The distinct MHC‐unrestricted immunobiology of innate‐like and adaptive‐like human γδ T cell subsets—Nature's CAR‐T cells

Distinct innate‐like and adaptive‐like immunobiological paradigms are emerging for human γδ T cells, supported by a combination of immunophenotypic, T cell receptor (TCR) repertoire, functional, and transcriptomic data. Evidence of the γδ TCR/ligand recognition modalities that respective human subsets utilize is accumulating. Although many questions remain unanswered, one superantigen‐like modality features interactions of germline‐encoded regions of particular TCR Vγ regions with specific BTN/BTNL family members and apparently aligns with an innate‐like biology, albeit with some scope for clonal amplification. A second involves CDR3‐mediated γδ TCR interaction with diverse ligands and aligns with an adaptive‐like biology. Importantly, these unconventional modalities provide γδ T cells with unique recognition capabilities relative to αβ T cells, B cells, and NK cells, allowing immunosurveillance for signatures of "altered self" on target cells, via a membrane‐linked γδ TCR recognizing intact non‐MHC proteins on the opposing cell surface. In doing so, they permit cellular responses in diverse situations including where MHC expression is compromised, or where conventional adaptive and/or NK cell‐mediated immunity is suppressed. γδ T cells may therefore utilize their TCR like a cell‐surface Fab repertoire, somewhat analogous to engineered chimeric antigen receptor T cells, but additionally integrating TCR signaling with parallel signals from other surface immunoreceptors, making them multimolecular sensors of cellular stress.

and antigen recognition requirements of the γδ T cell compartment have remained substantially unclear.
γδ T cells are proposed to perform diverse functions in immunity, including critical contributions to protection against bacteria and viruses, immunosurveillance against tumors, and immunoregulation and maintenance of epithelial surfaces. 5 However, a lack of understanding of both their fundamental immunobiology and the antigens they recognize through their γδ TCR has hampered our understanding of how γδ T cells perform these functions. Advances in a diverse range of techniques are now catalyzing discoveries in both of these areas. Such efforts should clarify our understanding of the niche γδ T cells occupy in the immune system and potentially explain why they have been retained throughout vertebrate evolution. A granular understanding of functional heterogeneity of the γδ T cell compartment, and increased knowledge of both γδ TCR ligands and mechanisms of antigen recognition, is also likely to be important in facilitating therapeutic exploitation of γδ T cells, a focus of increasing interest.
In this review, we will summarize some of our own and others' recent research findings in this context, which have shed light on fundamental immunobiological paradigms and novel γδ TCR ligands. Our focus will largely be on human γδ T cells but we include reference to work on other species where relevant. The picture that emerges is one of a compartment comprising functionally distinct subsets, collectively providing immunosurveillance capabilities that are distinct from αβ T cells, B cells, and Natural Killer (NK) cells, mediated by the γδ TCR, enabling fragment antigen binding (Fab)-like recognition of intact, non-MHC antigens on the surface of target cells.

| PAR ADIG MS
In recent years, there has been a drive to understand the paradigms that best apply to human γδ T cells, and consequently their niche in the immune system relative to other lymphocyte compartments.
A major focus of discussion has been whether γδ T cells align best with innate or adaptive biology. Although γδ T cells have often been viewed or analyzed en masse as a homogenous unconventional lymphocyte compartment, and typically referred to as either "innate-like T cells" or "at the interface between innate and adaptive immunity," recent studies clearly demonstrating considerable intracompartmental functional and phenotypic heterogeneity 6-10 reveal this assessment is out of date. In fact, multiple immunobiological paradigms may likely be required to describe the compartment, such is this heterogeneity, particularly when different γδ T cell subsets but also different species are taken into account. This would mirror the αβ T cell compartment, which contains both innate-like 11 and conventional adaptive subsets.
The recurring question of innate versus adaptive immunobiology remains highly relevant to paradigms invoked for human γδ T cell subsets. However, since these terms are used in different senses by different researchers, it is important to define our own usage of these terms here. We employ "adaptive-like" and "innate-like" to refer to a collection of features. Hence, adaptive-like immunobiology typically involves (a) diversity within an initial immune receptor repertoire, which provides potential for subsequent antigen-driven clonotypic focussing; notably αβ T cell and B cell antigen receptor diversity enables a diversity of targets to be recognized; (b) lymphocytes adopt an initial naïve phenotypic status prior to a given challenge, but transition upon challenge to effector status, and have the potential to impart altered homing capability; (c) this transition to effector status can occur throughout life, whenever antigen is en- Although such features may not all apply to a given unconventional T cell subset, as outlined below, specific γδ T cell subsets do appear substantially polarized toward either innate-like or adaptive-like biology. Of note, the ability to delineate such distinct immunobiologies is ideally addressed by a multimodal approach, enabling parallel characterization of immunophenotype, TCR repertoire, cellular function, and potentially transcriptional profile. New advances in single-cell methodologies mean it is possible to assess some such features in parallel. Therefore, the distinction between innate-like and adaptive-like paradigms remains an important and useful one, is increasingly feasible to assess, and provides a useful framework through which to investigate the contribution of such functionally distinct subsets to host defense, and ultimately to consider therapeutic avenues.

| Innate-like Vγ9Vδ2 T cells
Vγ9Vδ2 T cells, which are the dominant γδ T cell population in human peripheral blood, currently represent the strongest candidate for an innate-like human γδ T cell subset. Although rare and phenotypically naïve (CD27 + CD45RA + ) in cord blood, they expand in number and transition to a CD45RO + phenotype in early childhood, 6,12 upregulating expression of granzymes and perforin 6 to become fully mature effector cells relatively early in life, 13 most probably driven by microbial exposure. 14,15 Crucially, mature effector Vγ9Vδ2 T cells universally respond in a TCR-dependent fashion to host and pathogen-derived phosphoantigens (pAgs), which include the highly potent microbially derived compound (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP). 16 An intermediate in the non-mevalonate pathway of isoprenoid synthesis absent in mammals, 16 HMBPP is an essential metabolite for many pathogenic bacteria and could be arguably regarded as a pathogenassociated molecular pattern (PAMP). Recognition of pAg-exposed target cells can result in potent cytokine secretion and cytotoxicity.
The Vγ9Vδ2 T cell repertoire in the peripheral blood of most adults consists of semi-invariant Vγ9 chains with relatively minimal nontemplated (N)/pallindromic (P) region nucleotide addition, paired with relatively diverse Vδ2 chains. 6,7 In some cases, the same Vγ9 rearrangement is detected paired to multiple Vδ2 sequences, suggesting several common Vγ9 chain sequences can be recombined multiple times independently. 6,15 The fact that Vγ9Vδ2 T cells respond universally to pAgs and that both Vγ9 and Vδ2 chains are essential for this TCR-dependent reactivity, strongly suggests recognition of non-polymorphic ligands, one of which has recently been established as BTN2A1. 17,18 Along with their semi-invariant TCR repertoire, Vγ9Vδ2 T cells exhibit transcriptional similarities to invariant Natural Killer T cells (iNKT) and mucosal associated invariant T cells (MAIT) cells, such as expression of the transcription factor PLZF (encoded by the ZBTB16 gene), 10,19 a hallmark of innate-like lymphocytes. 20 Notably, Vγ9Vδ2 T cells can also be activated in a TCR-independent manner by IL-12/IL-18 stimulation, 6 another hallmark of innate-like lymphocytes; notably, this ability is not shared by human adaptive-like γδ T cells. 6,7 Although the features outlined above illustrate that Vγ9Vδ2 T cells adopt a broadly innate-like biology featuring Vγ9-JγP rearrangements and hydrophobic CDR3δ residues that are essential for pAg recognition, it is clear that their semi-invariant TCR repertoire allows the potential for clonal expansions and phenotypic differences in some individuals. 6 These are currently poorly understood but conceivably may relate to differences in CDR3-mediated antigen discrimination (see below) and are worthy of further investigation. Such clonal expansions may relate to phenotypic heterogeneity observed 10 in Vγ9Vδ2 T cells across donors and are also consistent with the findings that both CDR3γ and the highly diverse CDR3δ region of the Vγ9Vδ2 TCR are critical for pAg recognition. Moreover, phenotypic and clonotypic changes are likely to relate to either the current or past infection state, and consistent with this, Vγ9Vδ2 T cells have been shown to exhibit long-lived expansions in response to several pathogens. 16 These observations emphasize that the immunobiology of Vγ9Vδ2 T cells is best described as "innate-like" rather than "innate" and may in reality reflect a blend of a limited number of characteristics more typically associated with adaptive immunity, with numerous innate features.
Irrespective of which terminology is applied to this subset, it is clear the immunobiology of the Vγ9Vδ2 T cell subset is fundamentally different to those of "adaptive-like" subsets outlined below.

| Adaptive-like subsets
Vδ2 neg γδ T cells represent a minority (normally around 20%) of γδ T cells in peripheral blood, but are enriched relative to Vγ9Vδ2 T cells in solid tissues. In both settings, Vδ1 + T cells typically comprise the dominant proportion. Previous studies have highlighted the involvement of the Vδ2 neg subset in response to several viral infections, including cytomegalovirus (CMV), 21 human immunodeficiency virus (HIV), 22 hepatitis C virus (HCV), 23 and Epstein-Barr virus (EBV). 24,25 In the context of CMV, studies have highlighted CMV-driven expansions, 26,27 both in immunocompromised settings and healthy individuals. 28 In immunosuppressed patients, Vδ2 neg T cell expansions were concomitant with the start of resolution of infection, 29 and ex vivo analyses revealed Vδ2 neg cytokine and cytotoxicity in response to CMV-infected target cells, and suppression of viral infectivity in vitro. 27 Despite this extensive body of work, until recently the fundamental immunobiology of Vδ2 neg T cells remained substantially unclear. However, compelling evidence now suggests that human Vδ2 neg T cells, at least in peripheral blood, display an adaptive-like immunobiology. 30 Recent studies on Vδ2 neg T cells have shed considerable light on both their TCR repertoire, phenotype, and how these link with cellular function. Based on analyses of cord blood γδ T cells, studies have shown that at the start of life Vδ2 neg TCR repertoire is initially highly diverse and features numerous Vγ chains, unlike pAg responsive Vδ2 T cells, which uniformly express Vγ9 chains with limited sequence diversity. 6,7 There is extremely high diversity within Vδ2 neg TCR CDR3 sequences, in both TCRγ and TCRδ chains, owing to a high level of exonuclease activity, a large number of N/P nucleotide additions, and for the Vδ chain, multiple Dδ-segment usage. 7 The resultant Vδ2 neg TCR repertoire incorporates Vγ and Vδ CDR3 loops that are highly diverse in length and sequence, but is also overwhelmingly private, 7 in stark contrast to the semi-invariant repertoire of Vγ9Vδ2 T cells, which has restricted CDR3 lengths and incorporates a large proportion of public Vγ9 clonotypes. 31 Importantly, by adulthood, the peripheral blood Vδ2 neg TCR repertoire frequently contains dominant clonal expansions. While these can occur in response to CMV infection following SCT, 32 they are also detected in healthy CMV-seropositive and CMV-seronegative individuals, 7 suggesting multiple immune stimuli, most probably other infectious challenges, can induce clonal expansion within the compartment. While the full range of infections that can drive such clonal expansions is unclear, available data suggest these may also include viral infections such as EBV, 24,25 as well as parasitic infections such as P. falciparum. 33 Importantly, Vermijlen and colleagues have shown fetal responses to congenital CMV infection in utero were also heavily clonotypically focussed. 34 However, substantial differences have emerged in the underlying mechanisms in the fetal setting, as the dominant Vγ8Vδ1 TCRs exploited near-germline-encoded CDR3 regions, in contrast to postnatal responses. Subsequent work has suggested this highly TCR focussed prenatal response reflects low fetal thymic expression of terminal deoxynucleotidyl transferase (TdT), resulting in a low diversity TCR repertoire, and unlike postnatal Vδ1 responses involved intrathymic acquisition of effector function. 35 Assessment of Vδ2 neg subset phenotype also provides evidence of adaptive features. The dominant Vδ1 pool displays a naïve phenotype in cord blood alongside its unfocussed TCR repertoire. 7 By adulthood, there are clearly distinct T naive and T effector states evident in the Vδ1 compartment, with considerable inter-individual variation in the relative dominance of each. 7 This contrasts with the Vγ9Vδ2 T cell compartment, which is effector like from an early age. 13 Phenotypically Vδ1 T naive and T effector states appear to align relatively closely with CD8 T naive and T emra subtypes, respectively. 7 36 In addition, available evidence suggests that expanded clonotypes present within the T effector pool are relatively long-lived and can persist for years. 6,7,32 The considerations above indicate remarkable parallels between adaptive-like human γδ T cell populations and CD8 T cell biology, and are suggestive of an analogous but distinct branch of unconventional adaptive immunobiology. Clearly, the transition from T naive to T effector status in such subsets is not inevitable, but likely dependent upon environmental exposure to specific challenges including infection, as is well established for conventional adaptive αβ and B cell compartments. Further studies are required to explore the range of infectious and non-microbial stimuli that might drive such responses. Of strong current relevance, a recent study of COVID-19 patients determined higher levels of CD27 neg Vδ1 + T cells in peripheral blood, suggestive of an adaptive γδ T cell response to SARS-CoV2. 37 Based on homing marker expression, it is highly likely that T naive cells can recirculate between blood and secondary lymphoid organs, whereas T effector populations are likely equipped to migrate into tissues 30 (see below). Also, the long-term persistence of expanded adaptive-like γδ TCR clonotypes, combined with retention of sensitivity to CD3 stimulation in peripheral blood, strong upregulation of cytotoxicity marker expression, and cytokine production capability, suggests the probability that T effector populations make long-term contributions to antigen-specific immunosurveillance against recurrently encountered or chronically persistent pathogens. 21,30,31 While in general the alignment between such subsets and mouse γδ T cell subsets is unclear, of note, CMV challenge in mice led to expansion of γδ T cells that populated a range of peripheral tissues and protected from disease pathology. 38,39 Although the processes governing adaptive differentiation from T naive to T effector status in γδ T cells are poorly understood, the strong link between clonal expansion and transition to T effector state suggests that like CD8 T cells, TCR triggering most likely plays a key role. As outlined below, this has implications for γδ TCR ligand identification approaches.
Additionally, the transcriptional pathways that regulate this unconventional adaptive differentiation process are poorly understood and must be addressed by future studies.
In summary, recent studies on human peripheral blood γδ T cells have indicated remarkable parallels between Vδ1 + and Vγ9 neg Vδ2 subsets and conventional adaptive CD8 T cell populations. Important data are now emerging that suggest how human tissue-associated γδ T cells may integrate with such populations.

| Tissue-associated human γδ T cells 2.2.1 | Toward a holistic understanding of the human γδ T cell compartment
While Vγ9Vδ2 T cells are the predominant γδ T cell subset in peripheral blood, in contrast Vδ2 neg T cells, and especially Vδ1 + T cells, are dominant in solid tissues. 40 However, our understanding of such human tissue-associated γδ T cells lags far behind that of peripheral blood populations. Also, our knowledge of the interconnection between (and reciprocally, the divergence of) γδ T cell functionality and responses in blood versus tissues is currently poor. Nevertheless, a number of studies have made important advances for several human solid tissues. One of the first tissue-associated γδ T cell subsets to be heavily studied were dendritic epidermal T cells (DETC), a murine γδ subset localized to epidermis. 41 Although absent in humans, they are important to consider briefly as a prototypic tissue-associated γδ T cell subset.
DETC are the first γδ T cells to develop in the mouse thymus during embryonic development, and subsequently home to epidermis. 41 DETC appear highly specialized for their local microenvironmental niche, and as their name suggests adopt a dendritic shape, allowing their many processes to contact nearby keratinocytes, thereby enabling immunosurveillance of the epithelium for signs of damage or infection 41 ( Figure 1A). DETC are not only self-renewing, but are also potent effectors, which express cytokines and cytotoxic markers. These effector functions can be elicited by TCR-dependent triggering, but also by TCR-independent signals, the latter including ligation of NKG2D on their surface by target cell-expressed NKG2D ligands (NKG2DL). 42,43 They can kill infected and tumor cells, yet can also produce growth factors such as KGF and IGF-1 44,45 and other molecules to support epithelial maintenance and wound healing. A striking feature of DETC is their universal expression of an invariant Vγ5Vδ1 TCR 46 with no junctional diversity, which is likely to be critical for thymic selection, regulating skin homing, and cutaneous immunosurveillance. These functions are dependent on the B7-like molecule Skint1. 47 In summary, multiple features of DETC suggest they represent a canonical innate-like tissue-associated subset, against which γδ T cells in different sites, as discussed below, can be compared.

| Hepatic γδ T cells
A recent study focussed on the phenotype, TCR repertoire and function of human hepatic γδ T cells ( Figure 1B), which were dominated by Vδ2 neg T cells, in relation to comparable peripheral blood subsets. 9 Although it was perhaps not surprising that there were considerable similarities between the two compartments given the strong link between the liver and the peripheral circulation, there were nevertheless some telling distinctions. Liver γδ T cells were dominated by Vδ1 + and Vδ3 + lymphocytes paired with diverse Vγ chains, and as for peripheral blood Vδ2 neg T cells the associated TCR repertoire was highly private, with evidence for extensive exonuclease activity, N/P nucleotide addition, and use of multiple Dδ segments. 9 However, unlike peripheral blood (PB) Vδ2 neg T cells, hepatic Vδ2 neg subsets were invariably highly clonally expanded (irrespective of whether the liver was normal or diseased), with dominance of a relatively small number of prevalent clonotypes. 9 Moreover, CD27 hi T naive Vδ2 neg cells were largely absent in the liver, with the majority of Vδ2 neg cells adopting a CD45RA hi /CD27 lo/neg phenotype that matches that of peripheral blood Vδ2 neg T effector cells. 9 These observations suggest that such populations are probably adaptive-like T effector cells that were originally centrally primed and expanded from antigen-inexperienced T naive cells, with changes in homing marker expression following adaptive differentiation probably critical in enabling hepatic homing. 9 Strongly supporting this, not only did such CD45RA hi /CD27 lo/neg T cells express CX 3 CR1 as well as granzymes and perforin, but intra-individual single cell TCR analyses indicated substantial overlap in expanded TCR clonotypes present in matched liver/blood samples. 9 F I G U R E 1 Tissue-associated γδ T cell subsets and their described functional capabilities in health and disease. A, Dendritic epidermal T cells (DETC) produce IGF-1 and KGF, thought to function in epithelial maintenance and in wound healing, but can also respond to increased expression of NKG2DL on keratinocytes. B, Two populations of Vδ2 neg γδ T cells can be found in the liver: a CD45RA + circulating population similar to peripheral blood Vδ2 neg cells, and a CD45RA lo potentially liver resident population. C, Vγ4Vδ1 + IELs in human may contribute to epithelial maintenance, while cytotoxicity and NKR-responsivity may be important in infection or tumorigenesis. D, Proposed γδ subsets in human skin compartments may express distinct TCR chains based on studies of normal γδ T cells and γδ cutaneous T cell lymphoma cells from dermis/epidermis and subcutaneous fat layers. A second study also highlighted the presence of Vγ9 neg Vδ2 T cells in the liver, 6 which like hepatic Vδ1 + T cells also incorporate a diverse range of Vγ chains. In stark contrast to the comparable Vγ9 neg Vδ2 subset in the blood of most healthy individuals, hepatic Vγ9 neg Vδ2 lymphocytes adopted a T effector -like phenotype 6 that resembles both the majority of hepatic Vδ1 + T cells, 7 and T effector Vγ9 neg Vδ2 populations that arise after viral infection. 6 In addition to the populations outlined above, Hunter et al pre- sented evidence for a clonotypically distinct, tissue-resident CD45RA lo , CD69 + , CXCR6 + Vδ1 + hepatic subset, 9 which in contrast to CD45RA hi cells could be activated by IL-12/18 alone. Interestingly, upon stimulation, this CD45RA lo population produced IFNγ and TNFα but not Granzymes, whereas the CD45RA hi population expressed cytotoxic markers and TNFα, but did not produce IFNγ. 9 Collectively, these studies support a strong interconnection between adaptive-like γδ T cell biology in blood and liver, a picture quite different from the distinct, tissue-restricted biology of the DETC subset. They suggest that following appropriate stimulation, most likely including infectious challenge, circulating adaptive-like γδ T cells can give rise to T effector populations that home to or traffic through at least some peripheral tissues, including the liver. 30,31 In support of this, studies on mice have highlighted the potential of CMV challenge to drive expansions of γδ T cells that subsequently home to a diverse range of peripheral tissues, including the liver. 39 The finding that the prevalence of Vδ1 + T cells in liver was correlated with CMV seropositivity 9 suggests that CMV might drive a similar population of γδ T cells to peripheral tissues in humans. Of note, Hunter et al, focussed on HCV and HBVnegative livers, 9 and others have also highlighted the potential of HCV to boost the frequency of hepatic γδ T cells, which may contribute to chronic inflammatory pathology. 48 The long-term nature of adaptive γδ expansions and strong effector capacity of such populations suggests long-term contributions to tissue immunosurveillance. Importantly, the relative contribution of such peripheral blood-linked adaptive-like responses to the γδ T cell compartment in different tissues, including those with a greater degree of anatomical separation from peripheral circulation than liver, is an important issue to be addressed by separate studies. In addition, the CD45RA lo population of hepatic Vδ1 + T cells reported by Hunter et al, 9 which may represent a new liver resident subset, also emphasizes the potential for distinct tissue-associated γδ T cell immunobiology to emerge, even in tissues with strong links to the peripheral circulation. While this subset appears to be significantly distinct from CD45RA hi counterparts, both clonotypically, in its more cytokine-oriented effector profile, and in its potential for extra-TCR stimulation, many questions remain concerning its origin and role in hepatic immunosurveillance and immunoregulation.

| Cutaneous γδ T cells
Human epidermis lacks a γδ T cell population analogous to murine DETC; however, Vδ1 + T cells have been described in the human dermis and epidermis and may contribute to wound healing. 49,50 A recent study of primary γδ cutaneous T cell lymphomas (CTCLs) and γδ T cells in corresponding normal tissue reported that γδ CTCLs derived from the dermis and epidermis express diverse Vδ1 TCRs, while panniculitic γδ CTCLs found in the subcutaneous fat layer, express Vγ9 neg Vδ2 TCRs 51 ( Figure 1C). The authors hypothesize that γδ CTCL subtypes develop in situ from the tissue-associated γδ T cell subsets normally present, a possibility currently under investigation. While phenotypic similarities between γδ CTCL populations and equivalent normal cutaneous γδ T cell populations are unclear, the former often expressed cytotoxic markers such as perforin and granzymes, and the transcriptional phenotype of the Vγ9 neg Vδ2 panniculitic CTCL indicated they expressed IL-12R/IL-18R, suggesting differences from adaptive-like Vγ9 neg Vδ2 equivalents in peripheral blood, including after acute CMV. 31 In summary, this study raises the strong possibility that as in mouse, human skin-associated γδ T cell subsets may exhibit TCR-linked differences in tissue localization under normal conditions. They also highlight the presence of Vγ9 neg Vδ2 T cells, like Vδ1 + T cells, in multiple solid tissues. 31,51 However, further studies on the function, phenotype, and TCR repertoire of cutaneous Vδ1 + and Vγ9 neg Vδ2 T cells is required. This should resolve the immunobiology of these skin-associated subsets in normal physiology and in γδ CTCL, clarify the degree of interconnection with or functional distinction from equivalent peripheral blood populations, and also establish the degree of functional similarity to the mouse DETC subset.

| Intestinal γδ T cells
The existence of an intestinal intraepithelial lymphocyte (IEL) compartment has long been known in mouse, and this is enriched for Vγ7 + cells. 8 In contrast, the human IEL counterpart has only recently been reported to contain a conserved Vγ4 + Vδ1 + subset 8  NKp46, 56 and also granzymes A and B ( Figure 1D), although interestingly they were reported not to secrete IFNγ or TNFα upon in vitro stimulation. 52 However, conversely, mouse Vγ7 + IELs also express molecules involved in tissue homeostasis such as Keratinocyte Growth Factor (KGF) 44 ; similarly, human Vγ4 + IELs were reported to express the EGF family member Amphiregulin (Areg), Wnt10a, and Jagged1. 52 The discovery of a human butyrophilin family-reactive tissue-associated subset that appears to align closely with an analogous population in mouse is a significant step forward.

| Breast-associated γδ T cells
Vδ1 + T cells have recently been described in normal human breast tissue and breast tumors. 58 Vδ1 + T cells in healthy breast tissue displayed an effector phenotype, featuring NKG2D expression, production of IFNγ and TNFα (but not IL-17, as opposed to murine breast γδ T cells 59 ), and cytotoxic functionality. In addition, as observed for hepatic Vδ1 + T cells, 9 the TCR repertoire was relatively clonal, consistent with a T effector phenotype, and included diverse Vγ chains. 58 Some differences in TCR clonality were reported between tumors and healthy breast tissue, with the latter harboring less clonally focussed Vδ1 + populations for reasons that are unclear. Interestingly, Vδ1 + breast T cells were activated by IL-12/IL-18, and unlike CD8 T cells from the breast, in vitro assays indicated a minority of Vδ1 + T cells could be stimulated by NKG2DL alone, raising authors to suggest an innate-like biology. 58 In our view, some caution should be exercised in this assumption. Several features mirror those of hepatic Vδ1 + T cells, which themselves appear to be closely aligned with equivalent adaptive-like populations in the blood. 9 Such features include T effector status, private clonal expansions, and IL-12/IL-18-mediated activation, which was also observed for CD45RA lo hepatic Vδ1 + populations. 9 Moreover, given the high level of expression of NKG2D on breast Vδ1 + T cells, it was striking that only a fraction of Vδ1 + T cells responded to NKG2DL alone and notable that anti-CD3/NKG2DL combination resulted in an additive effect on activation. 58 Although one possibility is that the Vδ1 + compartment has "dual functionality," operating as an adaptive subset in some scenarios and in an innate-like fashion in others, an alternative interpretation is that such breast Vδ1 + T cells might have arisen in the circulation by adaptive processes and subsequently undergone functional changes in breast tissue, resulting in a population with altered IL-12/IL-18 responsiveness relative to equivalent circulating subsets, and the ability to be controlled independently either by TCR or (in some cases) NKG2D. A similar mechanism has been proposed to explain differentiation of hepatic CD45RA lo Vδ1 + T cells, which bear several features of tissue-resident T cell populations, based partly on analyses of matched liver/ blood samples. 9 Similar analyses on breast Vδ1 + T cells may help to resolve these issues.

| LI G AND RECOG NITI ON
How γδ T cell antigen recognition occurs in molecular detail has remained one of the more impenetrable aspects of γδ T cell immunobiology, and until the last 10 years, the γδ TCR could be regarded as an "orphan" receptor. 60 However in recent years, substantial progress has been made. This includes the central questions of the identity of direct ligands recognized by the γδ TCR, and the molecular basis of such interactions. Our aim here is not to provide a comprehensive overview of proposed ligands, particularly given previous reviews on this topic. 60,61 Instead, we will highlight those exemplars we believe are likely to be most instructive, their molecular features, and how these most likely relate to the underlying physiological biological paradigms (   contact keratinocytes. These processes contain sites known as phosphorylated-Tyrosine rich aggregates located on projections (PALPs), in which is concentrated both the TCR and constitutively phosphorylated CD3ζ and ZAP70, suggesting that TCR is constitutively engaging ligand and triggering at these sites. 70 It is therefore possible that the changes observed upon DETC activation in response to stress stimuli, including loss of PALPs, cell rounding, 71 and in some cases increased cytokine production, 41 might be initiated by diminished expression of such a TCR ligand. Therefore, it is conceivable that TCR ligand may have a role in "normality sensing" in the steady state. 72 Alternatively, it has been proposed that tissue damage leads to expression of a DETC TCR ligand, as probed by TCR tetramer staining. 73 This ligand may be distinct from the homeostatic ligand discussed above.
Although no direct DETC TCR/Skint1 binding data have been published, Skint1 and conceivably other family members remain plausible candidate ligands for the Vγ5Vδ1 TCR, a possibility that justifies further exploration.

| BTNL3 and Btnl6 interaction with human Vγ4 and murine Vγ7 TCRs
After the discovery that skint1 expression in the thymus is required for Vγ5 + Vδ1 + DETC selection, 47 Figure 2D).
These highly concordant studies significantly advance our understanding of how Vγ9Vδ2 T cell recognition takes place and confirm BTN2A1 represents the elusive chromosome 6-encoded "Factor X." Nevertheless, at least two major issues remain resolved. The first relates to whether a direct interaction between BTN3A1, the pAg sensor, and BTN2A1, a direct ligand for the Vγ9 chain, is required for the sensing mechanism. However,

Rigau et al used FRET to suggest proximity (≤10 nm) between
both proteins at the cell surface, 18  levels on the target cell surface, which allows interaction with an as-yet-unidentified ligand on the T cell, thereby helping to establish a stable immune synapse, in a process that involves RhoB. 88 Of note, other molecules have been identified as being involved in pAg recognition, such as periplakin 89 and ABC transporters, 90 but the role they play is currently unclear. Clearly, additional work will be required to clarify the molecular mechanisms involved.
In summary, recent studies firmly establish BTN2A1 as one critical component of an activator ligand complex for Vγ9Vδ2mediated pAg recognition, but the full molecular nature of this complex is yet to be defined, although the involvement of an as-yet-unidentified CDR3-recognized ligand appears likely. 3.2 | Adaptive-like reactivities and candidate ligands

| General considerations
The identity of physiologically relevant γδ TCR ligands for adaptivelike γδ T cell populations (eg the human Vδ2-negative T cell subset) and the immune recognition paradigms they reflect, including the relative importance of host-encoded versus foreign antigenic targets, are major questions that remain substantially unresolved. 60 However, advances in our understanding of the immunobiology of relevant subsets and of T cell recognition in general provide important clues that both help contextualize ligands reported for adaptive-like subsets and will shape future investigations.
From this perspective, four points are particularly pertinent and will inform our discussion of proposed ligands below. Firstly, studies on Vδ1 + , Vδ1 neg Vδ2 neg , and Vγ9 neg Vδ2 + subsets suggest they possess a very diverse TCR repertoire, which underlines the observation that γδ CDR3 loop lengths bear more similarity to antibodies than to αβ T cells. 91 The obvious implication of this is that there may be huge potential in the Finally, studies on αβ T cell recognition, including in the context of the kinetic segregation model, 92 have highlighted that TCR ligand size imposes constraints on ligand-driven activation, with smaller ligands generally more likely to be conducive to efficient TCR triggering. 93 It is entirely possible that similar constraints operate for γδ T cell recognition.  96 but was not peptide-dependent, and recognition did not involve polymorphic α1α2 residues which were critical for αβ TCR recognition. 98 Instead, recognition was dependent on a several residues at one end of the α1α2 platform which did not affect αβ TCR recognition and may be influenced by a glycosylation site. 99 Human γδ T cell clones that recognize classical MHC class I and class II molecules have also been described (reviewed by Haas 100 );

Non-classical class I MHC molecules
Some of the early mouse γδ T cell clones generated by mixed lymphocyte reaction were not reactive to classical MHC molecules, but instead were focussed on polymorphic non-classical MHC molecules encoded within the H-2 locus, specifically H-2T10/T22. 103,104 H-2T molecules adopt an MHC class I fold and are β2m associated but do not present peptide. Although clones were generated against allogeneic T10/T22, use of T22 tetramers showed that up to ~0.4% of γδ T cell splenocytes and IELs bound autologous T22 in addition to foreign T22, and T22-reactive γδ T cells developed even in mice lacking the genes for T22. 105 Tetramer-positive γδ T cells were isolated and TCRs sequenced, revealing diverse Vγ and Vδ chains were used, but CDR3δ usually contained the W…(S)EGYEL motif. 106 Strikingly, the crystal structure of the G8 TCR bound to T22 showed that germline- although the tetramer used to identify the cells can be loaded with antigen, in some cases the γδ TCRs isolated do not discriminate antigen and/or can recognize empty molecules. 75,111 In structural studies of γδ TCR/CD1 interaction ( Figure 3C,D), CDR3δ, by far the most sequence diverse of the two CDR3 regions, has often been shown to be involved and frequently appears to mediate the majority of contacts. 109,111 Although not yet supported by structural data, recent data on Vδ1 + T cells specific for CD1b suggest that interaction is similarly dominated by the Vδ1 CDR3 loop. 75 For MR1, in addition to PBMC, a number of other tissues were tested for the presence of tetramer-reactive populations, and somewhat higher levels of staining were obtained in some cases. 108 Moreover, intriguingly, structural ( Figure 3E) and mutagenesis studies revealed at least two potential modes of γδ TCR/ MR1 interaction, one involving a highly unexpected interaction of the γδ TCR with the underside of the MR1 platform, which was mediated by the Vδ1 region, and as for Vδ1 recognition of CD1c/d recognition, heavily involved the Vδ1 CDR3 region. Of relevance, Reijneveld et al also recently characterized diverse Vδ1 CDR3mediated TCR binding modes for CD1b, 75 with one hypothesized to involve the underside of the CD1b antigen-binding platform as for MR1. 108 The studies above demonstrate unequivocally that some Vδ1 + (and in the case of CD1d, Vδ3 + ) T cells are able to interact with CD1 or MR1, and outline the molecular basis of this recognition. [108][109][110][111][112] However, the significance of these interactions remains unclear.
Intriguingly, the γδ T cells involved invariably derive from the highly TCR-diverse Vδ2 neg compartment, for which there is now substantial evidence for adaptive-like immunobiology, at least in peripheral blood, and they typically involve its most variable molecular feature, the CDR3δ region. 113 This contrasts with non-classical MHC recognition by at least some αβ T cell populations, including T cells is either lacking or limited, these low percentages suggest that in many cases the populations involved may be drawn from the TCR-diverse T naive compartment, which is most likely antigen-inexperienced. What these rare γδ T cell populations could contribute to immunity in addition to the existing relatively large CD1d-and MR1-reactive iNKT and MAIT populations is currently unclear.
However, one possibility is that following immune stimuli such as infection or inflammation, relevant CD1 or MR1 molecules could become loaded with as-yet-unknown antigens that drive expansions of responding Vδ2 neg T cell clonotypes. In line with this, MR1 tetramer was shown to stain 41% in the γδ IELs of a single celiac disease patient. 108 Future investigations will be required to explore the potential immunological significance of such populations.

| Non-MHC-like molecules
A disparate collection of non-MHC-like proteins have been proposed and in several cases confirmed as direct γδ TCR ligands (Figure 4).

Annexin A2 and other host-encoded ligands
Additional studies have shed light on a number of other host-encoded TCR ligands that potentially play a role either in the γδ T cell response to CMV, or to transformed cells. These include Annexin A2, which was found to be a ligand for the 73R9 Vγ8Vδ3 TCR 116 using a blocking antibody approach similar to that used for identification of EPCR. 74 A predominantly intracellular protein that binds phospholipids in a Ca 2+dependent manner ( Figure 4A), Annexin A2 can translocate to the cell surface as part of a heterotetrameric complex with S100A10. The 73R9 T cell clone from which this TCR derived was generated from healthy donors by mixed lymphocyte culture of PBL in the presence of Burkitt lymphoma cells. Transfection studies showed the 73R9 TCR could confer reactivity of reporter lines to specific target cells. This reactivity correlated with staining of target cells by a blocking antibody hypothesized to recognize the putative target ligand. Immunoprecipitation studies ultimately identified Annexin A2 as the putative ligand and direct TCR/ Annexin A2 binding was confirmed using surface plasmon resonance.
Interestingly, exposure of target cells to a number of stress stimuli, including hypoxia, heat shock, and high confluence growth, increased Annexin A2 translocation to the surface and consequent 73R9 reactivity, most probably partly via increases in oxidative stress linked to ROS.

In addition, CMV infection of target cells also increased Annexin A2
cell-surface expression and 73R9 reactivity. These observations suggest that Annexin A2 translocation to the cell surface could represent a unified stress signal for the 73R9 TCR. Although two other Annexin A2-specific T cell clones were reported by Marlin et al, 116 and Annexin A2 was reported to induce proliferation of a small population of Vδ2 neg T cells, it is unclear whether these cells align to antigen-inexperienced T naive or antigen-experienced T effector subsets, or whether they reflect clonotypes expanded in vivo. Therefore, the full significance of Annexin A2 as a focus of adaptive γδ T cell responses, as opposed to a potential in vivo reactivity, is unclear. In addition to Annexin A2, Vδ2 neg TCR reactivity to the receptor tyrosine kinase EphA2, 101 a well-established tumor associated antigen, as well as to abnormal forms of class I MHC molecules, 117 has also been proposed in the context of CMV and/or tumor cell recognition, however these specificities await full description.

Aminoacyl transfer RNA synthetases in autoimmune myositis
Autoimmunity has also been proposed as an alternative context in

Phycoerythrin and other foreign antigens
Phycoerythrin (PE) is a red pigmented protein belonging to a family of light-harvesting proteins called phycobilins present in algal species. Exploited previously as a model foreign immunogen to study induction of antigen-specific B cell memory in mice, 121 Zeng et al used PE to study antigen-specific γδ T cell recognition 122 ( Figure 4C). PE was found to stain γδ T cells in both naïve mice and human peripheral blood, at frequencies (0.04% in mice, ~0.025% in human T cells), broadly comparable to B cells in naïve animals (~0.1%). PE-specific to identify similar percentages of reactive γδ T cells in humans and mice, which as for PE reactivity mapped to the Vδ1 T cell subset. 123 The findings outlined above are important in highlighting the potential of the human peripheral blood adaptive-like compartment (but notably not the semi-invariant Vγ9Vδ2 subset, which is numerically dominant in peripheral blood) to contain specificities for foreign, microbially derived antigens. This parallels previous studies defining γδ T cell specificities capable of recognizing herpes simplex virus (HSV) glycoprotein I (gI). 124 In addition, the impact of immunization of mice with PE aligns with subsequent studies on human adaptive-like T cells, which also suggest the potential for an immune stimulus (eg CMV infection) to induce differentiation of antigen-specific effectors with increased potential for cytokine production and altered homing capabilities. 6,32 The findings also emphasize recurring themes of CDR3-dependent recognition, a hallmark of adaptive antigen-specific recognition, and, as for HSV gI and other γδ TCR ligands, recognition of intact antigen. While likely highly relevant to foreign antigen recognition, an important caveat of Zeng et al's results 122,123 is that they also highlight the potential to detect antigen-specific γδ T cell populations for ligands that are unlikely to ever be encountered physiologically, based on the extreme CDR3-focussed sequence diversity present within adaptive TCR repertoires.

| IMMUNOB IOLOG IC AL AND E VOLUTIONARY " NICHE "
The retention of γδ T cells as distinct lineage alongside αβ T cells and B cells during 500 million years of vertebrate evolution suggests they provide important non-redundant contributions to vertebrate immune defense. A full appreciation of the niche(s) occupied by γδ T cells in vertebrate immunity will require a more complete picture of how they utilize their TCR to recognize target cells. Nevertheless, the current evidence regarding their immunobiology and recognition capabilities suggests some possibilities.
The distinct modes of antigen recognition they employ is an obvious but critical feature that distinguishes γδ T cells from not only αβ T cells, but also B cells and NK cells. Use of a unique somatically recombined γδ TCR is highly likely to provide the immune system with evolutionarily advantageous recognition capabilities. Like antibodies but unlike the αβ TCR, the γδ TCR enables MHC-independent recognition of unprocessed antigen. However like αβ T cells and unlike antibodies, γδ TCR-mediated interactions inherently occur in the context of cell-cell recognition. Therefore, from a simplistic extreme, the γδ TCR could arguably be regarded as arming γδ T cells with a cell-surface "Fab-like" repertoire, rendering γδ T cells arguably analogous to "Nature's CAR-Ts" ( Figure 5). However, crucially, NK cells appear particularly susceptible to pathogen immune evasion, since they ultimately rely on germline-encoded receptors 131,132 to recognize virally infected target cells. In contrast, the highly diverse TCR repertoires of somatically recombined adaptive-like γδ T cell subsets are likely to prove substantially less susceptible to immune evasion, potentially enabling responses against either host-encoded or pathogen-encoded targets, including those which may display high variability.
In summary, the ability of γδ T cells to exploit a somatically recombined antigen receptor to initiate powerful cell-mediated effector responses in a way that is not dependent on target-expressed MHC molecules is likely to provide a major evolutionary advantage when present in combination with αβ T cells, NK cells, and B cells.
The unique antigen recognition modes and distinct immunobiologies of different γδ T cell subsets provide the vertebrate immune system with unique immunosurveillance and immunoregulation capabilities in the face of highly divergent immune challenges. responses to non-microbial stimuli, for example via recognition of endogenous antigens altered or upregulated in disease states. 134 Although these likely occur via CDR3-mediated recognition of intact cell-surface antigens as for Fab/ligand interaction, they are orders of magnitude lower in affinity. Furthermore, for Vγ9Vδ2 T cells, it seems likely that the TCR can act as a "smart" receptor by engaging BTN2A1 alongside CDR3-mediated recognition of separate moieties as part of a composite ligand. 17,18 It comes as no surprise that evolution has resulted in a level of sophistication in the γδ TCR that exceeds current CAR approaches, which largely exploit single-chain fragment variable (scFv) specificities for single antigenic targets. Nevertheless, despite these differences, the analogy with CAR-T cells does emphasize how the defining feature of γδ T cells, namely their somatically recombined γδ TCR, provides them with a unique niche in the immune system relative to αβ T

cells, B cells, and NK cells, and is a useful perspective from which
to plan future studies.
For innate-like subsets, it will be crucial to establish to what extent Vγ-restricted recognition of specific BTN/BTNL/B7like proteins extends beyond blood and gut. 8,17,18,54 Based on the predominant Vγ9 subset in the peripheral blood and Vγ4/ Vγ7 population in gut, the prevalence of particular Vγ regions might provide clues as to the distinct innate-like subsets present in different sites. Furthermore, it will be important to assess if parallel innate-like and adaptive-like γδ arms operate in many tissues around the body, as appears to be the case in blood. 15,30 In addition, a major question going forward is if BTN/BTNL rec-

CO N FLI C T O F I NTE R E S T
I have no conflict of interest with regard to the topics discussed in this review manuscripts.