Recasting Human Vδ1 Lymphocytes in an Adaptive Role
Authors: Martin S. Davey, Carrie R. Willcox, Alfie T. Baker, Stuart Hunter, Benjamin E. Willcox
Journal: Trends in Immunology (2018)

Vδ1 T cells are the predominant tissue-associated γδ T cell subset in humans, and can recognise signs of cellular dysregulation, including viral infection and transformation. They are often assumed to be innate-like effectors. The Vδ1 TCR repertoire is initially diverse, yet a few clonotypes typically expand heavily over time. Such expanded clonotypes are diverse in sequence both within and between individuals. Clonal expansion occurs concurrently with differentiation from Vδ1 Tnaïve to Vδ1 Teffector status, alongside a switch from lymphoid to peripheral homing receptors, and upregulation of cytotoxic pathways. This concurrent expansion and differentiation suggests an adaptive-like response, likely driven by diverse stimuli, including microbial pathogen infection. γδ T cells are unconventional lymphocytes commonly described as 'innate-like' in function, which can respond in both a T cell receptor (TCR)-independent and also major histocompatibility complex (MHC)-unrestricted TCR-dependent manner. While the relative importance of TCR recognition had remained unclear, recent studies revealed that human Vδ1 T cells display unexpected parallels with adaptive αβ T cells. Vδ1 T cells undergo profound and highly focussed clonal expansion from an initially diverse and private TCR repertoire, most likely in response to specific immune challenges. Concomitantly, they differentiate from a Vδ1 T cell naïve (Tnaïve) to a Vδ1 T cell effector (Teffector) phenotype, marked by the downregulation of lymphoid homing receptors and upregulation of peripheral homing receptors and effector markers. This suggests that an adaptive paradigm applies to Vδ1 T cells, likely involving TCR-dependent but MHC-unrestricted responses to microbial and non-microbial challenges. γδ T cells are unconventional lymphocytes commonly described as 'innate-like' in function, which can respond in both a T cell receptor (TCR)-independent and also major histocompatibility complex (MHC)-unrestricted TCR-dependent manner. While the relative importance of TCR recognition had remained unclear, recent studies revealed that human Vδ1 T cells display unexpected parallels with adaptive αβ T cells. Vδ1 T cells undergo profound and highly focussed clonal expansion from an initially diverse and private TCR repertoire, most likely in response to specific immune challenges. Concomitantly, they differentiate from a Vδ1 T cell naïve (Tnaïve) to a Vδ1 T cell effector (Teffector) phenotype, marked by the downregulation of lymphoid homing receptors and upregulation of peripheral homing receptors and effector markers. This suggests that an adaptive paradigm applies to Vδ1 T cells, likely involving TCR-dependent but MHC-unrestricted responses to microbial and non-microbial challenges. γδ T cells have coevolved alongside αβ T cells and B cells for at least the past ∼450 million years of vertebrate evolution [1Hayday A.C. γδ cells: a right time and a right place for a conserved third way of protection.Annu. Rev. Immunol. 2000; 18: 975-1026Crossref PubMed Scopus (956) Google Scholar, 2Hirano M. et al.Evolutionary implications of a third lymphocyte lineage in lampreys.Nature. 2013; 501: 435-438Crossref PubMed Scopus (160) Google Scholar], each distinguished by related but distinct somatically recombined antigen receptors. However, our understanding of these different lineages is strikingly imbalanced. Critical to our understanding of αβ T cell and B cells is the classical adaptive paradigm (Box 1). Within this, seminal discoveries have established the core function of the αβ T cell lineage: to enable immune responses to target cells based on the presence on their surface of antigenic peptide in the context of MHC molecules; similarly, we understand that B cells, which underpin humoral immunity, enable the production of soluble antibodies capable of recognising a diverse range of antigenic targets in native, 3D conformation. In keeping with Burnet's suggestion that 'receptor occupation' is key in driving the activation and clonal selection of adaptive lymphocytes [3Burnet F.M. A modification of Jerne's theory of antibody production using the concept of clonal selection.Aust. J. Sci. 1957; 20: 67-69Google Scholar], structural studies have confirmed both the involvement of clonotypically unique hypervariable loops in αβ TCR/peptide-MHC and B cell receptor (BCR)/antigen engagement, and the significance of such interactions in regulating multiple facets of their immunobiology (Box 1).Box 1Hallmarks of Classical Adaptive ImmunityNotably, αβ T cells and B cells share key hallmarks of classical adaptive immunity.Generation of a Diverse Antigen Receptor Repertoire and Tolerance MechanismsBoth αβ T cell and B cell lineages feature somatically recombined TCRs and BCRs, with repertoires featuring high diversity in their hypervariable complementarity-determining region loops, particularly CDR3. For both lineages, selection events during lymphocyte development are critical for immune tolerance. αβ T cells undergo positive and negative selection in the thymus; B cells, in the bone marrow, undergo both antigen-independent positive selection, based on tonic BCR signalling, and processes that eliminate or mitigate autoreactive specificities, including negative selection and anergy induction.Clonal Expansion from a Diverse Immune Receptor RepertoireThe selection of individual clonotypes from within the diverse naïve immune receptor repertoire allows expansion of specific αβ T cell and B cell clonotypes bearing receptors that critically enable amplified responses to specific immune challenges, such as pathogen infection.Differentiation into Long-Lived EffectorsConcurrent with clonal expansion, both αβ T cell and B cell lineages not only undergo differentiation to effectors, but also permit the maintenance of long-lived clonotypically expanded populations, enabling immunological memory, whereby faster and more potent immune responses are induced in response to secondary antigenic challenge.Critical Importance of Antigen Receptor–Ligand InteractionsDiverse studies highlight the central role for TCR–pMHC and BCR–ligand interactions in directing αβ T cell and B cell development, maintenance, clonal amplification and activation, and memory formation, emphatically validating the concept that 'receptor occupancy' is a central driver of adaptive lymphocyte biology. Notably, αβ T cells and B cells share key hallmarks of classical adaptive immunity. Generation of a Diverse Antigen Receptor Repertoire and Tolerance Mechanisms Both αβ T cell and B cell lineages feature somatically recombined TCRs and BCRs, with repertoires featuring high diversity in their hypervariable complementarity-determining region loops, particularly CDR3. For both lineages, selection events during lymphocyte development are critical for immune tolerance. αβ T cells undergo positive and negative selection in the thymus; B cells, in the bone marrow, undergo both antigen-independent positive selection, based on tonic BCR signalling, and processes that eliminate or mitigate autoreactive specificities, including negative selection and anergy induction. Clonal Expansion from a Diverse Immune Receptor Repertoire The selection of individual clonotypes from within the diverse naïve immune receptor repertoire allows expansion of specific αβ T cell and B cell clonotypes bearing receptors that critically enable amplified responses to specific immune challenges, such as pathogen infection. Differentiation into Long-Lived Effectors Concurrent with clonal expansion, both αβ T cell and B cell lineages not only undergo differentiation to effectors, but also permit the maintenance of long-lived clonotypically expanded populations, enabling immunological memory, whereby faster and more potent immune responses are induced in response to secondary antigenic challenge. Critical Importance of Antigen Receptor–Ligand Interactions Diverse studies highlight the central role for TCR–pMHC and BCR–ligand interactions in directing αβ T cell and B cell development, maintenance, clonal amplification and activation, and memory formation, emphatically validating the concept that 'receptor occupancy' is a central driver of adaptive lymphocyte biology. Originally identified serendipitously during studies defining αβ TCR genes [4Chien Y.H. et al.A new T-cell receptor gene located within the alpha locus and expressed early in T-cell differentiation.Nature. 1987; 327: 677-682Crossref PubMed Scopus (354) Google Scholar, 5Saito H. et al.A third rearranged and expressed gene in a clone of cytotoxic T lymphocytes.Nature. 1984; 312: 36-40Crossref PubMed Scopus (364) Google Scholar] γδ T cells have by contrast remained somewhat mysterious both in terms of the immunological niche they occupy and the key reason(s) for their evolutionary preservation as a third lymphocyte lineage within vertebrate immunity. Moreover, although γδ T cells are implicated in a range of immune settings, including antimicrobial immunity, antitumour immunity, and tissue homeostasis (reviewed in [6Vantourout P. Hayday A. Six-of-the-best: unique contributions of gammadelta T cells to immunology.Nat. Rev. Immunol. 2013; 13: 88-100Crossref PubMed Scopus (834) Google Scholar]), the central paradigms that govern their development and antigen recognition functions are unresolved. Finally, despite remaining a focus of ongoing interest, the closely related issue of the importance and exact role of γδ TCR occupation in γδ T cell biology remains a central question. One concept emerging from mouse studies of γδ T cells is that certain γδ T cell subsets, instead of functioning via conventional adaptive paradigms, may instead act as 'innate-like' lymphocytes. Notably, murine γδ T cells express distinct TCRγ and TCRδ combinations at different anatomical sites, and often display semi-invariant TCR repertoires, in some cases featuring highly restricted CDR3 regions [7Asarnow D.M. et al.Distinct antigen receptor repertoires of two classes of murine epithelium-associated T cells.Nature. 1989; 341: 60-62Crossref PubMed Scopus (202) Google Scholar, 8Asarnow D.M. et al.Limited diversity of gamma delta antigen receptor genes of Thy-1+ dendritic epidermal cells.Cell. 1988; 55: 837-847Abstract Full Text PDF PubMed Scopus (449) Google Scholar, 9Itohara S. et al.Homing of a gamma delta thymocyte subset with homogeneous T-cell receptors to mucosal epithelia.Nature. 1990; 343: 754-757Crossref PubMed Scopus (462) Google Scholar]. They can be preprogrammed during thymic development to differentiate into discrete effector populations producing either interleukin-17 (IL-17) or interferon-gamma (IFN-γ) [10Jensen K.D. et al.Thymic selection determines gammadelta T cell effector fate: antigen-naïve cells make interleukin-17 and antigen-experienced cells make interferon gamma.Immunity. 2008; 29: 90-100Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 11Turchinovich G. Hayday A.C. Skint-1 identifies a common molecular mechanism for the development of interferon-gamma-secreting versus interleukin-17-secreting gammadelta T cells.Immunity. 2011; 35: 59-68Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar]. More recently, intra-epithelial lymphocyte populations have been shown to be selected in tissues after birth, dependent on the expression of particular butyrophilin-like molecules (BTNLs) [12Di Marco Barros R. et al.Epithelia use butyrophilin-like molecules to shape organ-specific gammadelta T cell compartments.Cell. 2016; 167 (e217): 203-218Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar]. Such populations of 'activated-but-resting' unconventional lymphocytes are thought to be capable of reacting directly to dysregulated target cells without the need for clonal expansion and differentiation. These data align with the idea such subsets may recognise a limited range of host-encoded stress ligands [13Janeway Jr., C.A. et al.Specificity and function of T cells bearing gamma delta receptors.Immunol. Today. 1988; 9: 73-76Abstract Full Text PDF PubMed Scopus (502) Google Scholar], and suggest that their TCRs act like surrogate pattern recognition receptors (PRRs) for molecular signals of microbial/non-microbial stress. In humans, the γδ T cell subset that aligns most clearly to this biology is characterised by a Vγ9/Vδ2 chain pairing, and represents the predominant peripheral blood subset (1–10% of T cells) [14De Libero G. et al.Selection by two powerful antigens may account for the presence of the major population of human peripheral gamma/delta T cells.J. Exp. Med. 1991; 173: 1311-1322Crossref PubMed Scopus (189) Google Scholar]. Based on their restricted TCR Vγ and Vδ gene segment usage and CDR3 lengths, presence of common CDR3 motifs, foetal generation, polyclonal production of IFNγ and Tumour Necrosis Factor-α (TNFα) following exposure to pyrophosphate antigens (P-Ags), and strong dependency of TCR-mediated recognition on the BTN3A1 Ig-like protein, this subset arguably conforms to such an innate-like functional paradigm, although the exact mechanisms underlying its recognition of target cells remain unclear. To some extent, the features of these γδ T cell subsets mirror those of unconventional αβ T cell subsets [e.g., mucosal-associated invariant T cells (MAITs) and invariant natural killer cells (iNKTs)], which also feature highly restricted TCR repertoires [15Dellabona P. et al.An invariant V alpha 24-J alpha Q/V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8− T cells.J. Exp. 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Gammadelta T cells and the lymphoid stress-surveillance response.Immunity. 2009; 31: 184-196Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar], which postulates that such effectors, by circumventing the requirement for clonal selection and differentiation, may provide protection from microbial or non-microbial stress challenges during the initial phase of the response, before adaptive immune responses have been generated. A key finding in this area has been that such subsets can be activated not only via their TCR, but also independently by TCR-extrinsic signals. For example, mouse dendritic epidermal T cells (DETC, a subset of γδ T cells present in murine skin) can be activated directly via NK receptor (NKR)-mediated recognition of stress ligands expressed on stressed epithelium, independently of the TCR [18Strid J. et al.Acute upregulation of an NKG2D ligand promotes rapid reorganization of a local immune compartment with pleiotropic effects on carcinogenesis.Nat. Immunol. 2008; 9: 146-154Crossref PubMed Scopus (206) Google Scholar]. Moreover, while human Vγ9/Vδ2 T cells exhibit potent TCR-dependent recognition of P-Ag-exposed target cells, they can also be activated by NKG2D–ligand interactions and are responsive to cytokines such as IL-12/IL-18 [19Garcia V.E. et al.IL-15 enhances the response of human gamma delta T cells to nonpeptide [correction of nonpetide] microbial antigens.J. Immunol. 1998; 160: 4322-4329PubMed Google Scholar, 20Garcia V.E. et al.IL-18 promotes type 1 cytokine production from NK cells and T cells in human intracellular infection.J. Immunol. 1999; 162: 6114-6121PubMed Google Scholar, 21Rincon-Orozco B. et al.Activation of V gamma 9V delta 2 T cells by NKG2D.J. Immunol. 2005; 175: 2144-2151Crossref PubMed Scopus (236) Google Scholar]. In addition, recent studies on mouse skin and gut γδ T cell subsets suggest that TCR signals, potentially mediated via interactions with BTNLs, are required for the development, homing, and establishment of their effector program [11Turchinovich G. Hayday A.C. Skint-1 identifies a common molecular mechanism for the development of interferon-gamma-secreting versus interleukin-17-secreting gammadelta T cells.Immunity. 2011; 35: 59-68Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 12Di Marco Barros R. et al.Epithelia use butyrophilin-like molecules to shape organ-specific gammadelta T cell compartments.Cell. 2016; 167 (e217): 203-218Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar]; however, these cells can become hyporesponsive to TCR signals and function in an TCR-independent manner to respond to signs of cellular stress [22Wencker M. et al.Innate-like T cells straddle innate and adaptive immunity by altering antigen-receptor responsiveness.Nat. Immunol. 2014; 15: 80-87Crossref PubMed Scopus (156) Google Scholar]. In addition to Vγ9/Vδ2 T cells, a second human γδ T cell compartment exists, bearing Vδ2-negative TCRs, of which the Vδ1 component is dominant. Vδ1 T cells are the most prevalent subtype of γδ T cells at birth [23Morita C.T. et al.TCR usage and functional capabilities of human gamma delta T cells at birth.J. Immunol. 1994; 153: 3979-3988PubMed Google Scholar], and the dominant γδ T cell subtype in peripheral tissues in adults, such as the gut [24Deusch K. et al.A major fraction of human intraepithelial lymphocytes simultaneously expresses the gamma/delta T cell receptor, the CD8 accessory molecule and preferentially uses the V delta 1 gene segment.Eur. J. Immunol. 1991; 21: 1053-1059Crossref PubMed Scopus (233) Google Scholar, 25Halstensen T.S. et al.Intraepithelial T cells of the TcR gamma/delta+ CD8- and V delta 1/J delta 1+ phenotypes are increased in coeliac disease.Scand. J. Immunol. 1989; 30: 665-672Crossref PubMed Scopus (300) Google Scholar] and skin [26Bos J.D. et al.T-cell receptor gamma delta bearing cells in normal human skin.J. Invest. Dermatol. 1990; 94: 37-42Abstract Full Text PDF PubMed Scopus (90) Google Scholar]. Vδ1 T cells have remained very much an enigma in terms of the fundamental paradigms underlying their biology. Based on their predominant effector phenotype, their potent cytotoxicity/cytokine production, combined with their ability to recognise both virally infected and also cancerous cells, and their expression of NKRs [27Halary F. et al.Shared reactivity of V{delta}2(neg) {gamma}{delta} T cells against cytomegalovirus-infected cells and tumor intestinal epithelial cells.J. Exp. Med. 2005; 201: 1567-1578Crossref PubMed Scopus (189) Google Scholar, 28Lafarge X. et al.Expression of MHC class I receptors confers functional intraclonal heterogeneity to a reactive expansion of gammadelta T cells.Eur. J. Immunol. 2005; 35: 1896-1905Crossref PubMed Scopus (25) Google Scholar, 29Pitard V. et al.Long-term expansion of effector/memory Vdelta2-gammadelta T cells is a specific blood signature of CMV infection.Blood. 2008; 112: 1317-1324Crossref PubMed Scopus (154) Google Scholar], they have often been assumed to act in an innate-like fashion, similar to NK cells, potentially enabling recognition of diverse cellular stress signals in target cells. However, here we review recent data that have revised this picture, and suggest instead that Vδ1 T cells exhibit a radical new adaptive immunobiology. These data highlight some of the most significant questions in γδ T cell biology, including the importance and exact role of the γδ TCR that defines the lineage, but ironically, is so poorly understood. The advent of next-generation sequencing (NGS) approaches has allowed in-depth analyses of the TCR/BCR repertoire within the αβ and B cell lineage, respectively [30Woodsworth D.J. et al.Sequence analysis of T-cell repertoires in health and disease.Genome Med. 2013; 5: 98Crossref PubMed Scopus (122) Google Scholar, 31Yaari G. Kleinstein S.H. Practical guidelines for B-cell receptor repertoire sequencing analysis.Genome Med. 2015; 7: 121Crossref PubMed Scopus (136) Google Scholar]. Furthermore, application of these technologies to human peripheral blood γδ T cells has provided valuable information on clonal evolution within the γδ T cell lineage. The approaches used include either DNA-based or RNA-based methods, with the former highlighting the requirement to purify γδ T cells from αβ subsets to avoid contamination of TCRγ sequences recombined in mature αβ T cells [32Sherwood A.M. et al.Deep sequencing of the human TCRgamma and TCRbeta repertoires suggests that TCRbeta rearranges after alphabeta and gammadelta T cell commitment.Sci. Transl. Med. 2011; 3: 90ra61Crossref PubMed Scopus (106) Google Scholar]. One inherent challenge of these methods is the potential for polymerase chain reaction (PCR) and sequence errors [33Heather J.M. et al.High-throughput sequencing of the T-cell receptor repertoire: pitfalls and opportunities.Brief. Bioinform. 2017; 2017: bbw138Crossref Scopus (67) Google Scholar]. In the absence of in silico error correction and appropriate data handling, this can result in retention of erroneous sequences, often resulting from single-base errors of other higher frequency clonotypes. Therefore, caution should be applied when interpreting highly similar TCR base sequences. The availability of public software packages dedicated to the interpretation of NGS TCR sequencing data (MiTCR [34Bolotin D.A. et al.MiTCR: software for T-cell receptor sequencing data analysis.Nat. Methods. 2013; 10: 813-814Crossref PubMed Scopus (141) Google Scholar], MiXCR [35Bolotin D.A. et al.MiXCR: software for comprehensive adaptive immunity profiling.Nat. Methods. 2015; 12: 380-381Crossref PubMed Scopus (748) Google Scholar], and TcR [36Nazarov V.I. et al.tcR: an R package for T cell receptor repertoire advanced data analysis.BMC Bioinform. 2015; 16: 175Crossref PubMed Scopus (159) Google Scholar]), which provide robust error correction, should alleviate such challenges. Moreover, since RNA-based approaches are potentially vulnerable to bias, based on the overall level of RNA differing between cells in different activation and/or differentiation states, ideally parallel techniques, such as single-cell PCR-based TCR analysis, should be considered to validate NGS-based TCR frequencies correlate with cell number on a specific platform [37Guo X.Z. et al.Rapid cloning, expression, and functional characterization of paired alphabeta and gammadelta T-cell receptor chains from single-cell analysis.Mol. Ther. Methods Clin. Dev. 2016; 3: 15054Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 38Davey M.S. et al.Clonal selection in the human Vdelta1 T cell repertoire indicates gammadelta TCR-dependent adaptive immune surveillance.Nat. Commun. 2017; 8: 14760Crossref PubMed Scopus (150) Google Scholar, 39Ravens S. et al.Human gammadelta T cells are quickly reconstituted after stem-cell transplantation and show adaptive clonal expansion in response to viral infection.Nat. Immunol. 2017; 18: 393-401Crossref PubMed Scopus (156) Google Scholar]. Such studies have not only shed substantial light on Vδ1 T cell immunobiology, but equally importantly, have also highlighted features of the Vδ1 repertoire that contrast markedly with that of both peripheral blood Vγ9/Vδ2 T cells and thymically programmed mouse γδ T cells. First, based on analyses of cord blood samples, the Vδ1 TCR repertoire at the start of life, which features a variety of Vγ chain pairings, is clonotypically diverse, featuring a range of CDR3 lengths apparently unrelated in sequence. Importantly, these neonatal cord blood Vδ1+ repertoires are essentially entirely unfocussed: in other words, it appears that 'all clonotypes are created equal', with no single sequence exceeding 1–2% of the repertoire (Figure 1). This contrasts with the Vγ9/Vδ2 population, which, even in foetal and cord blood, includes relatively prevalent Vγ9-JγP clonotypes that are public (i.e., shared at either the nucleotide or amino acid level between individuals) and are present throughout life, consistent with selection of a semi-invariant repertoire preprogrammed in development for polyclonal P-Ag recognition [32Sherwood A.M. et al.Deep sequencing of the human TCRgamma and TCRbeta repertoires suggests that TCRbeta rearranges after alphabeta and gammadelta T cell commitment.Sci. Transl. Med. 2011; 3: 90ra61Crossref PubMed Scopus (106) Google Scholar, 40Cairo C. et al.Impact of age, gender, and race on circulating gammadelta T cells.Hum. Immunol. 2010; 71: 968-975Crossref PubMed Scopus (38) Google Scholar, 41Cairo C. et al.Human cord blood gammadelta T cells expressing public Vgamma2 chains dominate the response to bisphosphonate plus interleukin-15.Immunology. 2013; 138: 346-360Crossref PubMed Scopus (18) Google Scholar, 42Dimova T. et al.Effector Vgamma9Vdelta2 T cells dominate the human fetal gammadelta T-cell repertoire.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E556-565Crossref PubMed Scopus (137) Google Scholar]. A second important finding from such studies is that, in comparison to cord blood repertoires, adult Vδ1 repertoires are in general substantially more focussed, typically resulting from the presence of a relatively small number (e.g., <5) of heavily expanded clonotypes, and which often account for a large proportion of the total adult Vδ1 repertoire. Of note, parallel single-cell TCR analysis was used by Davey et al. [38Davey M.S. et al.Clonal selection in the human Vdelta1 T cell repertoire indicates gammadelta TCR-dependent adaptive immune surveillance.Nat. Commun. 2017; 8: 14760Crossref PubMed Scopus (150) Google Scholar] and Ravens et al. [39Ravens S. et al.Human gammadelta T cells are quickly reconstituted after stem-cell transplantation and show adaptive clonal expansion in response to viral infection.Nat. Immunol. 2017; 18: 393-401Crossref PubMed Scopus (156) Google Scholar] to confirm that these represented genuine numerical clonal expansions, and were not biased by RNA abundance. Strikingly, both adult and cord blood Vδ1+ TCR repertoires were overwhelmingly private (i.e., unique to an individual at both a nucleotide and amino acid level; even more so than TCRβ [38Davey M.S. et al.Clonal selection in the human Vdelta1 T cell repertoire indicates gammadelta TCR-dependent adaptive immune surveillance.Nat. Commun. 2017; 8: 14760Crossref PubMed Scopus (150) Google Scholar]), with the Complementarity determining region 3 (CDR3) lengths of expanded clonotypes highly diverse, and their sequences apparently unrelated, both within and between individuals [38Davey M.S. et al.Clonal selection in the human Vdelta1 T cell repertoire indicates gammadelta TCR-dependent adaptive immune surveillance.Nat. Commun. 2017; 8: 14760Crossref PubMed Scopus (150) Google Scholar]. These features appeared to stem from the addition of high levels of nontemplated (N) nucleotides (introduced by terminal deoxynucleotidyl transferase) or occasionally palindromic (P) nucleotides (a mean of 19 N/P nucleotides for Vδ1 CDR3) during variable (diversity) joining [V(D)J] recombination. Both the private nature of the Vδ1 repertoire and the presence of such dominant clonal expansions contrast markedly with the Vγ9/Vδ2 TCR repertoire, which displays less pronounced focussing and contains several public Vγ9 sequences in cord blood and adults [32Sherwood A.M. et al.Deep sequencing of the human TCRgamma and TCRbeta repertoires suggests that TCRbeta rearranges after alphabeta and gammadelta T cell commitment.Sci. Transl. Med. 2011; 3: 90ra61Crossref PubMed Scopus (106) Google Scholar, 38Davey M.S. et al.Clonal selection in the human Vdelta1 T cell repertoire indicates gammadelta TCR-dependent adaptive immune surveillance.Nat. Commun. 2017; 8: 14760Crossref PubMed Scopus (150) Google Scholar, 41Cairo C. et al.Human cord blood gammadelta T cells expressing public Vgamma2 chains dominate the response to bisphosphonate plus interleukin-15.Immunology. 2013; 138: 346-360Crossref PubMed Scopus (18) Google Scholar]. Moreover, compared with Vδ1 T cells, the Vγ9/Vδ2 T cell repertoire features restricted CDR3γ and CDR3δ lengths, with CDR3δ2 sequences generally considerably shorter than CDR3δ1 sequences. Similarly, TCRαβ repertoires also display CDR3 length restriction, which is likely imposed by the structural constraints of peptide–MHC recognition [43Rock E.P. et al.CDR3 length in antigen-specific immune receptors.J. Exp. Med. 1994; 179: 323-328Crossref PubMed Scopus (436) Google Scholar]. γδ TCR repertoire analyses have also highlighted factors driving such clonal expansions. In particular, Ravens et al. showed that acute cytomegalovirus (CMV) infection following stem cell transplantation (SCT) can drive expansion of Vδ2neg (predominantly Vδ1) TCR clonotypes [39Ravens S. et al.Human gammadelta T cells are quickly reconstituted after stem-cell transplantation and show adaptive clonal expansion in response to viral infection.Nat. Immunol. 2017; 18: 393-401Crossref PubMed Scopus (156) Google Scholar]; Vδ repertoires were also noted to be private. These findings build on numerous studies highlighting the importance of Vδ2-negative T cells in responses to CMV infection following kidney transplantation and in healthy donors [29Pitard V. et al.Long-term expansion of effector/memory Vdelta2-gammadelta T cells is a specific blood signature of CMV infection.Blood. 2008; 112: 1317-1324Crossref PubMed Scopus (154) Google Scholar, 44Dechanet J. et al.Implication of gammadelta T cells in the human immune response to cytomegalovirus.J. Clin. Invest. 1999; 103: 1437-1449Crossref PubMed Scopus (268) Google Scholar, 45Lafarge X. et al.Cytomegalovirus infection in transplant recipients resolves when circulating gammadelta T lymphocytes expand, suggesting a protective antiviral role.J. Infect. Dis. 2001; 1