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Immunobiology
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KT and MAIT invariant TCR� sequences can be produced efficiently by VJ geneecombination
ui Yee Greenawaya, Benedict Nga, David A. Priceb,c, Daniel C. Douekc, Miles P. Davenportd,∗,anessa Venturia,∗∗
Computational Biology Group, Centre for Vascular Research, University of New South Wales, Kensington, AustraliaInstitute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, Wales, UKHuman Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USAComplex Systems in Biology Group, Centre for Vascular Research, University of New South Wales, Kensington, Australia
r t i c l e i n f o
rticle history:eceived 27 January 2012eceived in revised form 28 March 2012ccepted 24 April 2012
eywords:nvariant T cell receptor
AIT cellKT cellublic T cell response
cell receptor cell repertoire
a b s t r a c t
Semi-invariant T cell receptors (TCRs) found on natural killer T (NKT) and mucosal-associated invariantT (MAIT) cells are characterized by the use of invariant variable (V) and joining (J) gene combinationsin the TCR �-chain, as well as ubiquitous canonical TCR� amino acid sequences that are dominant inmany individuals and similar across species. That they are so prevalent indicates that they occupy animportant niche within the immune system. However, these TCRs are produced by a largely random generecombination process, which seems a risky approach for the immune system to acquire these innate-likecells. We surveyed studies reporting NKT and MAIT TCR� sequences for six and four different species,respectively. Although the germline nature of the canonical human and mouse NKT and mouse MAIT TCR�sequences and an overlap of nucleotides between the mouse MAIT-related V� and J� genes have beennoted in previous studies, in this study we demonstrate that, for all reported species, the canonical TCR�amino acid sequences can be encoded by at least one germline-derived nucleotide sequence. Moreover,these nucleotide sequences can utilize an overlap between the V� and J� genes in their production,which enables them to be produced by a large variety of recombination mechanisms. We investigatedthe role of these TCR� features in the production of the canonical NKT and MAIT TCR� sequences. In
computer simulations of a random recombination process involving the invariant NKT and MAIT TCR�gene combinations for each species, the canonical NKT and MAIT TCR� sequences were the first or secondmost generated of all sequences with the CDR3� length restrictions associated with NKT and MAIT cells.These results suggest that the immune machinery enables the canonical NKT and MAIT TCR� sequencesto be produced with great efficiency through the process of convergent recombination, ensuring theirprevalence across individuals and species.
ntroduction
Semi-invariant T cell receptors (TCRs), like those found on nat-
ral killer T (NKT) cells and mucosal-associated invariant T (MAIT)ells, are present in multiple individuals and across multiple speciesKjer-Nielsen et al. 2006). Their prevalence and evolutionary
Abbreviations: TCR, T cell receptor; MAIT, mucosal-associated invariant T; NKT,atural killer T; CDR3, complementarity determining region 3; TRA, T cell receptorlpha; TRAV, T cell receptor alpha variable; TRAJ, T cell receptor alpha joining.∗ Corresponding author at: Centre for Vascular Research, University of New Southales, Kensington, NSW 2052, Australia. Tel.: +61 2 9385 2762; fax: +61 2 9385 1797.
∗∗ Corresponding author at: Centre for Vascular Research, University of New Southales, Kensington, NSW 2052, Australia. Tel.: +61 2 9385 8234; fax: +61 2 9385 1797.
conservation indicates that they must play a critical role in immunedefence, and one would assume that the most efficient and reliableway for them to be produced would be for them to be “hard-wired”into the immune system through genetic encoding. Indeed, theyare often referred to as “innate-like T cells” (Arrenberg et al. 2010;Gapin 2009; Le Bourhis et al. 2010; Mallevaey et al. 2011). However,TCRs are produced via a largely random process of gene rearrange-ment involving the variable (V), joining (J) and diversity (D; for�-chains only) genes. The removal and addition of nucleotides atthe gene ends generates additional diversity across the junctionalregion. Thus, TCRs are expected to exist in many and varied forms,and only a minority of TCRs are expected to be shared between any
two individuals (Quigley et al. 2010; Venturi et al. 2011). This begsan obvious question. If the NKT and MAIT cells bearing these semi-invariant receptors are so vital, why is their production left up tochance?
NKT cells are a specialized, highly effective subset of T cellshat recognize self and foreign lipid antigens presented by CD1dBendelac et al. 1995). They are able to rapidly release a wide rangef cytokines and chemokines, as well as regulate the actions of othermmune cells (reviewed in Matsuda et al. 2008). Their effects haveeen implicated in a broad range of immune responses, includingutoimmunity, tumour immunity, and allergy and inflammationreviewed in Godfrey and Kronenberg 2004, Van Kaer 2007 and
atsuda et al. 2008); thus, they are important immune cells toonsider in the design of vaccines and other immunotherapeuticreatments. MAIT cells have not been as widely studied as NKT cellsnd, consequently, are not so well understood. However, recenttudies suggest that these MR1-restricted T cells (Huang et al. 2005)ave antimicrobial (Gold et al. 2010; Gold and Lewinsohn 2011; Leourhis et al. 2010, 2011), immunoregulatory (Croxford et al. 2006;iyazaki et al. 2011; Shimamura et al. 2011a,b), and pathogenic
Chiba et al. 2012) properties.Type I NKT cells, often referred to as invariant NKT (iNKT)
ells, and MAIT cells typically display a semi-invariant TCR, con-isting of an �-chain comprised of an invariant gene combinationTRAV10/TRAJ18 for NKTs and TRAV1-2/TRAJ33 for MAITs inumans, and TRAV11/TRAJ18 for NKTs and TRAV1/TRAJ33 forAITs in mice (Dellabona et al. 1994; Lantz and Bendelac 1994;
orcelli et al. 1993; Tilloy et al. 1999)). The �-chains are not asestricted as the �-chains but exhibit limited V gene usage (TRBV25or NKTs and TRBV6, TRBV20 for MAITs in humans, and TRBV13-2,RBV1, TRBV29 for NKTs and TRBV13, TRBV19 for MAITs in miceDellabona et al. 1994; Kawachi et al. 2006; Lantz and Bendelac994; Martin et al. 2009; Matsuda et al. 2001; Porcelli et al. 1993;illoy et al. 1999; Treiner and Lantz 2006)). Furthermore, stud-es that have sequenced NKT and MAIT TCRs using the invariant-chain gene combinations in humans and mice have observedommon TCR� amino acid sequences across a vast majority of indi-iduals that are predominantly encoded by the germline genesDellabona et al. 1994; Kent et al. 1999; Koseki et al. 1990; Lantznd Bendelac 1994; Porcelli et al. 1993; Tilloy et al. 1999; Treinert al. 2003).
Many structural studies have focussed on the NKT cell recep-or, CD1d, and interactions between NKT cells and CD1d-restrictedntigens (reviewed in Godfrey et al. 2010). It has been shown thathe invariant TCR� dominates the interaction between NKT cellsnd CD1d (Borg et al. 2007, Pellicci et al. 2009). At the molecularevel, Scott-Browne et al. (2007) identified specific germline-ncoded residues within the CDR3� region that were requiredor CD1d-glycolipid recognition, indicating the importance ofhe germline-encoded NKT TCR� in recognizing a diversity ofntigens.
These structural studies enable us to better understand theactors that govern interactions between invariant NKT cells andD1d bound to antigen within any one individual. The questionemains however: how does the immune machinery ensure thatuch invariant TCR� sequences are present in the majority ofndividuals and across multiple species in the first place? Afterll, these TCRs cannot be selected for in an immune responsenless the basic building blocks already exist. The question of thebiquitous nature of NKT and MAIT TCRs has parallels in conven-ional �� T cells, where some “public” TCR �-chains are involvedn antigen-specific CD8+ T cell responses in multiple individuals.hese inter-individually shared TCRs exist at a higher precursor fre-uency (Quigley et al. 2010) and are easily produced by a processermed “convergent recombination” (Venturi et al. 2006, 2008a,b).he process of convergent recombination enables some TCRs to
e produced much more frequently than others, because mul-iple recombination events converge to produce the same TCRucleotide sequence, and multiple TCR nucleotide sequences con-erge to encode the same TCR amino acid sequence. Multiple
ology 218 (2013) 213– 224
TCR amino acid sequences can also converge to produce distinctTCR motifs (Venturi et al. 2008c). More recently, our studies havedemonstrated the role of convergent recombination in the con-struction of naïve CD8+ T cell repertoires in mice (Quigley et al.2010) and humans (Venturi et al. 2011). We showed that TCRsequences prevalent in the naïve repertoire are more efficientlyproduced by a random V(D)J recombination process and that thesesequences are observed across multiple individuals.
In the present study, we propose that the canonical NKT andMAIT TCR� sequences are efficiently made by the TCR gene recom-bination process. While the previously noted germline nature ofboth types of sequences is an important factor in their produc-tion, an overlap between the V and J genes that allows nucleotidesat the VJ junction to be contributed by either the V or J genesubstantially enhances the potential for these sequences to beproduced frequently in the thymus. That is, a germline-derivednucleotide sequence has the potential to be efficiently producedin the absence of an overlap between V� and J� genes by a sin-gle frequently re-occurring recombination mechanism involvingno nucleotide additions. However, an overlap between the V� andJ� genes enables a germline-derived nucleotide sequence to beproduced by a variety of recombination mechanisms involving nonucleotide additions, each of which has the potential to frequentlyre-occur during the VJ recombination process. These NKT and MAITTCR� canonical sequences therefore have the potential to exist inhigh numbers in the T cell pool of many individuals, ready to bedeployed in response to immune challenge, thus enabling their sta-tus as highly shared TCR sequences. We surveyed the literature forstudies reporting NKT and MAIT TCR� sequences and found thatthese mechanisms governing efficient NKT and MAIT TCR� pro-duction are not confined to a single species, but are apparent acrossmultiple species.
Materials and methods
TRA genes
Human and mouse TRAV and TRAJ genes were obtained fromthe international ImMunoGeneTics (IMGT) information system(http://imgt.cines.fr/). Analysis was performed with reference tothe *01 allele sequences, and IMGT nomenclature is used through-out for all TCR genes (Lefranc et al. 1999).
TRA genes were extracted from the rhesus macaque (Macacamulatta) (Gibbs et al. 2007), dog (Canis lupus familiaris) (Lindblad-Toh et al. 2005), pig (Sus scrofa) (Archibald et al. 2010) and cow(Bos taurus) (Elsik et al. 2009) genomes based on their homologywith the equivalent human genes. Using the BLAST (discontiguousmegablast) (Altschul et al. 1990) tools available on the relevantNational Centre for Biotechnology Information (NCBI) GenomeResources websites (http://www.ncbi.nlm.nih.gov/projects/genome/guide/rhesus macaque/, http://www.ncbi.nlm.nih.gov/genome/guide/dog/, http://www.ncbi.nlm.nih.gov/genome/guide/pig/, http://www.ncbi.nlm.nih.gov/projects/genome/guide/cow/),the full human TRAV (IMGT sequence regions L-PART1, V-INTRON,V-EXON and V-RS) and TRAJ genes (IMGT sequence regions J-RSand J-REGION) were queried against the reference genomes.Matches to both the TRAV10 and TRAJ18 genes were extractedfrom chromosome 7 of the rhesus macaque genome (Genbankaccessions NW 001121195.1 and NW 001121196.1), chromosome8 of the dog genome (Genbank accession NW 876327.1) and chro-mosome 7 of the pig genome (Genbank accessions GL042185.1 and
GL042186.1). Matches to TRAV1-2 and TRAJ33 were extracted fromavailable genomic scaffolds of chromosome 10 of the cow genome(Genbank accessions NW 001492801.2 and NW 001492803.1).The top BLAST match in all cases was used.
Similarly, TRA genes were extracted from the rat (Rattusorvegicus) genome (Gibbs et al. 2004) based on their homologyith the equivalent mouse genes using the BLAST (discontiguousegablast) tools available on the NCBI Rat Genome Resources web-
ite (http://www.ncbi.nlm.nih.gov/genome/guide/rat/). Matches tooth the mouse TRAV11 (IMGT sequence regions L-PART1, V-NTRON, V-EXON and V-RS) and TRAJ18 genes (IMGT sequenceegions J-RS and J-REGION) were found on chromosome 15 of theat genome (Genbank accession NW 047454.2). Only the top BLASTatches were used, though as reported (Kinebuchi and Matsuura
004), there appear to be multiple genes matching the mouseRAV11 gene in the rat genome.
The extracted exon sequences (LPART1 + V-EXON and J-REGION)rom the rhesus macaque, rat, dog, pig and cow genomes areeported in Supplementary Table 1 and compared with previouslyeported gene sequences (Matsuura et al. 2000; Kinebuchi andatsuura 2004; Yasuda et al. 2009; Reinink and Van Rhijn, 2009).
iterature survey
We surveyed the literature for studies reporting NKT TCRequences using the invariant gene combinations TRAV10/TRAJ18previously designated V�24/J�18 or V�24/J�Q) in humans (Briglt al. 2006; Davodeau et al. 1997; Dellabona et al. 1994; Demoulinst al. 2003; Exley et al. 1997; Han et al. 1999; Kent et al. 1999;orcelli et al. 1993; Sumida et al. 1995), TRAV11/TRAJ18 (previ-usly designated V�14/J�18 or J�281) in mice (Arrenberg et al.010; Behar et al. 1999; Dao et al. 2004; Imai et al. 1986;oseki et al. 1990, 1991; Lantz and Bendelac 1994; Makinot al. 1993; Nyambayar et al. 2007; Pyz et al. 2006; Shimamurat al. 1997, 2001; Yagi et al. 1999), TRAV10/TRAJ18(human)-ike in rhesus macaques (Gansuvd et al. 2008; Kashiwase et al.003), TRAV10/TRAJ18(human)-like in dogs (Yasuda et al. 2009),RAV10/TRAJ18(human)-like in cats (Looringh van Beeck et al.009), TRAV10/TRAJ18(human)-like in horses (Looringh van Beeckt al. 2009), TRAV10/TRAJ18(human)-like in pigs (Looringh vaneeck et al. 2009), and TRAV11/TRAJ18(mouse)-like in ratsMatsuura et al. 2000; Pyz et al. 2006). We also surveyed theiterature for studies reporting MAIT TCR sequences using thenvariant gene combinations TRAV1-2/TRAJ33 (previously des-gnated V�7.2/J�33, TCRAV7S2/TCRAJ33 and V�7.2/IGRJ�14) inumans (Gold et al. 2010; Han et al. 1999; Illes et al. 2004; Porcellit al. 1993; Tilloy et al. 1999), TRAV1/TRAJ33 (previously designated�19/J�33) in mice (Lin et al. 1998; Shimamura and Huang 2002;illoy et al. 1999; Treiner et al. 2003), TRAV1-2/TRAJ33(human)-ike in cows (Goldfinch et al. 2010; Ishiguro et al. 1990; Tilloyt al. 1999), and TRAV1-2/TRAJ33(human)-like in sheep (Goldfincht al. 2010). Sequences were included in this study if full genesage information was available, though other studies reportedCR� sequences without gene information (e.g. (Moss and Bell995)).
A CDR3 was considered to occupy the sequence region fromhe conserved cysteine in the V-region to the phenylalanineNKT) or tryptophan (MAIT) in the J-region, inclusive. NKT TCR�equences were required to span at least amino acid residues–5 (VSD in humans, rhesus macaques and dogs; VGD in micend pigs; VAD in rats) within the CDR3 region to qualify fornclusion. MAIT TCR� sequences were required to span at leastmino residues 3–4 within the CDR3 region to qualify for inclu-ion. Full NKT and MAIT CDR3� sequences were inferred assumingdentity within the gene-encoded regions of the CDR3 if thextracted sequences were truncated (see Supplementary Table 2).
oth amino acid and nucleotide sequence data (if available) werextracted. Inter-individual frequency was used instead of intra-ndividual frequency in this study because (i) the availability andontext of frequency data varied widely between studies using
ology 218 (2013) 213– 224 215
differing experimental designs; and (ii) selection/expansion alsocontribute to intra-individual frequency, whereas inter-individualfrequency provides a measure of the production of independentclones, albeit in different individuals. If it was not clear how manyindividuals contributed sequence data to a study, or in how manyindividuals a particular sequence was found, the number wasassumed to be one.
Simulations of an unbiased VJ recombination process
We simulated the production of TCR� sequences usingthe invariant gene combinations TRAV10/TRAJ18 and TRAV1-2/TRAJ33 in humans, TRAV10/TRAJ18(human)-like in rhesusmacaques, TRAV11/TRAJ18 and TRAV1/TRAJ33 in mice, andTRAV11/TRAJ18(mouse)-like in rats to estimate the relative pro-duction frequencies of the observed NKT and MAIT TCR� aminoacid sequences and their encoding nucleotide sequences. Algo-rithms similar to those used in previous studies (Venturi et al.2006, 2008a,b) were used to simulate a random gene recombina-tion process (Supplementary Fig. 1A). The simulations allowed for arandom number of deletions, between 0 and 12 inclusive, from the3′ end of the V gene and the 5′ end of the J gene. A random numberof nucleotide additions from 0 to 12 inclusive were then insertedinto the junction between the V and J genes, with the bases beingdetermined randomly. Parameters were chosen so as to be able togenerate a broad range of TCR sequences, including the majorityof the TCR sequences observed. Alignment of the observed MAITinvariant sequences with the TCR genes suggests that up to 2 p-additions can be involved in their production. We therefore alsoperformed simulations allowing for up to 4 p-additions to ver-ify that the overall conclusions of this study that are based uponthe simulation results are not dependent on the chosen simulationparameters. These simulations allowing for p-additions at the cod-ing ends of the V and J genes involved 4 p-additions being addedto the 3′ end of the V gene and the 5′ end of the J gene prior to thedeletion step (Supplementary Fig. 1B). Then a random number ofdeletions between 0 and 16 inclusive were simulated, followed bya random number of nucleotide additions from 0 to 12 inclusive.Simulations were performed using Perl v5.10.0.
Results
NKT TCR ̨ sequences in the literature
We surveyed the literature for studies reporting NKT TCR�sequences, particularly focusing on sequences using the invariantgene combinations (TRAV10/TRAJ18 in humans, TRAV11/TRAJ18 inmice). Nine studies reported human NKT sequences, 13 reportedmouse NKT sequences, two reported rat NKT sequences, tworeported rhesus macaque NKT sequences, and one study andone Genbank entry reported pig NKT sequences. NKT sequencesfor each of dog, cat and horse have also been reported inone study. A summary of the CDR3� amino acid sequencesobtained is shown in Supplementary Table 2. Thirteen uniqueNKT TCR� amino acid sequences using the TRAV10/TRAJ18 genecombination were observed across the studies reporting humansequences. Fifteen unique NKT TCR� amino acid sequences usingthe TRAV11/TRAJ18 genes were observed across the mouse studies.NKT TCR� sequences from all species were highly length restrictedto 15 amino acid residues between the conserved cysteine inthe V-region and the phenylalanine in the J-region, inclusive
(Supplementary Table 2).
Although it is possible for many different TCR amino acidsequences to be derived from the invariant �-chain genes associ-ated with NKT cells, particular amino acid sequences are commonly
Table 1Sharing of canonical NKT TCR� amino acid sequences and encoding nucleotide sequences in human, mouse, rat, rhesus macaque, dog and pig studies.
Species CDR3 regiona,b Number of individualswith sequencec/totald
HumanTRAV10/TRAJ18
C V V S D R G S T L G R L Y F 32/32tgt gtg gtg agc gac aga ggc tca acc ctg ggg agg cta tac ttt 26/26tgt gtg gtg agt gac aga ggc tca acc ctg ggg agg cta tac ttt 1/26tgt gtg gtg tcc gac aga ggc tca acc ctg ggg agg cta tac ttt 1/26tgt gtg gtg agc gat cga ggc tca acc ctg ggg agg cta tac ttt 1/26
Mouse TRAV11/TRAJ18 C V V G D R G S A L G R L H F 24/25tgt gtg gtg ggc gat aga ggt tca gcc tta ggg agg ctg cat ttt 10/19tgt gtg gtg gga gat aga ggt tca gcc tta ggg agg ctg cat ttt 7/19tgt gtg gtg ggg gat aga ggt tca gcc tta ggg agg ctg cat ttt 12/19tgt gtg gtg ggt gat aga ggt tca gcc tta ggg agg ctg cat ttt 7/19tgt gtg gtg ggt gac aga ggt tca gcc tta ggg agg ctg cat ttt 1/19
Rat TRAV11-like/TRAJ18-like (mouse) C V V A D R G S A L G K L Y F 2/2tgt gtg gtg gcc gat aga ggt tca gct cta ggg aag ctg tat ttt 2/2tgt gtg gtg gcg gat aga ggt tca gct cta ggg aag ctg tat ttt 1/2
Rhesus MacaqueTRAV10-like/TRAJ18-like (human)
C V V S D R G S T L G K L Y F 2/2tgt gtg gtg agc gac aga ggc tca acc ctg ggg aag cta tac ttt 1/1
Dog TRAV10-like/TRAJ18-like (human) C V V S D R A S A L G K L H F 1/1tgt gtg gtg agc gac aga gcc tca gcc tta ggg aaa ctg cac ttc 1/1
Pig TRAV10-like/TRAJ18-like (human) C V V G D R G S R L G R L Y F 1/2
a Nucleotide sequences are shown with nucleotide additions (estimated so as to minimize the total number of nucleotide additions) in bold-underline.b When a canonical sequence has not been defined previously, the most shared and/or most gene-encoded sequence was chosen as the canonical sequence.
that rs as so
oi211iOascassKe
Ce
utTiidnwha
delThBA
c Number of individuals with a nucleotide sequence is calculated over all studiesd Total individuals for amino acid and nucleotide sequences differ in some specie
bserved to dominate the NKT cell clonal hierarchy in multiplendividuals both within and across many NKT studies (Dao et al.004; Dellabona et al. 1994; Demoulins et al. 2003; Kent et al.999; Koseki et al. 1991; Lantz and Bendelac 1994; Porcelli et al.993; Shimamura et al. 1997; Sumida et al. 1995). The term “canon-
cal” is commonly used to characterize these NKT TCR� sequences.ur survey confirms this sharing of canonical NKT TCR� sequencescross both mouse and human populations (Table 1). Rat and rhe-us macaque populations also showed evidence of these sharedanonical sequences (Table 1). Moreover, the canonical NKT TCR�mino acid sequences are closely related across all of the surveyedpecies (Table 1), confirming previous comparisons among variousubsets of the species surveyed in this study (Gansuvd et al. 2008;ashiwase et al. 2003; Kjer-Nielsen et al. 2006; Looringh van Beeckt al. 2009; Pellicci et al. 2009; Pyz et al. 2006; Yasuda et al. 2009).
anonical NKT TCR ̨ sequences have the potential to be producedfficiently
That canonical NKT TCR� sequences exist in multiple species leds to ponder the mechanisms by which these different immune sys-ems ensure the presence of these highly inter-individually sharedCRs. Our previous studies have demonstrated that TCR frequencyn the naïve repertoire and the extent of inter-individual TCR shar-ng are related to differential production frequencies, which areetermined on a probabilistic basis by the process of V(D)J recombi-ation (Quigley et al. 2010; Venturi et al. 2006, 2008a,b, 2011). Thus,e considered whether production efficiency could account for theigh frequency and sharing of the canonical NKT TCR� sequences,nd whether such effects were evident in multiple species.
One indicator of TCR sequences that have the potential to be pro-uced efficiently via gene recombination is the extent of germlinencoding, as sequences with fewer nucleotide additions containess of a random element (Fazilleau et al. 2005; Venturi et al. 2008c).
he germline nature of human and mouse NKT TCR� sequencesas been noted in previous studies (Koseki et al. 1990; Lantz andendelac 1994; Porcelli et al. 1993; Scott-Browne et al. 2007).cross all the studies surveyed that reported sequences at the
eport nucleotide sequences.me studies did not report nucleotide sequences.
nucleotide level, we found that the most prevalent nucleotidesequence encoding the human canonical NKT TCR� amino acidsequence was indeed germline-encoded (Table 1, Fig. 1A). For themouse canonical NKT TCR� amino acid sequence, both germline-encoded nucleotide sequences and nucleotide sequences requiringone nucleotide addition were prevalent (Table 1, Fig. 1B). Investi-gation of the extent of germline gene encoding of the NKT TCR�sequences for the other species has been hindered by the lack ofTCR gene sequence availability for these species. Here, we extractedTRA genes from the reported genomes for these other species thatare homologous to the human and mouse invariant NKT TRAVand TRAJ genes. The rat, rhesus macaque and dog canonical NKTTCR� sequences could also be encoded by at least one nucleotidesequence that could be produced without nucleotide additions(Table 1, Fig. 1C–E). Although no NKT TCR� nucleotide sequenceswere reported for the pig, we predicted that one of the reported pigNKT TCR� amino acid sequences could be completely encoded bygenes extracted from the pig genome that are homologous to thehuman TRAV10 and TRAJ18 genes (Fig. 1F).
A notable feature of the germline-encoded nucleotide sequencesthat code for the canonical NKT TCR� amino acid sequences acrossthe various species is the “overlap” between the V and J genes atthe VJ junction. That is, some of the nucleotides forming the codonsin the fourth and fifth positions of the CDR3� amino acid sequencecan be contributed by either the V or J genes (Fig. 1; yellow boxesindicate the nucleotides that can be contributed by either the Vor J gene, leaving the TCR in frame). This enables these germline-encoded NKT TCR� nucleotide sequences to be spliced together inseveral different ways that do not require nucleotide addition. InFig. 1, we demonstrate the variety of different recombination mech-anisms, involving different contributions from the V and J genes andeither zero or one nucleotide additions, that are predicted to pro-duce one of the observed germline-encoded nucleotide sequences,or, in the case of the pig, the predicted nucleotide sequence.
The variety of different nucleotide sequences observed toencode a TCR amino acid sequence (Venturi et al. 2008c) is anotherkey indicator of how efficiently a TCR sequence can be made. Thecanonical NKT TCR� amino acid sequences in humans and mice
tgt gtg gtg ggc gat aga ggttgt gtg gtg gga gat aga ggttgt gtg gtg ggt gat aga ggttgt gtg gtg ggg gat aga ggttgt gtg gtg ggt gac aga ggt
tgt gtg gtg ggc gat aga ggctgt gtg gtg ggc gat aga ggc
tgt gtg gtg ggc gat aga ggctgt gtg gtg ggc gat aga ggc tgt gtg gtg ggc gat aga ggc
0
1
C V V S D R G STLGRLYF
tgt gtg gtg agc gac aga ggctgt gtg gtg agt gac aga ggctgt gtg gtg tcc gac aga ggctgt gtg gtg agc gat cga ggc
tgt gtg gtg agc gac aga ggctgt gtg gtg agc gac aga ggctgt gtg gtg agc gac aga ggc
tgt gtg gtg agc gac aga ggctgt gtg gtg agc gac aga ggc tgt gtg gtg agc gac aga ggctgt gtg gtg agc gac aga ggc
0
1
A B C C V V A D R G SALGKLYF
C V V S D R G STLGKLYF
tgt gtg gtg agc gac aga ggc
tgt gtg gtg gcc gat aga ggttgt gtg gtg gcg gat aga ggt
tgt gtg gtg gcc gat aga ggttgt gtg gtg gcc gat aga ggt
tgt gtg gtg gcc gat aga ggttgt gtg gtg gcc gat aga ggt tgt gtg gtg gcc gat aga ggt
tgt gtg gtg agc gac aga ggctgt gtg gtg agc gac aga ggctgt gtg gtg agc gac aga ggc
tgt gtg gtg agc gac aga ggctgt gtg gtg agc gac aga ggc tgt gtg gtg agc gac aga ggctgt gtg gtg agc gac aga ggc
Human Mouse Rat
Rhesus macaqueD
0
1
0
1
C V V S D R A SALGKLHF
tgt gtg gtg agc gac aga gcc
tgt gtg gtg agc gac aga gcctgt gtg gtg agc gac aga gcctgt gtg gtg agc gac aga gcc
tgt gtg gtg agc gac aga gcctgt gtg gtg agc gac aga gcc tgt gtg gtg agc gac aga gcctgt gtg gtg agc gac aga gcc
DogE
0
1
C V V G D R G SRLGRLYF
tgt gtg gtg ggt gat aga gcc
tgt gtg gtg ggt gat aga ggctgt gtg gtg ggt gat aga ggctgt gtg gtg ggt gat aga ggc
tgt gtg gtg ggt gat aga ggctgt gtg gtg ggt gat aga ggc tgt gtg gtg ggt gat aga ggctgt gtg gtg ggt gat aga ggc
PigF
0
1
Fig. 1. Convergent recombination in the canonical NKT TCR� sequences of various species. The various aspects of the convergent recombination process are demonstratedfor human (A), mouse (B), rat (C), rhesus macaque (D), dog (E) and pig (F) canonical NKT TCR� amino acid sequences. Shown at the top of panels A–D are the multiplenucleotide sequences observed to encode for the canonical amino acid sequences in humans, mice, rats and rhesus macaques. Shown at the top of panels E and F arenucleotide sequences predicted to encode for the canonical amino acid sequences in dogs and pigs. Alignments to V genes (purple) and J genes (orange) that give minimalnucleotide additions (black) are shown. In the middle of each panel are the multiple recombination mechanisms requiring 0 and 1 nucleotide addition that could produceone of the germline-encoded nucleotide sequences (black box). The relevant portions of the TRAV and TRAJ genes are shown at the bottom of each panel with the overlapr ig geni heir hos ltiple
wcasdoa
si“Vbp
Cc
sraa(swt
egion between the V and J genes highlighted in yellow. Rhesus macaque, dog and pnvariant NKT human genes. Rat genes were extracted from the genome based on tequences were identified as the most prevalent across studies or, in the case of mu
ere encoded in a number of ways, largely owing to the highodon degeneracy of one of the amino acids (serine, glycine, orlanine) at the junction between the V and J genes in all reportedpecies. In addition to the germline-encoded nucleotide sequencesiscussed above, other nucleotide sequences that required at leastne nucleotide addition were also observed to code for the humannd mouse canonical NKT TCR� amino acid sequences (Table 1).
Collectively, these features of the canonical NKT TCR� sequencesuggest that a process of convergent recombination may play anmportant role in the production of these sequences and that theease” with which canonical NKT TCR� sequences can be made byJ recombination may influence their prevalence, both within andetween individuals. Moreover, this mechanism appears to be atlay in the NKT repertoires of multiple species.
anonical NKT TCR ̨ sequences are produced efficiently byonvergent recombination
Thus far, our results suggest that the canonical NKT TCR�equences for all species are more likely to be generated by VJecombination as they require fewer nucleotide additions, featuren amino acid at the VJ junction with a high codon redundancy,nd can be produced by a variety of recombination mechanisms
Fig. 1). However, when analyzing a frequently expressed TCRequence, it is not possible to know the actual mechanisms byhich it was produced; it could have been made repeatedly by
he same recombination mechanism, or many times by a variety of
es were extracted from the respective genomes based on their homology with themology with the invariant NKT mouse genes. The canonical NKT TCR� amino acid
NKT TCR� sequences of equal prevalence, as the most germline-encoded sequence.
recombination mechanisms. Thus, we used computer simulationsof a random VJ recombination process to determine if the canoni-cal NKT TCR� sequences are produced more efficiently by unbiasedrecombination and whether this could account for their prevalencein multiple individuals and across multiple species. The computersimulations of a random VJ recombination process (SupplementaryFig. 1A) generated 107 in-frame TCR sequences using the invariantTRAV/TRAJ gene combinations associated with the canonical NKTTCR� sequences for each of human, mouse, rhesus macaque, andrat.
The human canonical NKT TCR� amino acid sequence wasthe most generated sequence of all reported human sequencesusing the invariant TRAV10/TRAJ18 gene combination (Fig. 2A).A total of 746 different recombination mechanisms (i.e. differ-ent gene and nucleotide addition contributions) contributed tothe efficient generation of this sequence in the simulations. Ofthe reported nucleotide sequences encoding the human canoni-cal NKT TCR� amino acid sequence, the most commonly observednucleotide sequence was also the most frequently generated (i.e.making up 72.3% of all simulated sequences encoding this aminoacid sequence; Fig. 3B). The other three nucleotide sequencesreported to code for the human canonical NKT TCR� amino acidsequence, each of which was observed in only one individual, were
produced much less efficiently in the simulations (i.e. <8% of sim-ulated sequences coding for the canonical NKT TCR� amino acidsequence). The mouse canonical NKT TCR� amino acid sequencewas produced with a similar level of efficiency, being the most
218 H.Y. Greenaway et al. / Immunobi
A
B
0 200 400 600 800 1000 1200
0 0.2 0.4 0.6 0.8 1
CVVSDRGSTLGRLYF (32)
CVVSRGSTLGRLYF (1)
CVVTDRGSTLGRLYF (1)
CVVNDRGSTLGRLYF (2)
CVVGDRGSTLGRLYF (1)
CVVRADRGSTLGRLYF (1)
CVVFDRGSTLGRLYF (1)
CVASDRGSTLGRLYF (1)
CVVARDRGSTLGRLYF (1)
CVSVDRGSTLGRLYF (1)
CVVSDKGSTLGRLYF (1)
CVVSATNRGSTLGRLYF (1)
CVVPGRLYF (1)
Number of recombination mechanisms (light)
Percent of simulated repertoire (dark)
Ob
serv
ed h
um
an N
KT
CD
R3
seq
uen
ces
0 200 400 600 800 1000
0 0.2 0.4 0.6 0.8 1 1.2
CVVGDRGSALGRLHF (24)
CVVGRGSALGRLHF (3)
CVVVDRGSALGRLHF (2)
CVVGLDRGSALGRLHF (1)
CVVGVRGSALGRLHF (1)
CVVGARGSALGRLHF (1)
CVVGADRGSALGRLHF (1)
CVVADRGSALGRLHF (6)
CVVIDRGSALGRLHF (2)
CVVGHRGSALGRLHF (1)
CVVGAGGSALGRLHF (1)
CVVGARRGSALGRLHF (1)
CVVGDGGSALGRLHF (1)
CVVGGERGSALGRLHF (1)
CVVARGDRGSALGRLHF (1)
Number of recombination mechanisms (light)
Percent of simulated repertoire (dark)
Ob
serv
ed m
ou
se N
KT
CD
R3
seq
uen
ces
Fig. 2. Simulated frequencies of all observed NKT TCR� amino acid sequences. (A)The percent of the simulated repertoire occupied by each human NKT TCR� aminoacid sequence observed in the literature in 107 computer simulations of a randomVJ recombination process using the TRAV10 and TRAJ18 genes (black), and the num-ber of simulated recombination mechanisms resulting in each sequence (grey). Thehuman non-germline-encoded sequence CVVPGRLYF is shown here for complete-ness but could not be made with the chosen simulation parameters. (B) The percentof the simulated repertoire occupied by each mouse NKT TCR� amino acid sequenceobserved in the literature in 107 computer simulations of a random VJ recombinationprocess using the TRAV11 and TRAJ18 genes (black), and the number of simulatedrecombination mechanisms resulting in each sequence (grey). Canonical NKT TCR�amino acid sequences are in bold, and numbers in brackets denote the numberop
gsasmestwnsmtio
TRAV1/TRAJ33 gene combination were obtained from four studies
f individuals each sequence was reported in. Results are based on simulationserformed without p-additions (Supplemental Fig. 1A).
enerated of all reported mouse TRAV11/TRAJ18 amino acidequences (Fig. 2B). A total of 593 different recombination mech-nisms contributed to the generation of this sequence in theimulations. The five nucleotide sequences reported to encode theouse canonical NKT TCR� amino acid sequence were all gen-
rated in the simulations (Fig. 3D). However, in contrast to theituation for humans, the hierarchy of simulated frequencies forhe nucleotide sequences in mice was not as precisely correlatedith the hierarchy in the number of mice sharing the observeducleotide sequences. That is, the most frequently made nucleotideequence in the simulations was the second most shared betweenice. A potential explanation for the difference in results between
he human and mouse studies is that the number of mice usedn each mouse study from which sequences were obtained wasften less clear than in the human studies. Thus, with fewer mouse
ology 218 (2013) 213– 224
studies reporting nucleotide sequences (Table 1), we have less con-fidence in the accuracy of the number of individuals sharing eachof the nucleotide sequences across the various mouse studies thanacross the human studies.
The human and mouse canonical NKT TCR� amino acidsequences are frequently produced compared with the otherobserved sequences in the NKT repertoires. We next asked howefficiently produced the canonical NKT TCR� amino acid sequencesare relative to other simulated sequences using the invariant genecombinations, regardless of whether or not they were observed inthe NKT repertoires. We first compared the in silico productionof the canonical NKT TCR� amino acid sequences against all sim-ulated TRAV10/TRAJ18 amino acid sequences conforming to the15 amino acid CDR3 length restriction associated with NKT TCR�sequences. The human and mouse canonical NKT TCR� amino acidsequences were the most generated, making up 5.92% and 6.01% ofthese same-length CDR3 amino acid sequences for humans (Fig. 3A)and mice (Fig. 3C), respectively. Moreover, even if we ignore thelength restriction and look at the total simulated TCR repertoire,the human canonical NKT TCR� amino acid sequence was the thirdmost generated amino acid sequence overall, making up 0.95% ofthe total simulated human TRAV10/TRAJ18 repertoire. The mousecanonical NKT TCR� amino acid sequence was the fifth most gener-ated amino acid sequence, occupying 0.96% of the total simulatedmouse TRAV11/TRAJ18 repertoire.
We also simulated TCR� repertoires using the appropriate rat,rhesus macaque, dog, and pig invariant V and J gene combinations.Insufficient data are currently available in the literature to performany meaningful analyses involving the prevalence of the reportedNKT TCR� sequences. However, we used the simulations to predictthe production efficiencies of the reported NKT TCR� sequences forthese other species relative to all simulated sequences using theinvariant gene combinations with a CDR3 length of 15 amino acids,based on the NKT CDR3� length restriction evident in the humanand mouse data. For all four of these other species, the most fre-quently generated amino acid sequence in the in silico repertoiresconforming to the 15 amino acid length restriction was the reportedcanonical NKT TCR� sequence for that species (SupplementaryTable 4).
Thus, we see that the canonical NKT TCR� amino acid sequencesin the various species are all produced efficiently via gene recom-bination; they are frequently produced in computer simulationsof an unbiased VJ recombination process, and the most frequentlyproduced of all sequences conforming to the 15 amino acid residuelength restriction associated with NKT TCR� sequences. In addi-tion, the most commonly reported nucleotide sequences encodingthe human canonical NKT TCR� sequences are also the most easilyproduced by a random recombination process. This is consistentwith our expectation that the hierarchy of nucleotide sequenceprevalence will be largely determined by the relative productionfrequencies, as the factors that shape the T cell repertoire subse-quent to the production of the T cell receptor are driven at thelevel of the amino acid sequence. Collectively, these results providestrong evidence for the influence of convergent recombination inthe observed inter-individual and inter-species prevalence of thecanonical NKT TCR� sequences.
MAIT TCR ̨ sequences in the literature
Twenty-seven unique human MAIT TCR� amino acid sequencesusing the TRAV1-2/TRAJ33 gene combination were obtained fromfive studies and 31 unique mouse MAIT TCR� sequences using the
(Supplementary Table 3). A single MAIT TCR� amino acid sequencewith high homology to the human sequence was obtained for eachof cow and sheep from three studies and one study, respectively.
C V V S D R G tgt gtg gtg agc gac aga ggc 72.3% 50 100%tgt gtg gtg agt gac aga ggc 7.3% 23 5%tgt gtg gtg tcc gac aga ggc 7.1% 27 5%tgt gtg gtg agc gat cga ggc 0.4% 13 5%Other 12.9%
C V V G D R Gtgt gtg gtg ggc gat aga ggt 50.5% 44 53%tgt gtg gtg gga gat aga ggt 28.9% 35 37%tgt gtg gtg ggg gat aga ggt 7.3% 29 63%tgt gtg gtg ggt gat aga ggt 7.3% 25 37%tgt gtg gtg ggt gac aga ggt 0.1% 10 5%Other 5.9%
Observed encodingnucleotide sequences
Observed encodingnucleotide sequences
% ofsimulated
% ofsimulated
A
C
Clonotypes occupying ≥ 0.5%and reported in studies
Clonotypes occupying < 0.5%
Clonotypes occupying ≥ 0.5%and reported in studies
Clonotypes occupying < 0.5%
B
D
Clonotypes occupying ≥ 0.5%
Clonotypes occupying ≥ 0.5%
% ofindividuals
% ofindividuals
No. recombination mechanisms
No. recombination mechanisms
Fig. 3. Composition of the simulated human and mouse NKT TCR� repertoires. (A) All simulated human TRAV10/TRAJ18 sequences conforming to the 15 amino acid (a.a.)residue CDR3 length restriction associated with NKT TCR� sequences. Dark and light grey segments represent clonotypes occupying ≥0.5% and <0.5% of the 15 a.a. simulatedrepertoire, respectively. Dark blue segments represent (in reverse order of size) the clonotypes CVVSDRGSTLGRLYF, CVVTDRGSTLGRLYF, and CVVNDRGSTLGRLYF, whichhave been observed experimentally. (B) Breakdown of the human canonical NKT TCR� amino acid sequence into the nucleotide sequences reported in studies to encodefor it. The coloured bar indicates the percentage of the simulated nucleotide repertoire encoding the canonical NKT TCR� amino acid sequence occupied by each of theobserved nucleotide sequences. (C) All simulated mouse TRAV11/TRAJ18 sequences conforming to the 15 a.a. residue CDR3 length restriction associated with NKT TCR�sequences. Dark and light grey segments represent clonotypes occupying ≥0.5% and <0.5% of the 15 a.a. simulated repertoire, respectively. Dark red segments represent(in reverse order of size) the clonotypes CVVGDRGSALGRLHF, CVVVDRGSALGRLHF, CVVGVRGSALGRLHF, CVVGARGSALGRLHF, CVVADRGSALGRLHF, CVVIDRGSALGRLHF, andCVVGHRGSALGRLHF, which have been observed experimentally. (D) Breakdown of the mouse canonical NKT TCR� amino acid sequence into the nucleotide sequencesr simuo tions
AfCtTohsi
Se
pthfD(smh
m
eported in studies to encode for it. The coloured bar indicates the percentage of theccupied by each of the reported nucleotide sequences. Results are based on simula
majority of the observed human MAIT TCR� sequences con-ormed to a 12 amino acid residue length restriction across theDR3 (inclusive of the conserved cysteine and tryptophan). Whilehere was greater CDR3 length variation among the mouse MAITCR� sequences, the most prevalent sequences had CDR3 lengthsf 12 amino acids. Both the cow and sheep MAIT TCR� sequencesad CDR3 lengths of 12 amino acids. Thus, similar to the NKT TCR�equences, MAIT TCR� sequences also have a CDR3 length bias thats evident across species.
hared MAIT TCR ̨ sequences have the potential to be producedfficiently
As was the case with the NKT TCR� sequences, there werearticular human and mouse MAIT TCR� amino acid sequenceshat appeared in more individuals than others (Table 2). The mostighly shared human MAIT TCR� sequence, CAVRDSNYQLIW, was
ound in 10 of the 17 individuals across all the human studies.espite a much greater diversity of mouse MAIT TCR� sequences
Supplementary Table 3), the most highly shared mouse MAITequence CAVRDSNYQLIW was observed in all but one of the 12
ice (Table 2). The cow MAIT TCR� sequence, CVVMDGNYQWIW,
as been observed in three cows (Table 2).It has been previously reported that the most highly shared
ouse MAIT TCR� sequence can be completely germline encoded
lated nucleotide repertoire encoding the canonical NKT TCR� amino acid sequence performed without p-additions (Supplemental Fig. 1A).
(Tilloy et al. 1999; Treiner et al. 2003) and that there is an overlap innucleotides between the mouse TRAV1 and TRAJ33 genes (Treineret al. 2003). In this study, the most highly shared human, mouse,and cow MAIT TCR� amino acid sequences were observed to beencoded by at least one nucleotide sequence that can be made with-out the need for nucleotide additions (Table 2, Fig. 4A–C). Moreover,for each species, an overlap between the MAIT-related V� and J�genes enables nucleotides from either the V� or J� genes to con-tribute to the formation of codons at the VJ junction, enabling thegermline-encoded MAIT TCR� sequences to be made by a varietyof recombination mechanisms (Fig. 4; yellow boxes indicate thenucleotides that can be contributed by either the V or J gene). Wealso observed that palindromic additions (i.e. p-additions) couldplay a role in the production of the MAIT TCR� sequences, effec-tively increasing the number of gene-templated nucleotides thatcould be contributed by either the V or J gene (Fig. 4). Both thegermline nature of the MAIT TCR� sequences and the variety ofrecombination mechanisms that can produce each sequence indi-cate that MAIT TCR� sequences have the potential to be producedefficiently.
We again used computer simulations to determine whether
the most highly shared MAIT TCR� sequences could be pro-duced efficiently by an unbiased VJ recombination process. In107 computer simulations of random gene recombination of thehuman TRAV1-2 and TRAJ33 genes, the most prevalent MAIT TCR�
Table 2The most highly shared MAIT TCR� amino acid sequences and the encoding nucleotide sequences in human, mouse, cow and sheep studies.
Species CDR3 regiona Number of individualswith sequenceb/totalc
Human TRAV1-2/TRAJ33 C A V R D S N Y Q L I W 10/17tgt gct gtg aga gat agc aac tat cag tta atc tgg 8/13tgt gct gta aga gat agc aac tat cag tta atc tgg 1/13
Mouse TRAV1/TRAJ33 C A V R D S N Y Q L I W 11/12tgt gct gtg agg gat agc aac tat cag ttg atc tgg 3/3tgt gct gtg aga gat agc aac tat cag ttg atc tgg 1/3
Cow TRAV1-2-like/TRAJ33-like (human) C V V M D G N Y Q W I W 3/3tgt gtt gtg atg gat ggc aac tat cag tgg atc tgg 1/1
Sheep TRAV1-2-like/TRAJ33-like (human) C A V M D G N Y R L I W 1/1
a Nucleotide sequences are shown with nucleotide additions (estimated so as to minimize the total number of nucleotide additions) in bold-underline. that rs as so
so(osf(
m
Fscwppt
b Number of individuals with a nucleotide sequence is calculated over all studiesc Total individuals for amino acid and nucleotide sequences differ in some specie
equence, CAVRDSNYQLIW, was the most generated sequencef all reported sequences using the invariant gene combinationFig. 5A). Similarly, computer simulations of the recombinationf the mouse TRAV1 and TRAJ33 genes revealed that the TCR�equence CAVRDSNYQLIW, observed in 11/12 mice, was the mostrequently produced of all observed mouse MAIT TCR� sequences
Fig. 5B).
We also compared the in silico production frequencies of theost prevalent MAIT TCR� amino acid sequences against all
TRAV1-2-liketgt gtt gtg ata gat
atg gat ggc aac TRAJ33-like
TRAV1-2tgt gct gtg aga gat
tg gat agc aac TRAJ33
C C V V M D G N YQWIW
tgt gtt gtg atg gat ggc aac
tgt gtt gtg atg gat ggc aactgt gtt gtg atg gat ggc aactgt gtt gtg atg gat ggc aac
tgt gtt gtg atg gat ggc aactgt gtt gtg atg gat ggc aactgt gtt gtg atg gat ggc aactgt gtt gtg atg gat ggc aac
Cow
A
0
1
C A V R D S N YQLIW
tgt gct gtg aga gat agc aactgt gct gta aga gat agc aac
tgt gct gtg aga gat agc aactgt gct gtg aga gat agc aactgt gct gtg aga gat agc aactgt gct gtg aga gat agc aac
tgt gct gtg aga gat agc aactgt gct gtg aga gat agc aactgt gct gtg aga gat agc aactgt gct gtg aga gat agc aactgt gct gtg aga gat agc aac
Human
0
1
B
ig. 4. Convergent recombination in the TCR� sequences of MAIT cells. The various aspehared human (A), mouse (B), and cow (C) MAIT TCR� amino acid sequences. Shown at thanonical amino acid sequences in humans, mice, and cows. Alignments to V genes (purpith possible p-addition contributions underlined. In the middle of each panel are the muroduce the most frequently seen nucleotide sequence (black box). The relevant portion-additions underlined and the overlap region between the V and J genes highlighted in yhe invariant MAIT human genes.
eport nucleotide sequences.me studies did not report nucleotide sequences.
the simulated sequences that had a CDR3 length of 12 aminoacids, which is the preferential CDR3 length among MAIT TCR�sequences. The most prevalent human and mouse MAIT TCR�amino acid sequences were the most frequently generated ofall simulated human TRAV1-2/TRAJ33 and mouse TRAV1/TRAJ33amino acid sequences with a CDR3 length of 12 amino acids, respec-
tively (Fig. 5C and D). The cow MAIT TCR� amino acid sequence,CVVMDGNYQWIW, which was observed in 3/3 cows, was the sec-ond most generated of all 12 amino acid length CDR3 sequences
TRAV1tgt gct gtg agg gat
tg gat agc aac TRAJ33
C A V R D S N YQLIW
tgt gct gtg agg gat agc aactgt gct gtg aga gat agc aac
tgt gct gtg agg gat agc aactgt gct gtg agg gat agc aactgt gct gtg agg gat agc aactgt gct gtg agg gat agc aactgt gct gtg agg gat agc aac
tgt gct gtg agg gat agc aactgt gct gtg agg gat agc aactgt gct gtg agg gat agc aactgt gct gtg agg gat agc aactgt gct gtg agg gat agc aactgt gct gtg agg gat agc aac
Mouse
0
1
cts of the convergent recombination process are demonstrated for the most highlye top of panels A–C are the multiple nucleotide sequences observed to code for thele) and J genes (orange) that give minimal nucleotide additions (black) are shown,ltiple recombination mechanisms requiring 0 and 1 nucleotide addition that could
s of the TRAV and TRAJ genes are shown at the bottom of each panel with possibleellow. Cow genes were extracted from the genome based on their homology with
Fig. 5. Simulated frequencies of all observed MAIT TCR� amino acid sequences. (A) The percent of the simulated repertoire occupied by each human (A) and mouse (B)MAIT TCR� amino acid sequence observed in the literature in 107 computer simulations of a random VJ recombination process using the TRAV1-2 (human)/TRAV1 (mouse)and TRAJ33 genes (black), and the number of simulated recombination mechanisms resulting in each sequence (grey). (C) All simulated human TRAV1-2/TRAJ33 sequencesconforming to the 12 amino acid (a.a.) residue length restriction associated with MAIT TCR� sequences. Dark and light grey segments represent clonotypes occupying≥0.5% and <0.5% of the 12 a.a. simulated repertoire, respectively. Dark blue segments represent (in reverse order of size) the MAIT TCR� clonotypes CAVRDSNYQLIW,CAVMDSNYQLIW, CAVLDSNYQLIW, CAVVDSNYQLIW, CAVTDSNYQLIW, and CAVKDSNYQLIW. (D) All simulated mouse TRAV1/TRAJ33 sequences conforming to the 12 a.a.residue length restriction associated with MAIT TCR� sequences. Dark and light grey segments represent clonotypes occupying ≥0.5% and <0.5% of the 12 a.a. simulatedrepertoire, respectively. Red segments represent (in reverse order of size) the MAIT TCR� clonotypes CAVRDSNYQLIW, CAVMDSNYQLIW, and CAVLDSNYQLIW. Results areb
uoT
sttaTf
ased on simulations performed without p-additions (Supplemental Fig. 1A).
sing the genes extracted from the cow genome that are homol-gous to the human TRAV1-2 and TRAJ33 genes (Supplementaryable 4).
As we noted from the gene alignments of the MAIT TCR�equences, it appears that p-additions could play a role inhe production of these sequences (Fig. 4). Thus, we under-
ook computer simulations allowing for up to four p-additionst the junctional ends of the TRAV1-2/TRAJ33 (human) andRAV1/TRAJ33 (mouse) genes (Supplementary Fig. 1B). Weound that the most prevalent human and mouse MAIT TCR�
sequences remained the most frequently simulated of all theexperimentally observed MAIT TCR� sequences listed inSupplemental Table 3 (data not shown). Moreover, when com-pared to all simulated sequences produced when allowing forp-additions, the most highly shared human, mouse and cowMAIT TCR� sequences maintained the highest in silico production
frequencies (Supplemental Table 4).
Collectively, these results suggest that the highly shared MAITTCR� sequences also have the potential to be produced efficientlyvia gene recombination. Accordingly, as for the semi-invariant
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KT TCR� sequences, we hypothesize that such production effi-iency allows MAIT TCR� sequences to be found in multiplendividuals and explains the observation of highly similar MAITCR� sequences across different species.
iscussion
Despite the number of studies focussing on NKT and, moreecently, MAIT cells, these T cell subsets remain enigmatic. Theolecular origins of NKT cells are unclear and their functions
re diverse, while the functions of MAIT cells are still being dis-overed. The sharing of identical NKT and MAIT TCR� sequencesetween many individuals, and the similarity of canonical NKTnd MAIT TCR� sequences across multiple species, is particularlyntriguing. The gene recombination process is capable of generat-ng an enormous diversity of T cell receptors in each individuali.e. >1015 different TCR�� (Davis and Bjorkman 1988)), only aroportion of which populate the peripheral T cell populationi.e. >106 in mice (Casrouge et al. 2000) and >107 in humansArstila et al. 1999)). How, then, do these crucial groups of immuneells with innate-like properties emerge with such widespreadxistence from the enormously diverse T cell repertoires acrossndividuals and across species? While the many structural stud-es can explain the dominant interaction of the canonical NKTCR� sequences with CD1d, and post-production selection pro-esses that shape the TCR repertoire could potentially explainhe prevalence of the canonical NKT and MAIT TCR� sequencesithin an individual, neither addresses the unlikelihood of the
ame TCR� sequence being produced, and paired with an appro-riate TCR�, in so many individuals. In this study, our analysisf previously reported NKT and MAIT TCR� sequences obtainedrom a variety of species, including human, mouse, rat, rhesus
acaque, dog, cow, and pig, demonstrates that the canonical NKTnd MAIT TCR� sequences for each species have the potential toe produced with extraordinary efficiency by the recombinationachinery.Consistent with our previous findings that TCR production fre-
uency is an important determinant of the inter-individual sharingf antigen-specific (Venturi et al. 2006, 2008a,b) and naïve (Quigleyt al. 2010; Venturi et al. 2011) TCR� sequences, the highly sharedKT and MAIT TCR� sequences have the potential to be producedfficiently by a process of convergent recombination involvinghe invariant NKT and MAIT TCR� V and J genes. For each of thepecies studied, we identified at least one nucleotide sequencencoding the canonical NKT and MAIT TCR� sequence that coulde constructed entirely from the germline genes, without theeed for nucleotide additions. The germline nature of the canon-
cal NKT TCR� sequence in humans and mice and the canonicalAIT TCR� sequence in mice has been noted in previous stud-
es (Dellabona et al. 1994; Kent et al. 1999; Koseki et al. 1990;antz and Bendelac 1994; Porcelli et al. 1993; Tilloy et al. 1999;reiner et al. 2003), with germline-encoded residues shown to bemportant in the interaction between NKT cells and CD1d-antigenomplexes (Scott-Browne et al. 2007). This germline nature of theanonical NKT and MAIT TCR� sequences also plays an impor-ant role in their efficient production in each species. However,he extraordinary efficiency of production of the canonical NKTnd MAIT TCR� sequences arises from the large variety of recom-ination mechanisms that can produce these germline-encodeducleotide sequences. This variety of recombination mechanisms
s largely attributed to an overlap between the invariant NKT
nd MAIT TCR� V and J genes, which enables contributions fromither the V or J genes to participate in the formation of theodons at the VJ junction. Moreover, this feature was observedor the canonical NKT and MAIT TCR� sequences in all the species
ology 218 (2013) 213– 224
studied. Simulations of a random recombination process involv-ing the invariant NKT TCR� V and J genes in humans, rhesusmacaques, rats, mice, dogs and pigs found the canonical NKTTCR� sequences to be the most frequently produced of TCR�sequences with a CDR3 length of 15 amino acids (i.e. the CDR3length bias among NKT TCR� sequences). Similarly, the canonicalMAIT TCR� sequences for humans, mice, and cows were either thefirst or second most frequently produced of all simulated sequencesusing the invariant MAIT TCR� V and J genes and displaying aCDR3 length of 12 amino acids (i.e. the CDR3 length bias amongMAIT TCR� sequences). These results suggest that TCR productionfrequency plays an important role in the prevalence of the canon-ical NKT and MAIT TCR� sequences both across individuals andacross species. Furthermore, this is supported by the observation ofTreiner et al. (2003) that 7% of all TRAV1/TRAJ33 sequences derivedfrom MR1-deficient mice (i.e. in the absence of MR1-restrictedselection/expansion) corresponded to the canonical MAIT TCR�sequence.
Thus far, we have considered primarily the �-chains of both typeI NKT cells and MAIT cells. Studies have shown that the �-chains ofboth types of cells in humans and mice have limited V gene usage(Dellabona et al. 1994; Lantz and Bendelac 1994; Matsuda et al.2001; Porcelli et al. 1993; Treiner and Lantz 2006) but highly diversejunctional CDR3 sequences (Behar et al. 1999; Lantz and Bendelac1994; Matsuda et al. 2001) and CDR3 lengths (Borg et al. 2007).While the NKT TCR �-chain dominates the interaction with CD1d(Borg et al. 2007; Kjer-Nielsen et al. 2006; Pellicci et al. 2009), it hasbeen suggested that the �-chain fine-tunes the sensitivity to CD1dand antigen (Mallevaey et al. 2009; Matulis et al. 2010), enablingNKT cells to recognize a wide range of antigens. One wonders iffuture studies of MAIT cell receptor structures might reveal a sim-ilar mechanism. Promiscuous TCR �-chain pairing of the canonicalNKT and MAIT TCR �-chains most likely plays an important rolein the prevalence of the canonical NKT and MAIT TCR� sequences.That is, if a TCR �-chain could successfully pair with only a few TCR�-chains, this would substantially reduce the chances of observingthis TCR �-chain in multiple individuals unless the correspond-ing TCR �-chains were also produced efficiently. However, giventhat the canonical TCR� are paired with a large variety of differentTCR� chains (Dellabona et al. 1994; Kawachi et al. 2006; Lantz andBendelac 1994; Martin et al. 2009; Matsuda et al. 2001; Porcelliet al. 1993; Tilloy et al. 1999; Treiner and Lantz 2006), this suggeststhat the TCR� chain is both frequently produced, and relativelypromiscuous with respect to its �-chain pairing. Indeed, the abilityof a highly conserved TCR� to pair with a variety of different TCR�chains may be one advantage for these receptors being produced byTCR gene rearrangement, rather than as hard-wired gene-encodedreceptors.
In summary, this study demonstrates that the canonical NKTand MAIT TCR� sequences in various species have the potential tobe produced efficiently by a process of convergent recombination,suggesting that a high production frequency facilitates the inter-individual and inter-species prevalence of these TCR� sequences.Thus, the immune system of a host, regardless of the individ-ual or species, can depend on the presence of particular T cellsubsets that bear distinct receptors, without the need for theseimmune cells to be hard-wired. Moreover, the role of the hierarchyof TCR production frequencies in the emergence of the innate-likeNKT and MAIT cell receptors from the enormously diverse T cellrepertoires of many individuals suggests the existence of otherinvariant receptors produced in a similar manner. This work elu-cidates the molecular origins of the undeniably important highlyshared invariant NKT and MAIT �-chain receptors and predictsthat TCR production frequency plays an important role in the
emergence of innate-like T cell subsets from the diverse T cellrepertoire.
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cknowledgements
This work was supported by the Australian Research CouncilARC) and Australian National Health and Medical Research CouncilNHMRC). HYG is supported by an Australian Postgraduate Award,AP is a Medical Research Council (UK) Senior Clinical Fellow, MPD
s an NHMRC Senior Research Fellow and VV is an ARC Future Fel-ow.
ppendix A. Supplementary data
Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/.imbio.2012.04.003.
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