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Dieli, F.

Meraviglia, S.

Serena Meraviglia

Francesco Dieli

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Cellular and Molecular Immunology

Clonal expansion shapes the human Vδ1T cell receptor repertoire

Meraviglia S

Dieli F.

Archivio istituzionale della ricerca - Università di Palermo

RESEARCH HIGHLIGHTClonal expansion shapes the human Vδ1T cell receptorrepertoireSerena Meraviglia and Francesco DieliCellular & Molecular Immunology advance online publication, 24 July 2017; doi:10.1038/cmi.2017.66The γδ cells are a unique population of Tlymphocytes that combine innate-likefeatures and adaptive-type responsesand play an important role in the earlyhost response to infections and malig-nancies. Different from αβ T cells, γδT cells recognize a limited set of antigens,which are shared by a variety of micro-bial pathogens and tumor cells in a non-MHC restricted manner;1 thus, thesecells use the TCR in a manner similarto a pattern recognition receptor (PRR).Moreover, whereas αβ T cells requireantigen- and cytokine-driven clonalexpansion, γδ T cells are equipped withimmediate effector functions.1 However,the potential γδ repertoire with junc-tional diversity is estimated at ∼ 1018,which is much greater than the αβrepertoire (∼1016), thus raising questionsconcerning the forces governing theselection of such a huge TCR repertoireduring ontogeny and whether and howthe γδ TCR repertoire is shapedunder physiological and pathologicalconditions.In a recent issue of Nature Commu-nication, Davey et al.2 used ampliconrescued multiplex (ARM)-PCR andnext-generation sequencing to investigatethe clonal selection of a γδ TCR reper-toire that was negative for the Vδ2 chainin healthy adults and in cord blood. Asexpected, in all individuals, the Vδ2− γδT cell population was dominated byVδ1+ T cells paired with diverse Vγchains and displaying a mixed terminallydifferentiated effector memory (CD27−CD45RA+) or naive (CD27+ CD45RA+)phenotype.3 Strikingly, in most (70%)adult individuals, remarkably strongfocusing (i.e., a reduction in diversity)of the Vδ1+ TCR repertoire toward asmall number of individual clonotypes(p10) was observed, and this effect wasevident for both TCRγ and TCRδ chains.In contrast to this focused adult sub-group, markedly less focused repertoireswere observed in seven individuals(30%); notably, this minority of indivi-duals, deﬁned as “diverse adult donors”,were primarily cytomegalovirus (CMV)seronegative and included the youngestmembers of the cohort, suggesting anage-dependent modiﬁcation of the Vδ1+TCR repertoire.To investigate how the Vδ1+ TCRrepertoire differed in early life, Daveyet al.2 conducted comparable TCR reper-toire analyses on the Vδ1+ populationobtained from cord blood. Vδ1+ cellsdominate the cord blood γδ repertoireand express Vδ1 paired with diverse Vγregions. The cord blood TCRδ1 andTCRγ CDR3 sequences were extremelyunfocused, in contrast to focused theadult Vδ1 repertoires but similar to theunfocused adult samples, and comprisednumerous low-frequency clonotypes, themost prevalent of which representedo1.30% and o2.17% of the totalunique CDR3s detected for TCRγ andTCRδ, respectively (Figure 1). Asexpected, detailed comparisons of theCDR3 length within and between indi-viduals indicated that in Vδ1+ T cells, themean CDR3δ1 length was substantiallygreater than that of CDR3γ (mean 54versus 33 nucleotides); strikingly, com-parisons of the ten most prevalent TCRγand TCRδ1 clonotypes (typicallyaccounting for 450% of the repertoire)from each donor revealed privatesequences (e.g., present only in oneindividual but absent in any other indi-viduals either at a nucleotide or aminoacid level). Therefore, the Vδ1 TCRrepertoire was overwhelmingly private,with different TCR clonotypes present ineach individual (Figure 1), and compar-ison of Vδ1 repertoire data with age- andsex-matched TCRβ repertoire datarevealed that Vδ1, as a repertoire, waseven more private than TCRβ. Moreover,the most frequent clonotypes weredetected in subsequent ARM analyzesconducted 12–18 months later, and inmost donors, the hierarchy of prevalentclonotypes was broadly conserved inCentral Laboratory of Advanced Diagnosis andBiomedical Research (CLADIBIOR) and Depart-ment of Biopathology and Medical Biotechnolo-gies (DIBIMED), University of Palermo, 90129Palermo, ItalyCorrespondence: Professor S Meraviglia or Pro-fessor F Dieli Central Laboratory of AdvancedDiagnosis and Biomedical Research (CLADI-BIOR) and Department of Biopathology andMedical Biotechnologies (DIBIMED), Universityof Palermo, Corso Tukory 211, Via del Vespro129, 90129 Palermo, Italy.E-mail: serena.meraviglia@unipa.it or francesco.dieli@unipa.itReceived: 12 June 2017; Accepted: 13 June2017Cellular & Molecular Immunology (2017) 14, 1–3& 2017 CSI and USTC All rights reserved 2042-0226/17 $32.00www.nature.com/cmiboth analyses, which clearly indicatedthat the clonotypic expansions prevalentin the Vδ1T cell repertoire are stablymaintained over time.Thus, these ﬁndings indicate thatVδ1T cells undergo profound expansionsof TCR clonotypes in the periphery froman initially completely unfocused and pri-vate repertoire, which is highly suggestiveof an adaptive-type immune response.Hence, selection of the Vδ1 TCRrepertoire differs in several key aspectsfrom that of Vγ9Vδ2T cells, which arethe major γδ population in peripheralblood and secondary lymphoid organs.In contrast to the Vδ1 population, theVγ9Vδ2 TCR repertoire data from Daveyand colleagues conﬁrmed the highlyrestricted CDR3 lengths, including pre-valent Vγ9-JP sequences of limited com-plexity that were common to multipleindividuals, with CDR3γ9 lengths of11–18 amino acids, and 450% of theCDR3γ9 contained a typical 14 aminoacid sequence in all donors. Moreover,analysis of the 10 most frequent TCRγand TCRδ clonotypes in Vδ2+ cells fromeach donor revealed that the CDR3γ9sequences were public and constrainedin length.4 Although CDR3δ2 sequenceswere relatively private compared withTCRγ9, more CDR3δ2 sequences thanCDR3δ1 sequences were shared betweendonors. Therefore, in stark contrast toVδ1+ T cells but analogous to iNKT andMAIT populations, the Vγ9Vδ2 popula-tion expresses a semi-invariant TCR.Consistent with these data, Dimovaet al.5 detected prevalent Vγ9 sequencesthat were present at birth in multipleindividuals, regardless of pathogen expo-sure. These observations are consistentwith a semi-invariant, innate-like biologyfor the Vγ9Vδ2 subset that is compatiblewith the polyclonal activation of thesecells via phosphoantigens.Importantly, Vδ1 clonal expansionwas concomitant with phenotypic differ-entiation involving the loss of secondarylymphoid homing markers and the upre-gulation of effector molecules. The vastmajority of clonal populations occurredin an effector memory/terminally differ-entiated CD27lo/neg CD45RA+ popula-tion, whereas clonotypes showing naive-type CD27hi CD45RA+ expression werediverse, suggesting that Vδ1 TCR reper-toire focusing was accompanied by atransition from a naive to an effectorphenotype.Davey and colleagues used other sur-face markers to better deﬁne the celldifferentiation state, and thus Vδ1CD27hi cells express other naive mole-cules, such as IL-7Rα, CD28, CCR7, andCD62L, which were absent in the CD27lo/neg compartment. Functional analysisshowed that the naive CD27hi subsetproliferated in response to IL-7 stimula-tion, while the CD27lo/neg required IL-15for proliferation, and this latter is atypical feature of terminally differentiatedeffector memory γδ T cell subsets,6 andboth populations proliferated in responseto anti-CD3/CD28 and anti-TCR γδmAb. Moreover, CD27lo/neg cells con-tained granzyme A, B and perforin incontrast to CD27hi cells and expressedCX3CR1. The overall analysis showedthat Vδ1 clonal expansion was accompa-nied by a phenotypic and functionaltransition: of note, cord blood and adultVδ1T cells with an unfocused TCRrepertoire had a naive phenotype anddid not express cytotoxic molecules,whereas clonally expanded Vδ1 popula-tions were preferentially effector cellsequipped with cytotoxic activity.Collectively, these ﬁndings reveal afundamentally adaptive behavior forVδ1T cells, which is likely governed bythe TCR, and strongly support a modelinvolving the clonal selection of naiveVδ1T cells expressing TCRs enablingresponses to yet-unknown antigens,accompanied by differentiation to a term-inally differentiated effector memory phe-notype. These authors argue against theidea that Vδ1 clonotypic focusing merelyreﬂects an immunological imprint of pastchallenges and instead are more sugges-tive of a long-lived, highly speciﬁc, func-tional γδ T-cell memory that enablesaugmented responses to recurrent chal-lenges, akin to classical immunologicalmemory, although importantly, notMHC restricted.This model shares several key tenetswith classical adaptive immunity butdiffers critically by being MHC-unrest-ricted, and it represents an unconven-tional mode of adaptive immunesurveillance. Thus, this study presentsmany questions. Although the identityof the forces underlying Vδ1 TCR reper-toire focusing is unknown, it mightinclude microbial antigens and/or self-molecules.7 Furthermore, although theprivate TCR repertoires may reﬂectresponses restricted to each individual,they do not formally exclude the like-lihood of the degenerate recognition ofconserved ligands by diverse TCRs.CMV infection, which has beenstrongly associated with Vδ2− T-cellresponses,8,9 is not relevant to the dataFigure 1 The neonatal (cord blood) TCR Vδ1 repertoire is extremely unfocused and private.In most adult individuals, the Vδ1+ TCR repertoire is strongly focused, with up to 50% ofthe repertoire comprising the 10 most abundant TCRγ and TCRδ1 clonotypes. Moreover,the Vδ1 TCR repertoire was overwhelmingly private, with different TCR clonotypes presentin each individual. The nature of the forces governing the peripheral clonal selection of thehuman Vδ1 TCR repertoire and the role of CMV remains to be determined.Research Highlight2Cellular & Molecular Immunologyof Davey and colleagues, since CMV-seronegative individuals also exhibitedextreme clonotypic focusing. Addition-ally, it is important to determine why~30% of the adults in the study of Daveyand colleagues retain a largely unfocused,naive Vδ1 repertoire.Moreover, the study of Davey andcolleagues exclusively analyzed Vδ1+T cells in the peripheral blood. Noevidence on tissue-resident Vδ1+ T cellshas been reported, although these cellsrepresent a majority of the γδ T cellpopulation.10 This ﬁnding is importantbecause residence in a non-lymphoidtissue, regardless of whether the tissueis normal or has undergone tumortransformation, serves as a major deter-minant of the phenotypic and functionalcharacteristics of tissue-resident Vδ1 andVδ2T cells (Meraviglia et al., unpub-lished results). Because relatively little isknown about human tissue-resident γδT cells, sequencing the TCR repertoire ofcells isolated from several tissues is there-fore necessary to better increase thecurrent knowledge of the selection ofthe γδ TCR repertoire and to determinethe nature and mode of antigen recogni-tion and the role of γδ T cells in tissues.These aspects may be translationallyrelevant and may provide novel thera-peutic avenues in anti-tumor immuneresponses.11–13CONFLICT OF INTERESTThe authors declare no conﬂict of interest.ACKNOWLEDGEMENTSThis work was supported by grants from theMinistry of Health “Ricerca Finalizzata 2007”to FD.1 Bonneville M, O’Brien RL, Born WK. γδ Tcell effector functions: a blend of innateprogramming and acquired plasticity. NatRev Immunol 2010; 10: 467–478.2 Davey MS, Willcox CR, Joyce SP, Ladell K,Kasatskaya SA, McLaren JE et al. Clonalselection in the human Vδ1T cell repertoireindicates γδ TCR-dependent adaptiveimmune surveillance. Nat Commun 2017; 8:14760.3 Dieli F, Poccia F, Lipp M, Sireci G,Caccamo N, Di Sano C et al. Differentiationof effector/memory Vδ2T cells and migratoryroutes in lymph nodes or inﬂammatory sites.J Exp Med 2003; 198: 391–397.4 Sherwood AM, Desmarais C, Livingston RJ,Andriesen J, Haussler M, Carlson CS et al.Deep sequencing of the human TCRγ andTCRβ repertoires provides evidence thatTCRβ rearranges after αβ, γδ T-cell commit-ment. Sci Transl Med 2011; 3: 90ra61.5 Dimova T, Brouwer M, Gosselin F,Tassignon J, Leo O, Donner C et al. EffectorVγ9Vδ2T cells dominate the human fetal γδT-cell repertoire. Proc Natl Acad Sci USA2015; 112: E556–E565.6 Caccamo N, Meraviglia S, Ferlazzo V,Angelini D, Borsellino G, Poccia F et al.Differential requirements for antigenor homeostatic cytokines for proliferationand differentiation of human Vγ9Vδ2naive, memory and effector T cellsubsets. Eur J Immunol 2005; 35:1764–1772.7 Halary F, Pitard V, Dlubek D, Krzysiek R,de la Salle H, Merville P et al. Sharedreactivity of Vδ2− γδ T cells againstcytomegalovirus-infected cells and tumorintestinal epithelial cells. J Exp Med 2005;201: 1567–1578.8 Déchanet J, Merville P, Lim A, Retière C,Pitard V, Lafarge X et al. Implication of γδT cells in the human immune response tocytomegalovirus. J Clin Invest 1999; 103:1437–1449.9 Pitard V, Roumanes D, Lafarge X, Couzi L,Garrigue I, Lafon ME et al. Long-term expan-sion of effector/memory Vδ2− γδ T cells is aspeciﬁc blood signature of CMV infection.Blood 2008; 112: 1317–1324.10 Hayday AC. γδ T cells and the lymphoidstress-surveillance response. Immunity2009; 31: 184–196.11 Silva-Santos B, Serre K, Norell H. γδ T cellsin cancer. Nat Rev Immunol 2015; 15:683–691.12 Lo Presti E, Dieli F, Meraviglia S. Tumor-inﬁltrating γδ T lymphocytes: pathogenicrole, clinical signiﬁcance, and differentialprograming in the tumor microenvironment.Front Immunol 2014; 5: 607.13 Wu D, Wu P, Qiu F, Wei Q, Huang J. HumanγδT-cell subsets and their involvement intumor immunity. Cell Mol Immunol 2017;14: 245–253.Research Highlight3Cellular & Molecular Immunology

Clonal expansion shapes the human Vδ1T cell receptor repertoire.

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Clonal expansion shapes the human V\u3b41T cell receptor repertoire.

Clonal expansion shapes the human V\u3b41T cell receptor repertoire.

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Archivio istituzionale della ricerca - Università di Palermo