Bohemian T cell receptors: sketching the repertoires of unconventional lymphocytes

Introduction

The immune system relies on a number of receptor families to discriminate non-self or “altered”-self from healthy self, with discretion between these states as the switch maintaining tolerance against autoimmunity and a warranted response against pathogens and cancer. The first point of recognition in the immune response is tasked to ancient families of germline-encoded receptors able to sense conserved molecular patterns generated by tissue damage and inflammation (damage-associated molecular patterns, DAMPs), and/or pathogen infection and replication (pathogen-associated molecular patterns, PAMPs). These molecular products sensed by the pattern-recognition receptors (PRRs) can be found across all kingdoms of life (i.e. nucleic acids, cell wall components, ATP), and their recognition of the molecules in context initiates inflammatory signaling pathways promoting the initiation of an immune response (1, 2). Another pathway modulated by PRR signaling pathways is antigen presentation - the processing and display of exogenous and self-antigens in major histocompatibility complexes (MHC) for their recognition by a more flexible set of receptors used by the adaptive immune system (3).

Adaptive immunity is a more recent evolutionary development relying on receptor families consisting of somatically rearranged gene products with tremendous potential for variability. The T cell receptor (TCR) is the product of the chromosomal rearrangement of hundreds of mobile genetic elements through the process of VDJ recombination; successful, in-frame rearrangements of variable (V), joining (J), and diversity (D) genes of four TCR loci (TCRα, TCRβ, TCRγ, TCRδ), with the heterotypic pairings TCRα with TCRβ, and TCRγ with TCRδ, broadly define the two major, though distinct, T cell lineages (i.e.αβ and γδ T cells, respectively). This massive amount of receptor diversity endows T cells with the capacity to see nearly any peptide able to be presented in MHC; the theoretical number of potential 9-mer and 14-mer peptides able to bind MHCI and II is estimated at 10¹⁰ and 10¹⁷, respectively (4). Furthermore, it has been estimated a single TCR is able to recognize upwards of 10⁶–10⁷ unique peptides (4). This combination of diverse antigens and receptors able to recognize them results in a flexible compartment of cross-reactive immune cells able to respond to new pathogens, and importantly, able to remember previous encounters. We refer to the vast universe of T cell clones within an individual as being their TCR repertoire. A conventional response by T cells involves an encounter between the host and a pathogen or tumor. New and abundant antigens with PRR-driven inflammation results in the recruitment and clonal expansion of antigen-specific T cells aimed at eliminating the threat through cytotoxic killing (1). The trade-off for this level of specificity however, is slow kinetics, taking several days to weeks between first contact with PRRs and a pool of antigen-specific T cells responding to the agent. While this targeted type of conventional T cell response against peptide presented by MHC in a canonical-manner is certainly the best understood in terms of mechanism, it is only one way the antigen-specificity of the TCR is harnessed by the immune system (5). It is well-appreciated that unconventional T cells with more delimited functions and specificities develop and are elicited in non-canonical ways; more recently we have begun to better understand how these unconventional populations sit on the spectrum of innate and conventional acquired immunity.

Many questions regarding the origin, specificities, and activities of these unconventional T cells remain unanswered. The fact that several types of unconventional T cells are selected on the older, non-classical MHCs presenting a limited array of non-peptide antigens suggests that these unconventional T cells appeared early in the evolution of the lymphocytes. Reflecting the need for recognizing conserved molecules outside the bounds of peptide:MHC, lymphocyte populations with rapid response kinetics using invariant TCRs are selected for their recognition of non-classical MHC class Ib (MHCIb) molecules, fulfilling their destiny as an innate-like T lymphocyte (6). The MHCIb genes, are the older, well-conserved members of the MHC family, sharing considerable structural similarity with most requiring β2-microglobulin (β2m) for their expression on the cell surface (7). It is unknown what the ligands are, if any, for a number of the MHCIb proteins, and lesser known still how they affect tolerance or shape the innate and adaptive specificities and responses, alike. What is clear however, is some of these MHCIb proteins present small molecules both exogenous and endogenous in origin recognized directly by the invariant TCRs of unconventional lymphocytes, and sizeable pools of the invariant clones capable of responding to their antigens are present and stable as a means of immune surveillance. The unconventional lymphocytes display a diverse array of antigen-driven effector functions, even within the same population, with some producing cytokines and helping B cells similar to CD4 T helper cells, while others acquire cytotoxic capacities like the CD8 and NK cells (8). Clearly, the unconventional lymphocytes play critical roles in bridging the gap between innate and adaptive immunity, but with so many of the MHCIb ligands unaccounted for, and with some ignorant to positive selection in the thymus, how many more populations of unconventional lymphocytes might be out there? Further complicating matters is that one of the unconventional T cell types (CD8αα+, double-negative IELs) we will discuss here are selected by both classical and non-classical MHCs; further, after selection via high-affinity interactions with self-peptides, differentiates into a specialized, tolerogenic population.

The repertoires for several of the unconventional lymphocyte populations have been well-identified for some time, however, advances in sequencing technology allowing for a survey of immune repertoires at unprecedented depth and resolution have begun to reveal a world of diverse TCRs targeted to the recognition of non-peptide antigens. In this review, we will highlight and discuss the TCR repertoires of the unconventional lymphocytes and the role they play in shaping and defining the functions of these populations. Patterns of fate, function, and location tied to TCR reactivity are clearly emerging within the unconventional lymphocytes, and we speculate further study of their repertoires will help paint a more complete picture of their place in immune surveillance and responsiveness. We will discuss what is historically known about each population’s repertoire, the influences shaping it, how TCR specificity entails a population’s lineage fate, and how new tools and techniques are uncovering previously unknown repertoire diversity and tuning within unconventional lymphocytes.

The γδ T lymphocyte, the founding member of the unconventional lymphocytes

The γδ T cells are a unique lineage of T cells integral in a number of immune-related processes: tissue function/homeostasis, response to pathogens and commensal microbes, and tumor surveillance. In both mouse and human, γδ T cells are widely distributed across a number of tissues but enriched within lymphoid and mucosal tissues. Their localization puts γδ T cells at the frontline for surveillance, and they are able to respond rapidly to damage and infection, helping mediate their repair, secreting antimicrobial peptides, and coordinating the escalation of the immune response if necessary through cytokine/chemokine production. The functions and contributions of γδ T cells to immune responses have been well-reviewed in a number of excellent articles (9–11). Here, we will focus our discussion on the repertoire landscape of γδ T cells to try and highlight the emerging pattern whereby the γδTCR seemingly directs cell-fate decisions in a clonotypic-manner. (We will use the Tonegawa nomenclature for γδ TCRs)

Like their αβ T cell brethren, γδ T cells develop in the thymus, undergoing the required recombination activating gene (RAG)-dependent VDJ recombination resulting in productive, paired TCRγ and TCRδ chains. During rearrangement the CDR3γ and CDR3δ regions are amenable to nucleotide insertion for tuning of antigen recognition. In fact, due to the presence of multiple diversity genes (D) of TCRδ capable of being translated across three reading frames, the theoretical CDR3 diversity of the γδ TCR due to germline gene rearrangements and excluding N-additions at the CDR3 is ~10¹⁸, higher than that of αβ T cells (~10¹⁶) and Immunoglobulins (Ig) (~10¹¹) (12), and thus are at no disadvantage for recognizing diverse antigens in this regard. The reality does not reflect this capability, however. Rather a major portion of the γδ T cell repertoire is occupied by invariant, frequent rearrangements between just a small number of genes within the TCRD/TCRG loci, and the pairings between the TCRδ and TCRγ chains are rather restricted, overall imparting a lower functional diversity compared to αβ T cells and B cells.

Unlike αβ T cells however, γδ T cells are not subject to positive selection based on their affinity for class Ia or II MHC proteins, and for a number of γδTCRs, their development does not require any sort of antigen-based selection in the thymus. However, this is not to say all γδTCRs develop without reactivity against thymic antigens, and in some cases thymic selection is required for the development of specialized subsets of γδ T cells. It is now well-appreciated that the differential ability of γδTCRs to interact with thymic antigens, and the strength of TCR signaling due to this interaction, has a profound effect on the fate of the developing cell and the overall γδ T cell landscape (13). Broadly, γδ T cells can be subset by their capacity to make either IL-17 or IFNγ, and the decision to produce one or the other is made during thymic development based on the amount of input received through the TCR. In examining γδ T cells specific for the non-classical MHCIb molecules T10 and T22, Jensen et al noted T10/22-specific γδ T cells that developed in wild-type mice displayed an antigen-experienced phenotype and produced IFNγ in response to TCR stimulation (14), but when developed in mice deficient in β2m, an accessory protein required for presentation of T10/22 on the cell surface, T10/22-specific γδ T cells were still able to develop, but now had an antigen-naïve phenotype and produced IL-17A. Conversely, the specialized Vγ5Vδ1+ dendritic epidermal T cells (DETCs) found in mouse epidermis require the expression of Skint1 by thymic epithelial cells to acquire the ability to make IFNγ, and deletion of Skint1 directs DETCs towards the IL-17+ fate (15). These observations determined the default developmental program for γδ T cells is IL-17-production, but strong TCR signaling diverts the developing cell towards the IFNγ-producing fate. Thus, the ability to recognize thymic antigens does not appear to be required or biasing in selecting γδ T cells undergoing development, but rather a mechanism of education directing their effector functions.

An interesting framework within the γδ T cell populations has begun to emerge whereby the highly public, invariant TCRs seemingly provide an orderly distinction between the innate-like γδ T cells, and other clonotypes more tuned to conventional adaptive immunity. A number of invariant γδTCR clonotypes have been discovered across multiple species, and in each case a public, easily-generated γδTCR commits the developing T cell to a discrete fate and localization within specific tissues (mucosa, lymphoid organs, dermis) (16). This provides a useful mechanism for sorting different subsets of γδ T cells to their intended tissue where they establish residency and provide surveillance and response in tune with the tissues requirements. The high preponderance of semi-invariant, oligοclonal, and public γδTCRs found in the IL-17-producing populations, and their ability to respond to unidentified, though presumably conserved antigens, has led their categorization as innate-like lymphocytes. Formation of large clonal pools of γδ T cells using a fixed repertoire of receptors capable of responding rapidly to a shared molecular structure without the need for prior clonal expansion is reminiscent of the PRRs used by the innate immune system. These invariant γδ T cell populations are highly public across individuals of a given species, providing a fixed specificity within their repertoire dedicated to recognizing conserved molecular patterns at the organismal-level. This public specificity suggests that the γδ T cells evolved to address signals of stress elicited, and shared, by many forms of insult including: infection, tumorigenesis, autoimmunity, tissue damage, or any other inflammatory process giving them the flexibility to respond to a broad set of situations rather than restricted to a cognate antigen particular to a certain microbe.

Indeed, the ubiquitous nature of the few known antigens for γδ TCRs reflect this possibility. For example, the predominant human blood subset of γδ T cells, Vγ9Vδ2+, are highly public and semi-invariant across individuals (17). They recognize conserved diphosphate structures called phosphoantigens. One known phosphoantigen is isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway used by both eukaryotes and archaebacteria for cholesterol biosynthesis. Another is hydroxymethyl-but-2-enyl-pyrophosphate (HMBPP), an intermediate of the alternative, non-mevalonate pathway of cholesterol used by some Eubacteria, and importantly, the malaria-causing agent Plasmodium falciparum (18). Vγ9Vδ2+ cells rapidly produce IFNγ in response to P. falciparum-infected red blood cells (iRBCs), and proliferate, making up to 20% or so of peripheral lymphocytes (19, 20). Individuals clinically tolerant to P. falciparum were found to have a lower frequency of Vγ9Vδ2+ T cells along with qualitative loss of their functionality, suggesting they play an important role in immunity to the parasite (21). Presentation of phosphoantigens is not mediated by any classical or non-classical MHCs, rather they require butyrophilin 3A1 (BTN3A1) for their recognition by Vγ9Vδ2+ T cells. BTN3A1 is a ubiquitously expressed cell surface protein containing extracellular immunoglobulin domains, and a B30.2 domain in the intracellular portion. Intracellular IPP and HMBPP directly bind with the B30.2 domain of BTN3A1, and is necessary and sufficient for the activation of Vγ9Vδ2+ T cells, possibly through their recognition of allosteric alterations of BTN3A1’s extracellular domains elicited by phosphoantigen binding to the B30.2 domain (22, 23). This mechanism is in many ways analogous to how sensing of pathogens by PRRs stimulates innate immune responses; i.e. increased levels of PAMPs and DAMPs are sensed by BTN3A1, and this in turn stimulates its interaction and activation of Vγ9Vδ2+ T cells. Their conserved specificity to a PRR modified in response to DAMPs/PAMPs, coupled to their rapid responses, makes the case that Vγ9Vδ2+ T cells are more akin to innate cells than what we consider a classic adaptive response.

A classic example of tissue residency as dictated by the TCR specificity, is the IFNγ-producing Vγ5Vδ1+ DETCs (mentioned above). The Vδ1 (Vδ1-Dδ2-Jδ2) chain is a germline rearrangement without nucleotide insertion, which pairs to a near-invariant Vγ5 (Vγ5-Jγ1-Cγ1) chain (24, 25). DETCs are seeded from the earliest waves of thymic emigrants, developing at embryonic day 14 (E14), and are maintained throughout life through self-renewal. The transmembrane protein Skint-1, expressed exclusively in the thymic epithelial cells and keratinocytes, was identified as being required for the selection and maintenance of the DETCs expressing Vγ5Vδ1+. Importantly, there is no evidence the DETC TCR is able to directly interact with Skint-1, and while no other ligands for it have been identified, damaged keratinocytes are able to stimulate them through the TCR without MHC-restriction. Additionally, and this is a feature shared by a number of γδ T cell subsets, DETCs express multiple NK cell receptors that provide TCR co-signaling or alternative TCR-independent mechanisms of activation. Thus, DETCs are able to integrate TCR-dependent and independent signals from their environment to provide an appropriate response for the insult; e.g. engagement of junctional adhesion molecule-like (JAML) on DETCs by the coxsackie-virus and adenovirus receptor (CAR) induced in keratinocytes surrounding wound sites solicits DETC production of inflammatory cytokines and keratinocyte growth factor (KGF) needed for efficient wound resolution (26). In an analogous-manner, it was recently found the mouse Vγ7 and human Vγ4+ intraepithelial lymphocytes (IELs) residing in the gut mucosa are positively selected for their fate during early development by another family of transmembrane proteins known as butyrophilin-like (Btln) proteins. Embedded in the epithelium of the gut mucosa, γδ IELs are positioned to survey and respond to damaged epithelium to mediate its repair, and prevent penetration of commensal microbes into underlying tissues by providing immunomodulatory and antimicrobial factors (27–29). In mice, Vγ7Vδ4 + IELs are positively selected on Btln1 and Btln6 in a wave of prenatal, extrathymic development. Btnl1 and Btnl6 expression is confined to enterocytes providing continued TCR stimulation, driving their maturation and self-renewal (30). Human BTNL3 and BTNL8 appear to select for Vγ4+ IELs in a similar manner, suggesting a conserved mechanism exists for getting γδ IELs to the right location. It will be interesting to see if these butryophillin-like proteins identified as being required for γδ IEL development use a similar mechanism of intracellular sensing as BTN3A1 for γδTCR stimulation.

Taking a broader, systemic view of the γδ TCR repertoire has proven successful in identifying novel, innate-like clonotypes and adding new insight to old ones as well. Some of the first γδ T cells to develop during the early waves of prenatal thymic development are the so-called “natural” Tγδ17 cells that go on to seed a number of different tissues. Their name comes from their ability to rapidly produce IL-17A in response to a broad array of infectious and non-infectious agents. By sequencing the TCRδ genes paired to Vγ4+ cells, Kashani and colleagues found a high proportion of clones used a germline Vδ5-Dδ2-Jδ1 rearrangement, and this clone acquires an innate-like (IL-17A+ CCR6+ CD27−) phenotype with widespread tissue distribution, categorically placing them into the natural Tγδ17 cells (31). Consistent with this finding, single-cell TCR sequencing of the IL-23R+ natural Tγδ17 cells from multiple tissues identified the same Vγ4Vδ5+ clonotypes (32). This report also showed the repertoires of IL-23R+ versus IL-23R− γδ T cells are remarkably different in their diversity, with the IL-23R+ cells being mainly Vγ4Vδ5+ and Vγ6Vδ1+ cells, while the IL-23R− population is quite diverse. Their invariant, public nature would suggest the Vγ4Vδ5+ and Vγ6Vδ1+ TCR likely recognize well-conserved structures revealed in a number of situations, but for now their antigens remain unknown.

In addition to fixed specificity of the invariant, innate-like γδ T cells, it has been speculated for some time that γδTCRs with diverse gene rearrangements and CDR3 diversification were more adaptive-like in their nature. The finer points of γδ T cell antigen-specificity have been well reviewed elsewhere (13). With exception to those germline rearrangements of the innate-like γδ T cells, TCRδ chains in particular show extensive breadth of their CDR3 with clonal lineages appearing uniquely within a given individual, exhibiting what we would call TCRs with private specificity. On the other hand, TCRγ rearrangements are quite well-conserved and are generally public between individuals. Structural studies of the γδTCR interacting with its ligands show recognition of ligands occurs through the CDR3δ, and the CDR3δ is subject to extensive nucleotide addition making them quite long. While clearly γδ T cells can acquire antigen-specificity, technological limitations have hampered our understanding of the clonal dynamics within γδ T cells during antigen-specific responses and at the steady state. One of the outstanding questions regarding γδ T cell biology is, given that αβ T cells and B cells are activated and clonally expand upon recognition of their cognate antigens, do γδ T cells follow the same pattern? Most studies of γδ T cell repertoires have historically relied on antibody panels targeting specific TCRγ or TCRδ chains, but this method is limiting in that it is unable to identify clonal expansions or evidence of CDR3 selection. Single cell TCR sequencing drastically improved the ability to discern interesting receptor pairs and provide some of the first evidence of selection and expansion for γδ T cells, but until recent advances in single cell sequencing, it is a rather low-throughput approach with which to probe the repertoire at a population level at any meaningful depth. However, the advent of next-generation sequencing (NGS) technologies now allows one to sequence TCRs from vast numbers of cells for identifying their CDR3 and gene usage, providing a powerful tool for surveying immune repertoires and how they are shaped by antigenic environments.

Two recent works applying NGS methods to probe the γδ TCR repertoire of humans demonstrated that selection and expansion of particular clonotypes occurs naturally with age, as well as after allogeneic hematopoietic stem cell transplant (alloHSCT). In contrast to the semi-invariant Vδ2 chain discussed above, Davey et al showed human γδ T cells expressing the Vδ1 chain undergo clonal selection with age, where the Vδ1+ repertoire of neonatal cord blood was shown to be more polyclonal compared to peripheral blood from adults, which was dominated by clonal expansions (17). Clonally-expanded Vδ1+ cells display an effector phenotype (i.e.CD27low, granzymes, IFNγ+) and maintain their TCR sensitivity for a quick response to their cognate antigens. Studies by Ravens and colleagues yielded similar results where they found that while there is a high amount of diversity in γδ T cells there is also marked oligoclonality in the Vγ9− cells from adult blood, and far fewer clonal expansions in neonatal cord blood (33). When they compared the repertoires of γδ T cells from humans prior to and after alloHSCT therapy it was found that the repertoires of clonally-expanded γδ T cells after treatment were reconstituted quickly, stable, and fundamentally distinct from their pre-treatment repertoires. Cytomegalovirus (CMV) is a common human pathogen capable of establishing life-long latency, and is largely asymptomatic, however, CMV reactivation in patients undergoing HSCT therapy is a life-threatening complication. Human Vδ2+ γδ T cells are known to proliferate and acquire cytolytic capabilities in response to CMV reactivation, and this interaction with CMV can have profound effects on the γδ T cell landscape (34, 35). Whether the activation of Vδ2+ γδ T cells happens in a polyclonal manner with a number of reactive clones being pulled in, or if the repertoire simply becomes dominated by a few reactive clonotypes driven by a selecting antigen was heretofore unknown. Addressing this question, CMV reactivation in alloHSCT recipients induced the expansion of just a few distinct γδ T cell clonotypes, using mostly Vδ1 and Vδ3, and each expansion appeared privately within an individual making up a substantial frequency of their repertoire (33). This is the first report that the repertoire of γδ T cells, in part, undergoes selection in response to a virus infection, but where CMV-reactive γδ T cells fit into the effective immune response against the virus remains to be seen. A study of immune reconstitution in children undergoing HSCT for tumor malignancies found that patients that recovered their γδ T cell compartment more rapidly suffered fewer infections overall (36); no similar association was observed for αβ T cell recovery, suggesting an important role for γδ T cells in preventing mucosal infections in this vulnerable population.

In addition to their roles maintaining homeostasis in response to infection or injury, γδ T cells participate in the coordinated effort of tumor surveillance provided by the immune system. The presence of γδ T cells within tumors is associated with a more favorable prognosis for a number of diverse cancers (37). As discussed, they express a range of activating NK cell receptors capable of activating and licensing them for anti-tumor activity, but whether the TCR itself is able to recognize tumor antigens, or if the γδTCR repertoire is tuned to recognize certain antigens upregulated in adjacent tissue during tumorigenesis is unknown (10). Some of the first evidence that clonal selection of responding γδ T cell lineages is occurring in cancer comes from a recent study by Chen et al. (38). Here, the diversity of TCRδ, but not TCRγ, in peripheral blood γδ T cells from patients with lung carcinoma (LC) was significantly lower compared to the healthy controls due to high amounts of clonal expansion. Interestingly, the CDR3δ sequences from LC patients were more frequently shared between LC patients versus the controls, suggesting that a conserved set of antigens associated with tumorigenesis might be responsible for their selection. It is tempting to speculate these clonally-expanded responses are selected on intratumoral interactions between γδ T cells and antigen, but there is currently no evidence to support this possibility, and it cannot be ruled out that signals coming from the tumor might elicit their cognate antigens at peripheral sites, which in turn are the selecting factor. Future studies investigating the repertoires of γδTCR+ tumor-infiltrating lymphocytes (TILs) will help to further elucidate the mechanisms and locations of γδ T cell selection, and better resolve the nature of clones associated with anti-tumor immunity.

These studies of the γδ T cell repertoire and how it responds to strong selective pressures at the population level provide fascinating new insights into their biology building on the growing evidence the γδTCRs can be selected based on specific antigen reactivity and establish a pool of clones targeted for antigen recognition. At face value these data suggest γδ T cells maintain a diverse pool purposed with recognizing new antigens by virtue of their TCR reactivity, permitting new expansions with continued encounters, and the development of memory to prior infections accumulating along the way. However, it is important to consider where these selection events are taking place in time and space, and the antigens imparting it. With the notable exceptions of those positively selected based on their reactivity to non-classical MHCIb molecules (39), the γδ T cell repertoire is thought to be unbiased towards thymic antigens suggesting that clonal selection begins to occur in the periphery. Few antigens have been identified for γδTCRs, and far fewer proven to bind to them, but how big is the universe of potential γδ target antigens? This is much more difficult to predict than for αβ T cells, where MHC restriction provides a bound on the potential diversity of antigen recognition. Beyond that initial question, others include, are the antigens targeted foreign or self? Does each clone have one specificity against a unique antigen, or is it the case that multiple γδTCRs converge on a handful of antigens? The former case fits with our current model of adaptive responses, but the second possibility falls more in line with what we already know about the innate-like nature of γδ T cells. This type of selection mechanism would be useful for keeping clones capable of responding to a ligand expressed in response to stress and damage, thus providing a pool able to respond to a broad range of insults. Future studies focused on the biochemistry of antigen recognition will help resolve these ambiguities.

The unconventional αβ T cells

Conclusion

Studies of unconventional lymphocytes have demonstrated they arise during the first waves of T cell development due to a high affinity for self-antigen, and upon experiencing their antigens, expand to form large, stable pools of clones poised for responding quickly to conserved molecular structures as part of the immune response to infection and damage (Table 1). In the case of CD1- and MR1-restricted T cells, αGalCer and 5-OP-RU represent the immunodominant epitopes recognized by the invariant TCRs of iNKT and MAIT cells, respectively. The invariant TCRs are efficiently generated and optimized for their antigen specificity, and if not for the process of VDJ recombination required for their generation, they might easily be mistaken for a conventional PRR. On the other hand, deep sequencing the repertoires of MR1- and CD1-restricted T cells differing in their affinity to variants of the prototypical ligands, as well γδ T cells without known antigens, has found that invariant TCRs are not the only way T cells are able to gain specificity to non-peptide antigens. Rather, a proportion of lymphocytes restricted by a particular MHCIb molecule display a more diverse TCR repertoire better fitted to recognizing one variant over another with sufficient affinity and avidity to drive their differentiation into an unconventional lineage. This raises the question, with such large and dominating pools of MAITs and iNKTs, why bother? The fact the MR1T and dNKT cells are able to compete at all with their invariant counterparts for space might be telling of their importance – clones tuned to seeing variations of MR1 and CD1d ligands ensure functional avidity is maintained within the population. What now remains for the field is to develop better understanding of the in vivo pathological and physiological conditions leading to differential activation of the invariant and diverse unconventional lymphocytes in vivo, and how this contributes to either the resolution or continuation of those conditions. With new technologies allowing for paired transcriptome analysis coupled to TCR identity of a single cell, it is likely that new populations of unconventional lymphocytes endowed with targeted specificity will be discovered as we continue to fill in the atlas of TCR repertoires.