Evolution of the V, D, and J gene segments used inthe primate γδ T-cell receptor reveals a dichotomyof conservation and diversityAllison R. Kazena and Erin J. Adamsa,b,1

aDepartment of Biochemistry and Molecular Biology and bCommittee on Immunology, University of Chicago, Chicago, IL 60637

Edited by Peter Parham, Stanford University School of Medicine, Stanford, CA, and accepted by the Editorial Board June 14, 2011 (received for review March30, 2011)

γδ T cells are an immunological enigma in that both their functionin the immune response and the molecular mechanisms behindtheir activation remain unclear. These cells predominate in theepithelia and can be rapidly activated to provide an array ofresponses. However, no homologous γδ T-cell populations havebeen identified between humans and mice, and our understandingof what these cells recognize as ligands is limited. Here we take analternative approach to understanding human γδ T-cell ligand rec-ognition by studying the evolutionary forces that have shaped theV, D, and J gene segments that are used during somatic rearrange-ment to generate the γδ T-cell receptor. We find that distinctlydifferent forces have shaped the γ and δ loci. The Vδ and Jδ genesare highly conserved, some even through to mouse. In contrast,the γ-locus is split: the Vγ9, Vγ10, and Vγ11 genes represent theconserved region of the Vγ gene locus whereas the remaining Vγgenes have been evolving rapidly, such that orthology throughoutthe primate lineage is unclear. We have also analyzed the codingversus silent substitutions between species within the V andJ gene segments and find a preference for coding substitutionsin the complementarity determining region loops of many of theV gene segments. Our results provide a different perspective oninvestigating human γδ T-cell recognition, demonstrating that di-versification at particular γδ gene loci has been favored duringprimate evolution, suggesting adaptation of particular V domainsto a changing ligand environment.

nonsynonymous | synonymous | selection

Distinct from αβ T cells, γδ T cells represent an alternativelineage of T cells characterized by the expression of a het-

erodimeric T-cell receptor (TCR) composed of a γ- and a δ-chain. Despite substantial effort, the specific role of γδ T cells inhost responses against tumors, viruses, and pathogenic bacteriaremains unclear. γδ T cells represent a minority of circulatinglymphocytes in the blood, but are the majority of tissue-residentT cells in epithelial tissues such as the gut, lungs, and re-productive tract (1). Defined roles for γδ T cells are few; forexample, they have been shown to play an important role inwound healing in the mouse (2), and a particular population ofhuman γδ T cells expressing a Vγ9Vδ2 receptor are known torespond rapidly and potently to small pyrophosphate-containingmolecules, a response thought to function in pathogenic bacte-rial detection and elimination of tumor cells (3). However, theimmunological function of most γδ T cells remains unclear.The enigma of γδ T cells also extends to the molecular details

of how they respond to antigens through their TCRs. Detailedanalysis of ligand recognition by γδ T cells has primarily focusedon the mouse nonclassical class I molecule T22. γδ TCR rec-ognition of T22 is unlike that of conventional αβ TCR recog-nition of classical MHC molecules presenting peptidefragments, which use an assemblage of their complementaritydetermining region (CDR) loops to dock on the peptide/MHCcomposite surface. Instead, T22-reactive γδ T cells recognizeT22 through a specific amino acid motif in their CDR3δ loop

(W. . . EGYEL) (4, 5); this loop is necessary and sufficient forrecognition of T22 (6). However, the predominant use ofCDR3δ in ligand recognition does not seem to be ubiquitousacross all γδ T-cell populations, nor is it conserved betweenmouse and human. Other candidate ligands have been proposedfor γδ T cells; most of these are self-molecules such as classicaland nonclassical MHC proteins [i.e., CD1c (7), I-Ek (8), andMICA (9)], the aforementioned pyrophosphate-containingsmall molecules (3), and the F1 subunit of the mitochondrialATPase (10). However, a foreign protein, the glycoprotein Ifrom HSV-1, has also been characterized as a ligand for murineγδ T cells (11), suggesting that ligand targets are not exclusivelyself-molecules. Recognition of glycoprotein I was direct, with-out requirement for processing and presentation by MHCmolecules (12). Thus, γδ T-cell ligands represent a structurallyand chemically diverse array of molecules, suggesting that an-tigen recognition by these T cells may not have a conservedarchitecture like that seen with αβ TCR recognition of peptideMHC (13).In mice and humans, γδ T cells have a restricted repertoire of

V and J gene segments that are used for TCR rearrangement. Inhumans, the δ-locus is embedded within the α-locus on chro-mosome 14; only three true Vδ genes exist—Vδ1, Vδ2, and Vδ3—although particular Vα genes are infrequently used in δ-chainrearrangement and thus have a Vδ distinction: Vδ4 (Vα14), Vδ5(Vα29), Vδ6 (Vα23), Vδ7 (Vα36), and Vδ8 (Vα38) (14). Thehuman Vγ repertoire, located in the γ locus on chromosome 7, isalso small, with 12 Vγ genes, only seven of which are known to befunctional (Vγ1, Vγ5P, Vγ6, Vγ7, and Vγ10 are pseudogenes).The low diversity is also reflected in the number of J gene seg-ments for both δ and γ (four and five, respectively). This con-trasts with significant diversity at the α and β loci [Vα, 47; Vβ, 54;Jα, 58; and Jβ, 14 gene segments (15)] and has led to the sug-gestion that the apparent low diversity of γδ TCRs reflects theirrecognition of conserved self-proteins of low variability (16). Theadditional restriction of certain V segments in different tissuesfurther reduces the V domain diversity within γδ T-cell pop-ulations. In the mouse, the use of particular V domains maydictate the location and function of these cells (17).Contrasting with the low number of V and J gene segments

used in γδ TCR rearrangement is the ability of these receptors toincorporate multiple D segments during rearrangement (18).These segments can be incorporated in the forward or reverseframe, and in humans, most of the reading frames do not contain

Author contributions: A.R.K. and E.J.A. designed research, performed research, analyzeddata, and wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: ejadams@uchicago.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1105105108/-/DCSupplemental.

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stop codons. This ability to use multiple D segments makes theCDR3δ the most amino acid-diverse loop across all rearrangedreceptors, including αβ TCRs and preaffinity-matured antibodymolecules (19). The contrast between low variability of the Vdomains (which encode the CDR1 and CDR2 loops) with that ofthe extensive potential diversity of the CDR3δ loop posesa quandary for predicting the nature of the ligands recognized bythese TCRs. If recognition is centered on the CDR3δ, which wehave shown previously to be true for T22 recognition (4–6), thenare some γδ T cells recognizing highly variable antigens poten-tially derived from exogenous sources? Or are most γδ T-cellpopulations recognizing evolutionarily conserved ligands thatexhibit low polymorphism? Understanding the nature of γδ T-cell ligands has been a central focus in γδ T-cell biology; how-ever, despite substantial effort, few have been well characterized.We have taken an evolutionary approach to understanding li-gand recognition by γδ T cells by focusing on the evolutionarypressures that have shaped the repertoire of V, D, and J genesegments in humans and nonhuman primates. Previous work hasrevealed important similarities and differences between humansand chimpanzees at specific genes (20), but a comprehensiveanalysis of these loci across divergent nonhuman primatespecies has not been performed to our knowledge. Our analysisincorporates genomic location and arrangement, nucleotideand amino acid polymorphisms, and measurement of non-synonymous versus synonymous substitutions per site of theseloci across five species representing time points across approxi-mately 45 million years of primate evolution. We find that di-versification varies substantially across the γ- and δ-loci and thatsome variable gene segments exhibit signatures of strong selec-tion for coding changes at the regions encoding the CDR1 andCDR2 loops. Together, this analysis provides a foundation uponwhich to build our understanding of the nature of γδ TCR rec-ognition of ligand.

ResultsGenomic Organization of V, D, and J Gene Segments. Gene se-quences corresponding to human Vδ, Jδ, Dδ, Vγ, and Jγ homo-logues in four nonhuman primate species (Pan troglodytes, Pongopygmaeus,Macaca mulatta, and Callithrix jacchus) were identifiedbased on their homology to human, mapped to their genomiclocation, and are shown in comparison with human in Fig. 1.Identification of the Vδ5 gene from P. pygmaeus, which waslacking in the genome sequence, was done through amplifica-tion and sequencing by using gene-specific primers designedfrom human Vδ5; therefore, the exact position of this gene isundefined.In all species examined, the Vδ locus was embedded within the

Vα locus, as previously described in humans and mice. Strongconservation of this region across the five species was reflected inthe high similarity of gene content and organization. Dot plotanalysis, whereby long stretches of genomic sequence can becompared and their similarities and differences represented vi-sually on a dot-based matrix, reflects this high conservation (Fig.2A) as a mostly straight line with few gaps. Indeed, this conser-vation extends to the whole Vα locus (Fig. 2B). Three coreregions of high homology within the Vδ region were identified:one containing the Vδ4 genes; a central block containing theVδ1, Vδ5, Vδ6, Vδ7, and Vδ8 gene segments; and a third blockthat contains the Vδ2 and Vδ3 genes (Fig. 1A); these two genesalso flank the Dδ and Jδ segments (Fig. 1B). A second Vδ4(Vα14) gene, called Vδ4n2, was identified in all the nonhumanprimates species examined and thus appears to have been de-leted in the lineage leading to humans. Vδ4n2 appears to bea pseudogene in these four species based on the presence ofseveral stop codons throughout the predicted coding sequences.Vδ4 was not found in C. jacchus, perhaps because of a 45.7-kbdeletion between Vδ4n2 and Vδ6 relative to the human se-quence. Distance differences (represented in Fig. 1 as gray tri-angles with the indicated length difference in kilobases) between

A B

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Fig. 1. Genomic organization of the V, D, and J gene segments comprising the primate γδ TCR: (A) Vδ, (B) Jδ and Dδ, (C) Vγ and (D) Jγ from humans, Pantroglodytes (Patr), Pongo pygmaeus (Popy), Macaca mulatta (Mamu), and Callithrix jacchus (Caja). V and J genes are shown as arrows to indicate codingdirection. D segments are shown as blocks. Functional genes are shown in black, pseudogenes in gray. Scale for each locus is shown in the upper right of eachsection. Insertions and deletions are indicated as gray triangles with insertions shown as pluses and deletions as minuses, with distances in kilobases.

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the three blocks of genes was common, representing both in-sertion and deletion events in these regions. In addition, the Vδ3gene sequence in C. jacchus has several stop codons, and thusappears to be nonfunctional in this species. However, the orga-nization of the Vδ gene segments relative to humans was overallhighly conserved.Organization of the Dδ and Jδ gene segments was also very

highly conserved across the species examined, both in location(Fig. 1B) and sequence identity. Examination of the D segment

recombinational signal sequence (RSS) flanking regions alsoshows high identity across species (Fig. S1), suggesting the ma-jority of these segments are functional for V-D-J rearrangement.The exception is the Dδ1 sequences from M. mulatta and C.jacchus, in which an insertion of six nucleotides in the heptamer/nonamer 23 spacer region may result in this segment being un-able to be incorporated during V-D-J rearrangement in thesespecies. However, M. mulatta has an additional Dδ3 segment(Dδ3n2), located approximately 1.7 kb upstream from Dδ3. Se-

A B

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Fig. 2. Dot-plot analysis reveals a dichotomy of genomic evolution between the δ- and γ-loci. Dotplot analysis was applied to the genomic regions encodingthe Vδ (A), Vα (B), Vγ (C), and Vβ (D) genes between humans, P. troglodytes (Patr), P. pygmaeus (Popy), M. mulatta (Mamu), and C. jacchus (Caja) to determinethe relative stability of these regions across primate evolution. The relative positions of the V genes are shown with arrows, with black indicating functionalgenes and gray pseudogenes. Black dots on the plot correspond to regions with greater than 90% identity. Gray dots indicate greater than 80% identitybetween the two regions being compared. The position of the Vδ5 gene from P. pygmaeus is uncertain, indicated by a box. The size of the regions comparedare shown in base pairs after the species name. In D, pink boxes indicate the nature of the genes located in regions of repetitive sequences.

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quence identity with Dδ3 is high in the D segment region and theRSSs, suggesting this is a fully functional D segment that can beused for rearrangement. Overall, the gene segments used inrearrangement of the δ-chain appear to be remarkably conservedin these primate species.Contrasting with the evolutionary stability of the δ-locus, in-

vestigation of the γ-locus reveals a region that is one part stable,one part highly dynamic. Shown in Fig. 1C is the organization ofthe Vγ genes in the five species. The Vγ9, Vγ10, and Vγ11 genes[previously designated groups II, III, and IV, respectively (21),and here designated group 2 for simplicity] are present in apesand the Old World monkey representative,M. mulatta.However,Vγ11 was not found in C. jacchus, and a stop codon early in thecoding sequence of Vγ11 in M. mulatta suggests this gene isnonfunctional in this species. As noted previously for Pan trog-lodytes (22), the Vγ10 gene appears to be functional in all thesespecies except for human. The genomic distance separating Vγ9,

Vγ10, and Vγ11 varies little across the species, and phylogeneticanalysis (discussed here later) establishes the orthology of thesethree genes across these primate species. Vγ9, Vγ10, and Vγ11therefore appear to be evolutionarily conserved and stable inrelation to the other Vγ genes.Distinct differences between species at the remaining Vγ

genes [previously designated group I (21) and here designatedgroup 1] become evident even in P. troglodytes, in which the Vγ5Pgene is missing and Vγ10 is functional (22). Further distinction isclear in P. pygmaeus, in which orthology between gene segmentsbecomes unclear; this is also reflected to a greater degree in theorganization of M. mulatta and C. jacchus. Although the regionappears to have been evolving dynamically in each species, a grossorder to genes is still maintained. Vγ1 maintains a flanking po-sition in each of the species. Although Vγ1 is a pseudogene inhumans and P. troglodytes as a result of a deletion in the RSSsequence, this mutation is not present in the remaining primate

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Fig. 3. Phylogenetic relationships of the primate V, D, and J gene segments. Shown are neighbor-joining trees of Vδ (A), Vγ (B), Jδ (C), and Jγ (D). Bootstrapconfidence values are shown for most branches. Well supported groupings are indicated by colored shading. Identical sequences are enclosed in boxes. A scalefor distance is shown in the lower right of each tree.

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species, and therefore the Vγ1 gene in these species appearsfunctional. Genes with gross homology to Vγ2, Vγ3, Vγ5, andVγ6 generally occupy similar positions across the species, but it isclear that gene duplication and/or deletion—and potentially evenrecombination and gene conversion—frequently occurred in thisregion, as the orthology between genes is unclear.The evolutionary dichotomy of this region is strongly sup-

ported by dot-plot analysis of the Vγ-locus (Fig. 2C). Compar-isons of the region encoding group 2—Vγ9, Vγ10, and Vγ11—are represented by a single diagonal straight line, indicatinga linear orthology between species, whereas the region encodingthe group 1 Vγ genes has a hatched appearance on the plot,consistent with repeated homologous segments produced byduplication events. For comparison, a representative dot-plotanalysis of the β-locus of human and P. troglodytes is shown inFig. 2D. There are small regions that have a similar hatchedpattern (the duplicated block of Vβ6, Vβ7, and Vβ5 genes anda family of trypsinogen genes, TRY and PRSS), suggesting thatparts of the Vβ gene locus are evolving rapidly through a similarprocess to that of the Vγ locus. Overall, our genomic mappinganalysis has revealed distinct differences between the genomicand evolutionary forces that have shaped the gene repertoire atthe δ- and γ-loci.

Evolutionary Relationships as Revealed by Gene Phylogenies. Tounderstand the evolutionary relationships between the V and Jsegments characterized from the δ- and γ-loci, we generatedneighbor-joining phylogenetic trees by using the Jukes–Cantormethod and performed bootstrap analysis (500 replications) totest for confidence of groupings. Phylogenies for Vδ, Vγ, Jδ, andJγ are shown in Fig. 3 A–D, respectively. Included in the phy-logenies are the homologous mouse genes (Mumu) to identifywhether gene conservation has extended to the rodent lineage.Phylogenetic analysis of the Vδ genes (Fig. 3A) reflects the

conservation seen in genomic organization. There are cleargroupings of the species at each of the Vδ genes (Fig. 3A, shadedin blue), and the bootstrap confidence values for these groupingsare generally very high (98–100%). In addition, as noted pre-viously (23), there are also mouse Vδ genes that clearly groupwithin primate Vδ clades. For example, the four mouse DV6genes [International Immunogenetics (IMGT) project nomen-clature] group with 99% bootstrap confidence with the primateVδ1, mouse DV4 groups with 100% confidence with primate Vδ2,and mouse DV5 groups with 100% confidence with primate Vδ3.Clear mouse homologues also exist for primate Vδ4 (mouseDV11), Vδ6 (mouse DV8), and Vδ8 (mouse DV2-1 and -2).Mouse DV1, DV7, and DV10 group with primate Vδ7, but theconfidence for this grouping is weaker (66%). The Jδ gene seg-ments group similarly, forming four distinct clades with highbootstrap confidence values (83–100%). Within the Jδ1 and Jδ3clades group the TRDJ1 and TRDJ2 genes from mouse, re-spectively. Remarkably, the general organization of the mouse Vδand Jδ gene fragments is similar to that of primates, suggestingthese genes may be true orthologues.Neighbor-joining trees of the primate Vγ genes show a dis-

tinctly different morphology than those of the Vδ and Jδ genefragments but again reflect the bipolar nature of the Vγ locus(Fig. 3B). The group 2 Vγ genes—Vγ9, Vγ10, and Vγ11, whichare the three anchor genes similar in location across all primatetaxa—form distinct clades with long branch lengths supported bystrong bootstrap confidence. The group 1 Vγ genes, however,form a bush-like structure, with short branch lengths and lowbootstrap confidence values. Within the group 1 Vγ, the Vγ1,Vγ6, Vγ7, and Vγ8 form well supported subgroupings within thisregion. The Vγ2 and Vγ4 genes group together, as do the Vγ3and Vγ5 genes, but no clear subgroupings within these clades areevident, suggesting these two represent sets of paralogous genes,generated through a recent block duplication of a segment

containing the Vγ2 and Vγ3 ancestors, or genes that have fre-quently exchanged sequences through recombination or geneconversion. The hashed pattern corresponding to this regionidentified by dot-plot analysis supports this prediction. Rapidduplication also appears to have shaped the Jγ gene segments aswell; sequence analysis and phylogeny construction reveal thatJγ1 and Jγ2, despite being separate genes, are identical at thenucleotide level, and JPγ1 and JPγ2 are highly homologous. Theorganization of these genes, combined with their phylogenicsignature, is consistent with a block duplication of a Jγ1/JPγ1ancestor. Overall, our analysis of the γ-locus suggests it has beenevolving dynamically, with gene duplication (and presumablydeletion) generating new repertoires of genes even betweenclosely related species, such as the primates studied here. Sup-porting this hypothesis is the lack of clear homologues in mousefor any of the group 1 Vγ genes, or for the Jγ genes. All themouse Vγ cluster outside of this group, with only mouse TRGV1,TRGV2, TRGV3, and TRGV4 clearing grouping with one pri-mate gene, Vγ11, homology noted previously based on aminoacid sequences (24).

Pairwise Comparison of Amino Acid Sequences of Human Vγ and VδReveal a High Degree of Diversity. Because γδ TCRs have few Vgenes from which to rearrange their receptors, we sought toassess the functional diversity between the protein sequences ofthe human V domains and to compare them with amino acidsequences of human V domains from αβ TCRs and antibodymolecules. Shown in Fig. 4 are graphs of these data, binned forevery five differences for simplicity. Shown to the right of thechart legend are the numbers of sequences included in thisanalysis; all allelic forms of these genes, obtained from theIMGT database, were included in this analysis. We find that thedistribution of pairwise differences is much more extensive forthe Vδ and Vγ domains than for Vα, Vβ, VH, or Vκ, suggestingthat, although the number of gene segments may be few for γδ Vdomains, they are functionally very divergent from each other.The bimodal distribution in particular for the Vγ domains islikely a result of comparisons of within group (i.e., group 1 vs.group 1) and between groups (i.e., group 1 vs. group 2). The Vδ

Fig. 4. Vδ and Vγ domains exhibit high levels of amino acid diversity. Shownare histograms of pairwise amino acid differences: Vδ (blue) and Vγ (green;Top), Vα (purple) and Vβ (yellow; Middle), and IgK (pink) and IgH (orange;Bottom). The numbers of sequences used in this analysis are shown to theright of the legend labels. All sequences available (including allelic forms)were included in this analysis.

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domain pairwise differences demonstrate the divergence of everyVδ domain from each other. The maintenance of these genesthroughout the primate lineage suggests these distinctions likelyhave important functional consequences.

Coding Substitutions Outweigh Silent Substitutions in Key Regions ofthe V Genes. To understand the evolutionary forces that havegenerated diversity at the V and J genes of the γ- and δ-loci, wehave calculated the number of substitutions per site that result incoding (i.e., nonsynonymous; dn) versus silent (i.e.,synonymous;ds) mutations. This analysis, often represented as a ratio of dn to ds(i.e., dn/ds) has been very informative when applied to rapidlyevolving genes such as the classical MHC genes, demonstratingthat diversifying selection (i.e., dn/ds ratio > 1) has been acting onregions of the MHC genes encoding the peptide binding regions(25). Genomic regions under no selective pressure are consid-ered to be neutrally evolving (i.e., dn/ds ratio of 1), whereas thoseunder purifying selection typically have a dn/ds ratio lower than 1.Although caveats exist for this analysis, it can be generally in-formative for how different regions of a gene have been evolvingin relation to each other. For our analysis, we have comparedacross species within each Vδ, Jδ, Vγ, and Jγ gene classification.For the V gene analysis, we have further divided the V genesequences into four categories: the regions encoding (i) theCDR1 loop, (ii) the CDR2 loop, (iii) the framework region(excluding the CDR regions), and (iv) the entire V gene se-quence. Where clear orthologues exist, such as at the Vδ and Jδgene loci, comparisons were made within these genes. For the Vγloci in which orthology was ambiguous, such as the Vγ2/Vγ4 andVγ3/Vγ5 gene clusters, these genes were analyzed as a group andare thus represented as Vγ2/4 and Vγ3/5. Because Caja Vγ1,Vγ3, and Vγ7 formed their own clade in our phylogenetic

analysis, they have been grouped together for this analysis. Onlyfunctional genes (i.e., those capable of encoding a proteinproduct) were included.Results of our dn and ds calculations are shown in Fig. 5. The

highest dn and ds values in the variable genes were found in theregions encoding the CDR loops, the highest being a ds of 0.439for the Vδ6 CDR1 region (Fig. 5A). Averages for the frameworkregions ranged from 0.019 (Vδ8 dn) to 0.103 (Vδ4 ds). The dn/dsratios calculated for the framework and V domain regions werealmost always significantly lower than 1, consistent with the ac-tion of purifying selection (Fig. 5B). Although the short sequencelength and close evolutionary distance complicated dn/ds analysisof the CDR regions, several trends were noteworthy. In manycases, ds was 0, which made calculation of an informative dn/dsratio impossible; however, in a disproportionate number of thesecases, dn was greater than 0 despite the short sequence. In thesecases, the ratio is shown graphically with a maximum of 4, withan additional plus sign to highlight that only coding changes existin these particular regions. In some cases, such as the CDR2region of Vδ1 and CDR1 region of Vδ3, the average dn sub-stitution per site are among some of the highest averages noted(Fig. 5A). Overall, there appears to be a bias for dn/ds ratiosgreater than 1 in the regions encoding the CDR1 and CDR2loops for several of the V domains, suggesting that evolution hasfavored coding changes in these regions throughout the evolu-tion of the primate lineage. In contrast, dn/ds analysis at theJ gene segments revealed all ratios significantly lower than 1. Theimportant role that the J segment performs in encoding a regionof the rearranged TCR that is involved in chain hetero-dimerization may explain why coding changes have been selectedagainst in these gene segments. Because of the small codinglength of the D segments (Dδ1, 8 nt; Dδ2, 9 nt; and Dδ3, 14–15 nt)

Nonsynonymous (dn) Synonymous (ds)

V domainFrameworkCDR 1,2

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Fig. 5. Coding (dn) versus silent (ds) mutations are unequally distributed across the Vδ and Vγ genes. (A) Average dn (green) and ds (yellow) substitutions persite across the primate species are shown for the Vδ, Vγ, Jγ, and Jδ genes. Average substitutions per site were calculated for the regions encoding the CDR1loop (1), CDR2 loop (2), framework region (F), and the entire coding region (V) for the variable genes and for the entire coding region of the J gene segments.Error bars represent SE of these averages. (B) Ratio of dn to ds substitutions for each region calculated in A. A ratio of 1, indicated as a dashed purple line ineach plot, is consistent with neutral selection. Significant values greater than this are consistent with diversifying selection. Those lower than this are con-sistent with purifying selection. CDR1 and -2 loop regions are colored in red, framework in orange, and V domain in purple. For those values at which ds is 0,ratios are shown extending to the maximum value of 4 with a plus sign. (Significant at *P ≤ 0.05, **P ≤ 0.005.)

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and their ability to be read in multiple frames, dn/ds analysis wasnot performed on these sequences. Instead, they were aligned(with the flanking RSS regions as an aid; Fig. S1), and the Dsegment regions were translated in all three frames, both forwardand reverse. These are shown in Fig. 6; the human D sequencesare shown as consensus. The three D segments exhibit differentlevels of variation; all functional Dδ1 sequences are identical,even at the nucleotide level (Fig. S1), whereas Dδ2 and Dδ3differ between species to varying degrees. Identity at Dδ2 ispreserved in the great apes, with differences apparent in M.mulatta and C. jacchus. However, differences at Dδ3 are evidenteven in P. pygmaeus (an insertion and 2-nt differences) and ex-tend to the more distantly related species. This suggests that theD segments, despite the small genomic distance between them,are evolving at different rates. The average nucleotide pairwisedifferences for the functional Dδ1, Dδ2, and Dδ3 are 0.000,0.167, and 0.148 substitutions per site, respectively.

DiscussionOur analysis of the V, D, and J gene segments used in primate γδTCR rearrangement through use of primate genome sequencesprovides a comprehensive perspective on the evolutionary forcesthat have shaped these regions. We have found that distinctlydifferent evolutionary mechanisms have acted at the primate δ-and γ-loci, with the δ-gene segments exhibiting a grossly con-served nature across species (some extending to mouse) whereasmany of the γ-gene segments have undergone substantial di-versification within the primate lineage. Vγ9, Vγ10, and Vγ11represent the exceptions to this trend, and appear to be con-served within the primate species examined here. Vγ11, althoughabsent in the C. jacchus sequence, even appears to have ortho-logues in mouse (Fig. 3) (23). Dot-plot comparisons have pro-vided broad insight into the evolution of these regions,supporting the conclusions derived from the gene maps. Re-petitive elements (visualized as hashmarks) are apparent in thegroup 1 Vγ region, suggestive of recent, repeated duplications asthe source of the gene content of this region. Patterns such asthese are also apparent in regions of the Vβ locus (particularlyfor the Vβ6, Vβ7, and Vβ5 gene families) but do not appear tohave played a substantial role in the recent evolution of the Vαor Vδ loci. The rapid evolution of the group 1 Vγ genes, whichhave very high amino acid identity with each other and differ

mainly in the sequences of their CDR loops, suggests that theymay be evolving to keep up with a selective force, perhaps par-ticular ligands that may also be rapidly evolving (assuming theseγ-chains are used in ligand recognition). This analysis is alsoinformative for understanding the clear differences between theprimate species and the various model systems that have beenused to study the functions of γδ T cells. Our analysis supportsthe suggestion that human γδ T cells, at least those that use thegroup 1 Vγ domains, are not orthologous to those in mouse (23),and may explain in part the lack of clear functional γδ T-cellhomologues between the two species. Furthermore, primatespecies used as models for human research, such as M. mulatta,have different repertoires of the group 1 Vγ genes, and thereforecaution should be used in making direct comparisons of cellsexpressing these receptors within these species.Our dn and ds analysis reveals that the average substitutions

per site (both coding and silent) at many of the V gene CDRregions are higher than the average calculated in the frameworkregions. Furthermore, in several of the V gene CDR regions,substitutions causing amino acid mutations appear to be favoredover those that are silent. Indeed, many of these regions, asa result of their short sequence and relatively short evolutionaryseparation between species, had a ds of 0, making assigningsignificance to these dn/ds ratios impossible. However, this trendtoward coding level diversification of these CDR loop sequencessuggests that these regions may be coevolving with a similarlyrapidly evolving ligand. This insight may prove useful in the as-sessment of candidate γδ T-cell ligands to ascertain their con-servation between primate species. Our observation that thesilent substitution rate was elevated in several CDR loop regionsraises interesting questions regarding the mechanisms behind theevolution of these DNA stretches. Factors such as localized in-creased mutation rates, introduction of genetic variation throughgene conversion and/or codon use bias could be at play in thisregion; however, testing for their effects may be difficult or im-possible, so this remains speculative.Application of our results to known human γδ TCR chain

pairings such as the Vγ9Vδ2 γδ T-cell population found in theblood reveals that three of the four CDR loops have a dn/ds ratiogreater than 1 (Fig. 5B). This population of γδ T cells is known tobe stimulated in the presence of small phosphoantigens; how-ever, it is speculated that there is an antigen-presenting moleculerequired on the target cell for this stimulation to occur (26), andthat all CDR loops appear to be required for this recognition(27). Although certain residues within the CDR loops found tobe critical for phosphoantigen-mediated stimulation were con-served between humans and macaques, our analysis suggests thatpositive selection has favored alterations in these CDR loops,suggesting that selection is subtly adapting the Vγ9Vδ2 pop-ulation to a changing stimulus. This adaptation has not com-pletely abrogated cross-species reactivity, as chimeric TCRsbetween macaque and human maintain phosphoantigen re-activity (28), but not to the degree of WT, suggesting that dif-ferences between macaque and human coding regions canmodulate binding. Positive selection to keep up with evolvingligands may provide a reason for why this population has notbeen found outside of the primate lineage.The Vδ1 chain is commonly found in γδ T cells resident in the

intestine, paired with various Vγ chains. Some of these intestinalVδ1 positive cells recognize the MICA molecule, a nonclassicalMHC class I molecule that exhibits moderate polymorphism inthe human population, both through the natural killer cell-activating receptor NKG2D and through the γδ TCR (9). In ouranalysis, the CDR2 of Vδ1 exhibits a high nonsynonymous sub-stitution rate (and no synonymous substitutions) whereas theCDR1 loop appears to be evolving neutrally (Fig. 5). It is yetunclear which loops are involved in MICA recognition, and ithas not been explored whether nonhuman primates also have

Fig. 6. Diversity of the Dδ segments across primate species. Translations ofthe Dδ segments are shown relative to the human consensus. Readingframes are shown at the top in the forward or reverse orientation. Dashesindicate identity with the human consensus, and differences are shown assingle amino acid abbreviation. (*Stop codons; †Dδ segments that haveinsertions or deletions relative to human.) Segments deemed potentiallynonfunctional are shaded lavender.

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a MICA reactive population; however, a recent structure ofa human MICA-reactive Vδ1 γδ TCR suggests that the CDR3δmay not play a major role, and affinity measurements andcompetition assays suggest that the γδ TCR may be binding toa polymorphic region of MICA (29). Although Vδ1 chains arelikely also involved in recognition of other non–MICA-relatedantigens in other tissues, our observation of a rapidly evolvingCDR2 loop is consistent with recognition of a polymorphicsite on MICA. However, further mutational analysis will benecessary to test the significance of the CDR2δ loop on MICArecognition.The function and ligand recognition capacity of human γδ T

cells still remains much of a mystery, and unfortunately there areno clear model systems in the mouse to aid our understanding.Our investigation of the evolutionary forces that shape primateγδ TCRs is focused toward gaining a better understanding ofhow they interact with their environment through this receptor.Our work suggests that these “innate-like” lymphocytes are notstationary in evolutionary time. Indeed, the Vγ locus has beenrapidly evolving even within the primate lineage, such that sev-eral of the human Vγ genes do not have clear orthologues evenin P. pygmaeus. Within many of the V genes, the regionsencoding the CDR loops appear to be diversifying, implicatingtheir involvement in recognition of ligands that may also beunder pressure to change. Conversely, loops that appear con-served in other V domains may implicate these domains in rec-ognition of invariant ligands. These cells reside in the epithelialtissues and are implicated as a first line of defense (16) againstinvading pathogens and cancers. Selection on these cells istherefore likely strong; therefore, the patterns of diversification,both positive and negative, that we reveal in this analysis can beused to better understand the nature of the ligands that mediatetissue-specific and stimulus-specific reactivity.

Materials and MethodsGenomic Sequence Analysis. Genome sequences for P. troglodytes, P. pyg-maeus, M. mulatta, and C. jacchus were accessed through the University ofCalifornia, Santa Cruz, Genome Browser Web site (30). Human γδ TCR genesequences obtained from IMGT (15) were used to retrieve sequence matchesfor P. troglodytes (Chimpanzee Sequencing and Analysis Consortium),P. pygmaeus (Genome Sequencing Center at Washington University),M. mulatta (Baylor College of Medicine Genome Sequencing Center), andC. jacchus (Genome Sequencing Center at Washington University) by usingthe Genome Browser’s BLAST-Like Alignment Tool search algorithm.Sequences showing homology to the human Vδ (TRAV/DV), Vγ (TRGV), Jδ(TRDJ), Dδ (TRDD), and Jγ (TRGJ) genes were mapped according to theirchromosomal localization within each species by using a prerelease versionof the SnapGene molecular biology software (GSL Biotech), establishingtheir location, orientation, and genomic organization relative to each other.Duplicate/overlapping hits and sequences on irrelevant chromosomes wereeliminated. All nonhuman primate genes retrieved were named accordingto homology with the human gene sequence with which they were identi-

fied. Where available, bacterial artificial chromosome sequences and codingsequences derived from deposited cDNA and expressed sequence tag data-bases were used to validate the genome sequences used in this analysis.Species names were abbreviated according to that proposed for MHCnomenclature (31) and are used this way throughout the figures: Homosapiens, “Hosa,” P. troglodytes, “Patr,” P. pygmaeus, “Popy,” M. mulatta,“Mamu,” C. jacchus, “Caja,” and Mus musculus, “Mumu.” All gene namesused are according to IMGT nomenclature (15). Dot plots were generatedusing the Genome Shovel server (http://www-btls.jst.go.jp/cgi-bin/Tools/dotBLAST/index.cgi).

Phylogenetic Analysis. Sequence alignments were created using ClustalWalignment software, version 2 (32). Neighbor-joining, nucleotide phyloge-netic trees were constructed using Molecular Evolutionary Genetics Analysis(MEGA) software, version 5.02 (33), with direct p-distance measurements ofdistance. The confidence of the phylogenetic arrangement was confirmedby using bootstrapping analysis with 500 replicates; these values are shownon the most significant branches. Gene sequences from M. musculus wereobtained from IMGT for inclusion in the phylogenetic analysis.

PCR Amplification of Genes of Interest. Locus-specific PCR primers (IntegratedDNA Technologies) were designed from alignments of human γδ TCR genefragments. Genomic DNA samples from P. troglodytes and P. pygmaeuswere used as template for locus specific amplification. PCR products wereanalyzed via agarose gel electrophoresis, gel-purified (Qiagen), and clonedby using the TOPO TA cloning kit (Invitrogen), following 3′-adenosine ad-dition using Taq polymerase. Clones containing the proper size insert weresubcultured and DNA-purified with a commercial miniprep kit (Qiagen).Sequencing of these clones was conducted by the University of ChicagoCancer Research Center DNA Sequencing Facility.

Pairwise Difference and dn/ds Analysis. Pairwise amino acid differences foreach gene between species were calculated by MEGA software (33).Sequences included those retrieved from the genomic database and thoseisolated through molecular cloning using locus specific primers. The numberof differences between each pair of sequences was normalized over thetotal number of sites compared.

MEGA software was additionally used to conduct dn/ds substitution persite analysis. Counts of substitutions per site were performed using the Nei–Gojobori method with Jukes–Cantor correction, generating dn and ds. Themean dn and ds values and the SE for each gene segment across species werecalculated by using Excel. To assess the significance of the dn/ds ratios cal-culated, a two-tailed paired t test was conducted on all of the pairwise dn

and ds values calculated within each gene, with the null hypothesis was thatdn/ds ratio was equal to 1. All dn/ds ratios with a P value less than 0.05 wereconsidered significant. In cases of a ds of 0, statistical analysis was not per-formed; ratios in which the average ds was 0 are represented at the maxi-mum (ratio, 4) with a plus sign.

ACKNOWLEDGMENTS. The authors thank Dr. Ben Glick for use of a pre-release version of his SnapGene molecular biology software (GSL Biotech).A.R.K. was supported by Biological Sciences Division Summer Fellowships. Thisstudy was supported by a Searle Scholars Award through the KinshipFoundation (to E.J.A.) and National Institutes of Health Grant R01-AI073922 (to E.J.A.).

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