Understanding the complexity of γδ T-cell subsets in mouse and human

Abstract

γδ T cells are increasingly recognized as having important functional roles in a range of disease scenarios such as infection, allergy, autoimmunity and cancer. With this has come realization that γδ cells are not a homogeneous population of cells with a single physiological role. Instead, ever increasing complexity in both phenotype and function is being ascribed to γδ cell subsets from various tissues and locations, and in both mouse and human. Here, we review this complexity by describing how diverse γδ cell subsets are generated in the murine thymus, and how these events relate to subsequent γδ subset function in the periphery. We then review the two major γδ cell populations in human, highlighting the several similarities of Vδ1⁺ cells to certain murine γδ subsets, and describing the remarkable functional plasticity of human Vδ2⁺ cells. A better understanding of this spectrum of γδ cell phenotypes should facilitate more targeted approaches to utilise their tremendous functional potential in the clinic.

Introduction

The initial perception of γδ cells as innate cells with limited functional potential is now distinctly outdated. Instead, γδ cells display considerable subset heterogeneity, with complex patterns of effector function that range from T-cell help to antigen presentation. Understanding this heterogeneity, and how it develops, is central to understanding the role of γδ cells in complex diseases, and will provide an important foundation for harnessing these enigmatic cells for multiple future therapeutic opportunities in the clinic.

Commitment to the γδ cell lineage; pre-commitment and signal strength

In the murine thymus, commitment to the γδ lineage occurs at the immature CD4⁻ CD8⁻ double-negative (DN) stage of thymocyte development.¹ Early DN cells with potential to develop as either γδ or αβ T cells rearrange their T-cell receptor-γ (TCR-γ), TCR-δ and TCR-β loci in an attempt to generate a productive TCR-γδ or pre-TCR (TCR-β paired with the invariant pre-TCR-α chain) that compete to drive γδ or αβ T-cell development, respectively.^1–3 Despite initial suggestions that TCR-γδ and pre-TCR ‘instruct’ DN cells into their respective lineages, it now appears that signal strength, rather than identity of the expressed TCR complex, is the critical factor in fate determination; strong signalling committing DN cells to the γδ lineage; weak signalling committing cells to the αβ lineage.^4,5 Nonetheless, this effectively equates to an instructional model, as under normal circumstances the pre-TCR signals weakly while TCR-γδ signalling is stronger.

Although this strength-of-signal model is now widely accepted, there is significant evidence to suggest that factors operating before, or contemporaneous with, TCR rearrangement and signalling may also influence γδ/αβ fate determination. Thus, CD44⁺ CD25⁺ DN2 cells that express CD127 [interleukin-7 receptor-α (IL-7Rα) chain],⁶ or Sox13,⁷ appear more likely to enter the γδ lineage, whereas adult DN cells, or DN cells from the later CD44⁻ CD25⁺ DN3, or CD44⁻ CD25⁻ DN4 subsets appear biased toward αβ T-cell development.^3,8 Thus, commitment to a γδ fate requires strong signalling from successfully rearranged TCR-γδ complexes in DN cells that are permissive for entry into the γδ cell lineage.³

Thymic γδ subsets in mouse; the consequence of strong TCR-γδ signals

The earliest murine TCR-γδ⁺ thymic progenitors are CD24⁺ CD25⁺ CD27⁺ cells that express relatively low levels of surface TCR-γδ, but are highly proliferative.^9,10 At this stage, TCR-γδ signalling initiates, leading to CD25 down-regulation, up-regulation of TCR-γδ, and generation of cells with a CD24⁺ CD25⁻ CD27⁺ phenotype.⁹ These uncommitted cells probably represent precursors of several distinct mature CD24⁻ TCR-γδ⁺ thymocyte populations (Fig. 1a, and see below), that must arise from TCR-γδ signalling that exceeds a critical commitment threshold. Nonetheless, evidence now suggests that mechanisms of TCR-γδ signal initiation that underpin these commitment events may significantly differ.^3,11,12

Figure 1.

Mouse γδ cell subsets in the thymus and periphery. (a) Thymic γδ subsets described by surface expression of; yellow triangles – CD27; green triangles – CD24; blue triangles CD44; purple triangles – CD25; and red triangles – CD122. Proposed developmental relationships between subsets are indicated by arrows, and potential for cytokine secretion is shown. (b) Peripheral γδ subsets described by tissue location, potential for cytokine secretion and surface markers, as described for (a). Gut γδ cells (i.e. γδ intraepithelial lymphocytes) express only low levels of CD122. IL-17A, interleukin-17A; IFN-γ, interferon-γ.

Strong TCR signals of the type that commit DN cells to the γδ lineage are generally considered a consequence of agonist–ligand binding. However, a general TCR-γδ restricting element, analogous to MHC-I or MHC-II for TCR-αβ, has not been identified, and in mouse only one confirmed TCR-γδ-ligand, the MHC class Ib thymus leukaemia (TL) molecule, has been found.¹³ Nonetheless, there is reasonable evidence, although mostly indirect, for TCR-γδ-ligand binding during generation of thymic progenitors of at least three peripheral γδ cell subsets; dendritic epidermal T cells (DETC) that use TCR-γ chain variable region-5 (Vγ5) (nomenclature from ref. 14), and TCR-δ chain variable region-1 (Vδ1),¹¹ natural killer T (NKT) -like γδ cells that use a Vγ1Vδ6.3/6.4⁺ TCR,^15,16 and γδ cells whose TCRs recognize the TL molecules T10^b and T22^b (∼ 0·5–1·0% of all γδ cells).^17–20 In each case, the TCRs used by these subsets already show restricted CDR3 length and amino acid composition in the thymus, indicative of thymic ligand-driven TCR selection.^11,15,21 Moreover, these CD27⁺ thymic progenitors display a CD44⁺ CD62L⁻ CD122⁺ phenotype that is generally associated with ligand engagement (Fig. 1a).

A further subset of mature TCR-γδ⁺ thymocytes that express a Vγ6Vδ1⁺ TCR, have also been reported to show evidence of CDR3-mediated TCR selection.²¹ These cells are progenitors of those that populate the female reproductive tract and peritoneal cavity, and appear shortly after Vγ5Vδ1⁺ DETC progenitors at approximately embryonic day 16.¹ Notably, Vγ6Vδ1⁺γδ cells differ from DETC, NKT-like γδ cells and (ligand-selected) TL-specific γδ cells in their capacity to produce IL-17A.²² This IL-17A-secreting effector potential is shared by CD27⁻ CCR6⁺γδ thymocytes^9,23 (Fig. 1a), that are also CD44⁺ CD62L⁻, consistent with a previous TCR-γδ-ligand interaction. However, CD27⁻γδ cells do not express CD122, and conspicuously fail to develop in fetal thymic organ cultures supplemented with TCR-γδ antibodies that induce strong TCR signals.^9,23 Hence, any putative ligand interaction for the development of CD27⁻ IL-17A-secreting γδ cells must necessarily induce an attenuated (or at least qualitatively different) TCR signal that cannot readily be reproduced by classical antibody cross-linking of the TCR.^9,11,19

The development of IL-17A-secreting γδ cells has also been suggested to result from ligand-independent TCR-γδ signalling in the thymus, possibly as a result of oligomerization of TCR-γδ induced by the variable domain of TCR-δ.¹⁹ However, a TCR-γδ that lacks both Vγ and Vδ can still signal effectively in DN thymocytes, resulting in generation of γδ thymocytes with interferon-γ (IFN-γ) -secreting potential.¹² These cells evoke a subset of mature (i.e. CD24⁻) CD27⁺γδ thymocytes that show IFN-γ-secreting potential and display a ‘naive’ CD44⁻ CD62L⁺ phenotype consistent with an absence of previous TCR-γδ-ligand interactions (Fig. 1a).⁹ Thus, several distinct subsets of mature γδ thymocytes can be identified that are probably the result of distinct mechanisms of thymic TCR-γδ signal initiation.³

Mouse peripheral γδ subsets

Several discrete populations of peripheral murine γδ cells have now been identified (Fig. 1b). Perhaps the best studied are Vγ5Vδ1⁺ DETC from the murine (but not human) epidermis that display ‘innate-like’ properties, in that they are thought to respond en masse to relatively few stress-associated TCR-ligands.²⁴ These cells are CD44⁺ CD62L⁻ CD103⁺, and express CD122 consistent with their dependence on IL-15.^25,26 DETC secrete IFN-γ when activated, but can also drive IL-13-mediated T helper type 2 (Th2) -associated responses on recognition of NKG2D-ligands expressed on stressed epithelial cells.²⁷ A CD44⁺ CD62L⁻ CD122⁺ phenotype is also shared by a CD90^dull CD27⁺ NKT-like γδ subset that uses a restricted Vγ1Vδ6·3/6·4⁺ TCR and secretes both IFN-γ and IL-4 when activated,^15,28 and by IFN-γ-secreting TL-specific lymphoid γδ cells that develop in a T10^b/T22^b-expressing background.¹⁹

In contrast to DETC, which primarily make IFN-γ, γδ cells from the murine dermis predominantly secrete IL-17A. These cells are biased towards use of a Vγ4-containing TCR-γδ, are CD44⁺ CD122⁻, and express both CCR6 and SCART2.^25,29–31 This phenotype closely resembles that of IL-17A-secreting γδ cells from the female reproductive tract, tongue and peritoneal cavity, that are CD27⁻ CD25⁺ and predominantly use a Vγ6Vδ1⁺ TCR,^22,32 and also of CD27⁻γδ cells from secondary lymphoid organs⁹ (Fig. 1b). A common feature of these γδ subsets is their potent IL-17A secretion en masse in response to cytokines such as IL-1β and IL-23.^33,34 Indeed, this characteristic strongly predicts an innate-like role for these γδ subsets in diverse immune responses.

The capacity to secrete IFN-γ is also a feature of murine γδ cell populations that are found in secondary lymphoid tissues, and organs such as the lung.⁹ Unlike the IFN-γ-secreting DETC and NKT-like γδ subset, these CD27⁺γδ cells display a naive CD44⁻ CD62L⁺ CD122⁻ phenotype consistent with an absence of TCR ligation during development.^3,9 They also possess a polyclonal TCR repertoire (using mainly Vγ1 or Vγ4), and expand extensively when activated through TCR-γδ.^9,35 However, it remains to be determined whether these cells respond to environmental stimuli en masse in an innate-like manner, or whether their diverse TCR-γδ specificities and considerable proliferative potential allow adaptive-like TCR-driven clonal expansions in response to foreign antigen challenge.

A sizable yet enigmatic subset of γδ cells permanently resides in the epithelial layers of the gastrointestinal tract.³⁶ These γδ intraepithelial lymphocytes (IELs) can be generated in gut-associated lymphoid tissue (e.g. cryptopatches)³⁷ and are present (to ∼ 25% of normal levels) in athymic nude animals.³⁸γδ IELs use predominantly Vγ1⁺ or Vγ7⁺ TCRs with limited junctional diversity, although their TCR specificities remain unknown.³⁹γδ IELs lack expression of the conventional T-cell co-receptors CD4 and CD8αβ, but often express CD8αα.³⁶ The vast majority of γδ IELs are CD27⁺ CD122^lo CCR9⁺, but do not express either CD90 or CD2.^9,18,40,41 Functionally, γδ IELs are cytolytic effector cells with an immunoprotective role in the gut,^36,40 especially in young animals,⁴² and largely through the production of cytokines such as IFN-γ.⁴³ Nonetheless, γδ IELs are also immunomodulatory. For example, adult mice lacking γδ IELs display exaggerated intestinal damage in response to Eimeria vermiformis infection, as a result of failure to control αβ T-cell responses.⁴⁴

Human γδ cells

Human γδ cells, like their murine counterparts, are a minor population (1–10% of nucleated cells) in peripheral blood but are abundant in tissues, especially in epithelial layers.²⁴ For identification purposes, they are usually sub-divided based on use of one of two variable regions of TCR-δ; Vδ1 or Vδ2.¹ Vδ1⁺γδ cells are the predominant subset found at mucosal surfaces, and so share certain characteristics with murine γδ IELs (see below). By contrast, Vδ2⁺γδ cells (that are almost exclusively Vγ9⁺) largely dominate the peripheral blood (Vγ9 is often referred to as Vγ2 in an alternative nomenclature).^45–48 Indeed, γδ cells expressing a Vγ9Vδ2⁺ TCR-γδ can sometimes identify > 50% of blood leucocytes after certain bacterial or parasitic infections.⁴⁹

Vγ9Vδ2⁺γδ cells

A γδ population with the specific features of Vγ9Vδ2⁺ cells are found only in humans and higher primates (the absence of an equivalent subset in rodents making study of Vγ9Vδ2⁺ cells problematic). Vγ9Vδ2⁺ cells are unique in their recognition of low-molecular-weight non-peptide phosphoantigens; e.g. (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), an intermediate metabolite from the 2-C-methyl-d-erythritol 4-phosphate pathway of microbial isoprenoid biosynthesis.^50–52 Nanomolar concentrations of HMB-PP lead to rapid TCR-dependent activation of Vγ9Vδ2⁺ cells, enabling them to respond to a diverse range of pathogens, including Mycobacterium tuberculosis⁵³ and Plasmodium falciparum.⁵⁴ Vγ9Vδ2⁺ cells are also indirectly activated by aminobisphosphonates and alkylamines. These compounds inhibit farnesyl diphosphate synthase, an enzyme in the mevalonate pathway of isoprenoid synthesis (used by eukaryotic cells), leading to accumulation of the stimulatory phosphoantigen isopentenyl pyrophosphate (IPP).⁵⁵ Interestingly, elevated IPP levels are also characteristic of many human tumours, rendering them potential targets for Vγ9Vδ2⁺ cells.^56–58 Indeed, although the mechanism by which phosphoantigens activate the Vγ9Vδ2⁺ TCR remains unclear, administration of bisphosphonates such as zoledronate (plus IL-2) are generating encouraging Vγ9Vδ2⁺ cell responses against a range of tumours in the clinic.^57,58

Heterogeneity within the Vγ9Vδ2⁺ subset

Vγ9Vδ2⁺ cells are often sub-divided on surface expression of CD45RA and CD27 (Fig. 2a); markers more commonly used to identify the naive, effector or memory status of conventional αβ T cells.⁵⁹ Nonetheless, CD27 does not obviously identify a γδ subset in human comparable to the CD27⁺γδ subset in mouse (i.e. pre-committed to robust IFN-γ secretion). Instead, CD27 and CD45RA identify four Vγ9Vδ2⁺ subsets. ‘Naive’ (T_naive) CD45RA⁺ CD27⁺ Vγ9Vδ2⁺ cells generally comprise 10–20% of those in peripheral blood (addition of a further marker; CD11a, suggests a slightly lower percentage),⁶⁰ but are the major Vγ9Vδ2⁺ subset in lymph nodes, in keeping with their expression of CCR7 and CD62L but absence of CCR2, CCR5, CCR6 or CXCR3. T_naive cells proliferate at relatively high concentrations of IPP (10⁻⁴–10⁻³ m), but do not secrete IFN-γ.⁵⁹ After activation for 12 days with IPP+IL-2, T_naive cells become largely CD45RA⁻ CD27⁺. These ‘Central Memory’ (T_CM) cells are CD45RO⁺, but remain CCR7⁺ CD62L⁺ (Fig. 2a). In healthy individuals T_CM cells represent ∼ 25% and ∼ 50% of Vγ9Vδ2⁺ cells in lymph nodes and peripheral blood, respectively. T_CM cells appear to proliferate at much lower concentrations of IPP (10⁻⁶–10⁻⁷ m), but can secrete only low levels of IFN-γ.⁵⁹

Figure 2.

Human Vγ9Vδ2⁺γδ cells can be sub-divided using surface expression of CD45RA and CD27, and show remarkable functional plasticity after activation. (a) CD45RA⁺ CD27⁺ T_naive cells give rise to CD45RA⁻ CD27⁺ T_CM cells on activation with isopentenyl pyrophosphate + interleukin-2 (IPP+IL-2), but do not secrete interferon-γ (IFN-γ). Central memory T (T_CM) cells generate CD45RA⁻ CD27⁻ effector memory T (T_EM) cells after activation with IPP+IL-2, or CD45RA⁺ CD27⁻ T_EMRA cells in the presence of IL-15. T_EM cells can secrete abundant IFN-γ, whereas T_EMRA cells are mainly cytotoxic. Prol; proliferative capacity, Kill; cytotoxic capacity; red triangles indicate CD27 expression; blue triangles indicate CD45RA expression; green triangles indicate CD62L expression; (b) Vγ9Vδ2⁺ cells display extensive plasticity after activation with phosphoantigen [(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) or IPP] in the presence or absence of various cytokines as indicated. ‘?’ indicates uncertainty as to the potential of T_naive, T_CM, T_EM and T_EMRA to generate the indicated effector subsets. Expression of functionally relevant genes and characteristics are indicated. TGF-β, transforming growth factor-β; TNF-α, tumour necrosis factor-α.

After 12 days of activation with IPP+IL-2, T_CM cells generate CD45RA⁻ CD27⁻‘Effector Memory’ (T_EM) cells that are CD45RO⁺, CCR7⁻ CD62L⁻, but positive for the tissue-associated chemokine receptors CCR2, CCR5, CCR6 and CXCR3⁵⁹ (Fig. 2a). Unsurprisingly, T_EM cells are scarce in lymph nodes, but are readily detected in blood and inflammatory sites. T_EM cells secrete abundant IFN-γ and tumour necrosis factor-α (TNF-α) when activated with IPP+IL-2, but their capacity for proliferation is reduced compared with T_naive and T_CM cells. T_CM cells also appear to generate a CD45RA⁺ effector memory (T_EMRA) population when activated with IL-15.⁶¹ These cells are virtually absent from blood, are CD27⁻, CCR7⁻ and CD62L⁻, but express CCR5 and CXCR3, a phenotype shared by a subset of CD8⁺αβ T cells that display robust cytotoxic potential. Consistent with this, T_EMRA cells express abundant perforin, granulysin and N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT)-esterase, and readily display cytolytic activity (but little production of IFN-γ). They also express CD16, KIR2DL1-3 and NKG2A/CD94. However, T_EMRA cells are unresponsive to further TCR stimulation and have little proliferative capacity, a phenotype consistent with a terminally differentiated state.

Although T_naive, T_CM, T_EM and T_EMRA Vγ9Vδ2⁺ subsets can be identified, whether these represent true naive, effector and memory subsets, comparable to those observed for αβ T cells, is still unclear.⁶² Nonetheless, the assessment of Vγ9Vδ2⁺ cells on these criteria appears to correlate with objective clinical outcomes.⁶³ For example, in a phase I trial of patients with advanced solid tumours, increased proportions of T_CM and T_EM from patients’ peripheral blood was predictive for good cell expansion in vitro with zoledronate+IL-2, which in turn correlated with better clinical responses after subsequent adoptive transfer.⁶⁴

Notwithstanding the utility of CD45RA and CD27 to describe functional subsets of Vγ9Vδ2⁺ cells, further useful surface markers have also been identified. For example, cytotoxic potential appears to correlate with increased CD56 and CD16 expression following activation of Vγ9Vδ2⁺ cells with phosphoantigen and IL-2 for 10–14 days.⁶⁵ The IPP-expanded CD56⁺, but not CD56⁻, Vγ9Vδ2⁺ cells efficiently killed several tumour lines in a perforin/granzyme-dependent manner that also required NKG2D. Interestingly, tracking individual clones using Vγ9 CDR3 regions appeared to reveal that the capacity for CD56 expression was a stable pre-existing characteristic of individual Vγ9Vδ2⁺ cells that was unrelated to TCR specificity.⁶⁶ However, whether this reveals a cytotoxic lineage for Vγ9Vδ2⁺ cells remains uncertain.

Finally, approximately half the Vγ9Vδ2⁺ subset also expresses the skin homing receptor cutaneous lymphocyte-associated antigen (CLA).⁶⁷ Dermal CLA⁺γδ cells were recently implicated in psoriasis, in which they secrete abundant IFN-γ and TNF-α, and high levels of CCL3, CCL4, CCL5 and CXCL8. However, as with CD56 and CD16, it is still unclear how the use of CLA to identify Vγ9Vδ2⁺ subsets overlaps with the method defined by CD45RA and CD27.

Remarkable plasticity of activated Vγ9Vδ2⁺ cells

For a cell type generally considered innate, the Vγ9Vδ2⁺ subset displays remarkable functional plasticity upon TCR activation that is easily comparable with their more illustrious cousin – the CD4⁺αβ T helper cell. Such plasticity was initially demonstrated in vitro through polarization of IPP-activated Vγ9Vδ2⁺ cells; IL-12 and anti-IL-4 antibody generating IFN-γ-secreting Th1-like cells; IL-4 plus anti-IL-12 antibody generating IL-4-producing Th2-like cells.⁶⁸ An IFN-γ/TNF-α-secreting Th1-like phenotype is also generated following activation with HMB-PP plus IL-2, although the Th2-associated cytokines IL-5 and IL-13 are also produced⁶⁹ (Fig. 2b). By contrast, HMB-PP activation of Vγ9Vδ2⁺ cells in the presence of IL-21 promotes a follicular helper (T_FH) -like phenotype that is characterized by increased expression of IL-21R, CD244, CXCL10 and CXCL13, and trafficking to lymph node germinal centres.⁷⁰ Somewhat surprisingly, Vγ9Vδ2⁺ cells have also been reported to express Foxp3 and to display regulatory activity after IPP activation with IL-15 and transforming growth factor-β,⁷¹ while 18–24 hr IPP stimulation (alone) of tonsillar Vγ9Vδ2⁺ cells appears to induce considerable antigen-presenting cell-like activity, with accompanying surface expression of MHC-II, CD80, CD86, CD40 and CD54.⁷²

The production of IL-17A by human γδ cells, unlike for murine γδ cells, has been difficult to demonstrate.⁷³ Nonetheless, T_naive Vγ9Vδ2⁺ cells (especially those from neonates)⁷⁴ can adopt an IL-17A-secreting Th17-like phenotype if cultured in the presence of various combinations of IL-1β, IL-6, transforming growth factor-β and IL-23 in media containing aromatic hydrocarbons.⁷⁵ These IL-17A Vγ9Vδ2⁺ cells are CD161⁺ CCR6⁺ TRAIL⁺ FasL⁺ with a largely CD45RA⁺ CD27⁻ T_EMRA phenotype, and have been identified in psoriasis,⁶⁷ and in the cerebrospinal fluid of patients with bacterial meningitis.⁷⁵ However, these IL-17A-expressing T_EMRA cells appear distinct from the previously described cytotoxic T_EMRA cells because they do not express perforin or NKG2D.⁷⁵

The extensive plasticity of activated Vγ9Vδ2⁺ cells contrasts sharply with murine γδ cells that demonstrate considerable pre-commitment to cytokine production in the thymus.⁹ However, it is still unclear whether this plasticity relates equally well to all CD45RA/CD27-defined Vγ9Vδ2⁺ subsets,⁷⁵ or to what extent possible pre-commitment, for example to a CD56⁺ cytotoxic fate,⁶⁶ regulates subsequent effector function.

Human Vδ1⁺ cells; the cousin of murine γδ IELs?

Human Vδ1⁺ cells are the major γδ population at epithelial sites such as the intestine and skin.^76,77 Similar to murine γδ IELs, Vδ1⁺ cells frequently express CD8, and display a cytotoxic, Th1-like phenotype characterized by IFN-γ secretion.⁷⁶ This notwithstanding, Vδ1⁺ cells also appear to play a significant role in tissue homeostasis and repair, as demonstrated by insulin-like growth factor-1 production in wound healing.⁷⁸ Indeed, consistent with epithelial immunosurveillance,²⁴ Vδ1⁺ cells kill a range of epithelial tumours,^79,80 possibly through recognition of stress-induced MHC class I-related molecules MICA and MICB.^80,81 Vδ1⁺ cells are also known to respond to autologous and/or endogenous phospholipids presented by CD1,⁸² and display TCR-driven clonal expansions in response to cytomegalovirus (along with the minor Vδ3⁺ and Vδ5⁺ subsets),^83,84 and possibly HIV⁸⁵ and malaria.⁸⁶

Like Vδ2⁺ cells, Vδ1⁺ cells can be sub-divided based on CD45RA and CD27 expression.⁶⁰ In contrast to Vδ2⁺ cells, the majority of adult blood Vδ1⁺ cells are CD45RA⁺, being evenly split into an IL-2-secreting CD27⁺ CD11a^lo‘naive’ subset, and an IFN-γ-secreting CD27⁻ CD11a^hi‘non-naive’ population.⁶⁰ Unsurprisingly, ∼ 80% of cord blood Vδ1⁺ cells are naive (compared with ∼ 50–60% for Vδ2⁺ cells), dropping to ∼ 30–40% by 2 years of age.⁶⁰ By contrast, < 5% of Vδ2⁺ cells are naive by 1 year of age, reflecting significant expansion of a restricted number of phosphoantigen-reactive Vγ9Vδ2⁺ clones. The fact that the percentage of naive Vδ1⁺ cells remains relatively constant in peripheral blood until late middle age may suggest a constant thymic production.⁶⁰

In addition to expressing common surface markers such as CD2, ICAM-1 and NKG2D,⁸⁷ Vδ1⁺ cells also display several notable differences when compared with Vδ2⁺ cells. For example, they are CD5^dull CD28^lo, but express abundant CD57 that correlates with high perforin expression.⁸⁸ On activation of Vδ1⁺ cells through their TCR (e.g. with phytohaemagglutinin), in the presence of either IL-2 or IL-15, the natural cytotoxicity receptors NKp30, NKp44 and NKp46 are up-regulated, which correlates with potent tumour-directed cytotoxicity and CD56 expression.^89,90 By contrast, HMB-PP+IL-2-activated Vδ2⁺ cells do not express natural cytotoxicity receptors; instead they use the NKG2D pathway as their main mechanism of targeting tumours.^89,91

Concluding remarks

Recent studies have begun to characterize the subset complexity of mouse and human γδ cells. Interestingly, certain subsets, such as murine DETC or human Vγ9Vδ2⁺ cells, are restricted to certain species, whereas certain useful surface markers, such as CD27, do not appear to identify comparable subsets across species. Nonetheless, this methodical dissection of γδ cell repertoires has exposed the critically important innate-like, and possibly adaptive-like, functional roles for γδ cells in diverse disease settings. A further understanding of this largely unanticipated γδ cell biology should reveal much about the relationship between early tissue-associated immune surveillance and the powerful adaptive responses that follow and should perhaps provide unexpected therapeutic opportunities for the clinic.

Disclosures

The authors have no financial disclosures.