with Y-based signaling motifsre present in agnathans (Pancer et al., 2004; Haruta et al., 2006).he final assemblies of the lamprey and elasmobranch genomesill clarify whether such sequences are related to the paralogous

ets described above and whether these receptors represent ances-ors of the B7s.

In contrast to B7, the evolutionary links between the B7H genesnd the paralogous regions described earlier were more elusive.hile the human B7-H2 belongs to the linkage group located on

hromosome 21, B7-H2 appears to be missing in teleosts, thus pre-luding further speculation about the existence of the linkage groupo which it belongs (defined by DSCAM and NCAM2, see Fig. 8). For7-H1, B7-H3 and B7-H4, it was difficult to determine whether theyome from the linkage groups and were translocated, or whetherhey represent an older independent origin. Thus, we were not ableo track the common ancestor of the B7H genes, and our observa-ions simply show that the diversification of the B7H subset (into7-H1, B7-H3, B7-H4) occurred before the split between teleostsnd tetrapods (Fig. 9).

We therefore propose the following evolutionary scenario forignal 2 molecules: the common ancestor of fish and tetrapodsxpressed CD28, CTLA4, B7R and different B7H membrane recep-

ors with the B7R molecule binding both CD28/CTLA4 and B7-H1.D28-, B7- and B7H-clades further diversified during vertebratevolution in a class-specific way leading to unique binding oppor-unities between newly emerging members of the CD28 and B7Hlades. In this scenario, PD-1, B7-DC, B7.1 and B7.2 were produced

J.D. Hansen et al. / Molecular Immunology 46 (2009) 457–472 471

F ratesw s denog

bccrbgiBmiottgd2iitofmisIIdrTaadcccotP

taDit(ttlf

A

cctaiGRbSFNULiraSb

ig. 9. Schematic depiction of the hypothetical evolution of the B7 family in vertebhile 3R signifies the additional teleost-specific genome duplication event. Asterisk

enomes.

y gene duplication during tetrapod evolution and selected byoevolution with their ligands. When a duplication product wasompatible (i.e. binding resulting in a “signal”) with a pre-existingeceptor, it was co-opted into a new co-evolving unit constrainedy the previous interaction. Such a model would explain the sin-le B7-H1/DC ortholog in fish in the absence of PD-1. However,t is surprising that fish do not have a more diverse CD28 and7 families than mammals since they have undergone one orore additional rounds of whole genome duplication, thus provid-

ng additional opportunities for recruitment and/or specializationf the duplicated products. Therefore, it appears that most ofhe additional copies have been lost, probably reflecting a func-ional simplicity of the fish lymphocyte activation system or moreenerally, that gene loss is a common occurrence post-genomeuplication in teleosts (Postlethwait et al., 2004; Semon and Wolfe,007). Thus, the teleost B7R and B7H molecules potentially reflect

nternal constraints due to the (regulatory) structure of the fishmmune system. While mammals and birds have two B7 recep-ors with different functional properties, fish appear to have onlyne B7R per species possibly mirroring the primordial pathwayor delivering signal 2 that was established at the origin of cell

editated immunity. B7-H2 and its ligand ICOS were not foundn our teleost searches, but a B7-H2-related sequence is found inharks and Xenopus (scaffold 201; 753441) as well as a putativeCOS (Bernard et al., 2007) in Xenopus and in other tetrapods. SinceCOS delivers positive stimulatory signals that are important forifferentiation of T helper 1 and 2 cells, its absence in fish couldeflect a fundamental difference in the regulation of the Th1 andh2 differentiation. Finally, teleosts possess typical B7-H1, B7-H3nd B7-H4 genes that in mammals are involved in T-cell tolerancend inhibition. Such receptors are logical members of the primor-ial system for controlling adaptive immunity: the regulation of Tell activation and tolerance. Of note, the high degree of sequence

onservation of B7-H1 and B7-H3 suggests that they were tightlyonstrained by critical interactions of which potential mutationsr gene loss could be detrimental, thus implying direction selec-ion for these genes. Interestingly, while mammals possess twoD-1 ligands (B7-H1 and B7-DC), fish only encode one of which

A

i

. 2R reflects the genome duplication events that are common to all gnathostomes,te that partial sequences were found for B7-H1 and DC in the chicken and Xenopus

he ligand may be B7R. The recruitment of PD-1 as a B7-H1 lig-nd coupled with the differentiation of two receptors (B7-H1 andC) appears as a consequence of the step-wise expansion observed

n the adaptive immune systems in tetrapods. The differentia-ion of new dedicated lymphoid tissues and microenvironmentslymph nodes and true germinal centers) during tetrapod evolu-ion likely led to the creation of compartmentalization favorableo the step-wise expansion of the regulatory network of molecu-ar interactions, which is reflected in the history of CD28 and B7amilies.

cknowledgements

The authors would like to thank Pierre Pontarotti for criti-al comments in regard to the manuscript and for help withonserved synteny discovery. We also thank Olivier Chabrol forhe bioinformatic development of CASSIOPE and Yong-An Zhangnd Oriol Sunyer for sharing the trout B7R sequence prior tots release in GenBank. This work has been supported by U.S.eological Survey base funding and the Institut National de laecherche Agronomique. IMGT is supported by the CNRS andy the Ministère de l’Education Nationale, de l’Enseignementupérieur et de la Recherche (Université Montpellier II Plan Pluri-ormation, Genopole Montpellier-Languedoc-Roussillon, Agenceationale de la Recherche [ANR-06-BIOSYS-0005-01], Europeannion 6th PCRD ImmunoGrid project [IST-2004-0280069], Régionanguedoc-Roussillon). The use of trade, firm or corporation namesn this publication is for the information and convenience of theeader. Such use does not constitute an official endorsement orpproval by the U.S. Department of Interior or the U.S. Geologicalurvey of any product or service to the exclusion of others that maye suitable.

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at doi:10.1016/j.molimm.2008.10.007.

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