The Multifunctionality of Human Vγ9Vδ2 γδ T Cells: Clonal Plasticity or Distinct Subsets?

Authors


D. Kabelitz, Institute of Immunology, University of Kiel, Arnold-Heller-Str. 3, Haus 17, D-24105 Kiel, Germany. E-mail: kabelitz@immunologie.uni-kiel.de

Abstract

The dominant subset of γδ T cells in human peripheral blood expresses Vγ9 paired with Vδ2 as variable TCR elements. Vγ9Vδ2 T cells recognize pyrophosphates derived from the microbial non-mevalonate isoprenoid biosynthesis pathway at pico- to nanomolar concentrations. Structurally related pyrophosphates are generated in eukaryotic cells through the mevalonate pathway involved in protein prenylation and cholesterol synthesis. However, micromolar concentrations of endogenous pyrophosphates are required to be recognized by Vγ9Vδ2 T cells. Such concentrations are not produced by normal cells but can accumulate upon cellular stress and transformation. Therefore, many tumour cells are susceptible to γδ T cell–mediated lysis owing to the overproduction of endogenous pyrophosphates. This explains why Vγ9Vδ2 T cells contribute to both anti-infective and anti-tumour immunity. Ex vivo analysed Vγ9Vδ2 T cells can be subdivided on the basis of additional surface markers, including chemokine receptors and markers for naïve and memory T cells. At the functional level, Vγ9Vδ2 T cells produce a broad range of cytokines, display potent cytotoxic activity, regulate αβ T cell responses, and – quite surprisingly – can act as professional antigen-presenting cells. Thus, an exceptional range of effector functions has been assigned to a population of T cells, which all recognize invariant exogenous or endogenous pyrophosphates that are not seen by any other immune cell. Here, we discuss whether this plethora of effector functions reflects the plasticity of individual Vγ9Vδ2 T cells or can be assigned to distinct subsets.

Introduction

In addition to antibody-producing B-cells, T cells represent the second effector cell type of the adaptive immune system. Both cell populations harbour a myriad of lymphocytes carrying clonally variable antigen receptors (surface immunoglobulin and T-cell receptor [TCR], respectively) destined to recognize antigens with incredible precision. In addition, however, both lymphocyte populations include subsets with unconventional features. In the B-cell compartment, this refers to the so-called B1 lymphocytes which secrete natural IgM antibodies and polyreactive antibodies with recurrent idiotypes, and which display additional features of innate immune cells [1, 2]. Within the T cell compartment (i.e. lymphoid cells expressing a CD3-associated TCR heterodimer), at least two subsets of ‘unconventional’ cells have been identified, that is, natural killer-like T cells (NKT cells) and γδ T cells. The majority of NKT cells express an invariant TCR, which recognizes glycolipid antigens presented by CD1 molecules [3]. γδ T cells on the other hand express a CD3-associated γδ TCR heterodimer instead of the conventional αβ TCR. γδ T cells are a minor T cell subset in the peripheral blood, spleen and lymph nodes but account for a major proportion of intraepithelial lymphocytes in the intestine [4]. Species-specific differences exist with respect to the numbers of γδ T cells in the skin. While γδ T cells are rare in human skin [5], they form a dense network of dendritic-like cells, termed dendritic epidermal T cells (DETC) in the skin of mice [6].

γδ T cells differ from the majority of conventional αβ T cells in several aspects, most importantly with regard to the expression of CD4/CD8 co-receptors and the germline diversity of TCR genes. The majority of γδ T cells lack CD4 or CD8 expression and thus displays a ‘double-negative’ phenotype [4]. Owing to their role as co-receptors for MHC class I or MHC class II molecules, the expression of CD8 or CD4 dictates the MHC restriction pattern of the conventional αβ T cells. Even though low level CD8 expression is present on a subpopulation of γδ T cells and CD4 might be detected on rare γδ T cells, the double-negative phenotype of most γδ T cells is well in line with the lack of MHC restriction in their antigen recognition behaviour [4]. The diversity of the αβ TCR repertoire is largely due to the large (>50) number of Vα and Vβ gene segments in the germline genome, which can be used for TCR gene rearrangement during intrathymic T cell differentiation. In contrast, there are only a few Vγ and Vδ germline genes that can be used to construct functional γδ TCRs. In humans, 6 Vγ genes can be expressed (Vγ2,3,4,5,8,9) together with a similarly small number of Vδ genes [7]. Although the germline repertoire is small, non-germline-encoded mechanisms such as the insertion of N-nucleotides during the process of gene rearrangement are also operating in γδ T cells and contribute tremendously to the diversity of the γδ TCR repertoire. Interestingly, the available Vγ and Vδ genes are not randomly used. γδ T cells expressing certain Vγ and Vδ genes preferentially home to specific anatomical localisations such as the reproductive tract, intestine, skin or blood [8]. In the blood of healthy human adults, approximately 5% of the CD3+ T cells are γδ T cells (with a large degree of inter-individual variability). In most Caucasian donors, the blood γδ T cells are dominated by cells expressing a Vγ9Vδ2-encoded TCR, and Vγ9Vδ2 T cells can account for anywhere between 50% and more than 95% of the blood γδ T cell population [8, 9]. This dominance of the Vγ9Vδ2 TCR is not present from birth on but develops during childhood and then persists throughout adulthood [10]. A plausible explanation for this dramatic age-dependent alteration of the γδ TCR repertoire is a TCR shaping owing to the continuous exposure to environmental microbial ligands during childhood [10]. In the following, we will concentrate on this dominant human γδ T cell population, with a focus on their functional plasticity.

Antigens for Vγ9Vδ2 T cells

γd T cells share features of both innate and adaptive immunity as they sense cellular stress and infection through TCR-dependent recognition of conserved antigens without requirement for antigen processing and MHC-dependent presentation. It has long been known that human Vγ9Vδ2 T cells are rapidly activated by a broad range of bacteria, including Mycobacterium tuberculosis [11]. More importantly, a dramatic but transient increase of peripheral blood γδ T cell numbers takes place during the acute phase of many bacterial and parasitic infections [12]. The microbial antigens recognized by the Vγ9Vδ2 TCR have been identified as phosphorylated intermediates of the non-mevalonate (also called ‘Rohmer’-) pathway of isoprenoid biosynthesis, notably (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) [13]. Many bacteria and some parasites including Plasmodium malariae make use of the non-mevalonate pathway and secrete such phosphorylated molecules. HMB-PP and synthetic analogues such as bromohydrin pyrophosphate (BrHPP) activate Vγ9Vδ2 T cells at pico- to nanomolar concentrations. Structurally related pyrophosphates are also generated in the mevalonate pathway of isoprenoid synthesis involved in protein prenylation and cholesterol synthesis in mammalian cells. The mevalonate pathway intermediate isopentenyl pyrophosphate (IPP) is also recognized by the Vγ9Vδ2 TCR, but micromolar concentrations are required for activation of γδ T cells [14]. Recognition of such phosphorylated non-peptide ligands, collectively termed phosphoantigens, requires surface presentation in a species-restricted manner [15] but presenting molecules have not yet been unambiguously identified [16]. Subtle differences exist in TCR signalling stimulated by anti-CD3 mAb versus phosphoantigen in Vγ9Vδ2 T cells. While anti-CD3 mAb induces rapid but transient phosphorylation of kinases such as ERK1/2 and p38 associated with down-modulation of cell surface TCR expression, phosphoantigens induce sustained and prolonged protein phosphorylation and no TCR surface modulation [17, 18]. Importantly, the recognition of phosphoantigens occurs primarily via germline-encoded regions of the Vγ9Vδ2 TCR involving all CDR loops [19]. This implies that most if not all Vγ9Vδ2 T cells use their TCR as a pattern recognition receptor for sensing exogenous or endogenous pyrophosphates that signal danger associated with infection or cellular stress/transformation. A critical step in the regulation of IPP production in mammalian cells is the IPP-processing enzyme farnesyl pyrophosphate synthase (FPPS). Inhibition of FPPS by siRNA-mediated silencing or inhibition of enzyme activity by aminobisphosphonates results in increased levels of IPP associated with enhanced susceptibility of tumour cells to γδ T cell–mediated lysis [20–22] and monocyte-dependent stimulation of blood γδ T cells [23, 24]. Moreover, bacterial infection may also contribute to a dysregulation of the host mevalonate pathway pointing to both microbial and endogenous phosphoantigen stimulation of Vγ9Vδ2 T cells upon infection [25, 26]. Altogether, the recognition of structurally related pyrophosphates generated during infection or cellular dysregulation provides a rationale why the very same population of Vγ9Vδ2 T cells plays a pivotal role in anti-infective and anti-tumour immunity [8, 27, 28]. Given the strong and unique response of human (and primate) Vγ9Vδ2 T cells to microbial pyrophosphates, it is surprising to note that there is no homologous γδ TCR expressed in mice [4]. Apart from the evolutionary aspect, this implies that conventional mouse models cannot be used to investigate the role of pyrophosphate-recognizing γδ T cells in infection and tumour immunity.

Surface markers

In addition to the TCR, Vγ9Vδ2 T cells express numerous molecules potentially associated with different stages of differentiation, previous antigen exposure, homing pattern and function. αβ T cells are commonly classified into naïve (Tnaïve), central memory (TCM), effector memory (TEM) and terminally differentiated effector memory (TEMRA) cells based on CD45RA, CD27 and CCR7 surface expression [29]. Early studies indicated that most peripheral blood Vγ9Vδ2 T cells in adults express CD45RO, in line with their preactivated (‘memory’) phenotype [30, 31], whereas cord blood Vγ9Vδ2 T cells are largely CD45RO negative [32]. More recently, CD45RA and CD27 have been used to identify naïve (CD45RA+CD27+), TCM (CD45RACD27+), TEM (CD45RACD27) and TEMRA (CD45RA+CD27) subpopulations within circulating Vγ9Vδ2 T cells. CD27, a member of the TNF receptor family with known co-stimulatory activity, is expressed on a major proportion of Vγ9Vδ2 T cells [33]. CD45RA+CD27+ Tnaïve cells account for approximately 15–20%, CD45RACD27+ TCM for 40–60%, CD45RACD27 TEM for ≈30% and CD45RA+CD27 TEMRA for ≈7% of the Vγ9Vδ2 T cell population, with substantial inter-individual variability [34, 35]. While the Tnaïve and TCM subsets readily proliferate in response to phosphoantigen stimulation, the TEM and TEMRA subsets expand in response to homoeostatic cytokines, notably IL-15, but poorly to antigenic stimulation [35]. Alterations in the subset distribution are observed under pathophysiological conditions. As an example, CD45RACD27 TEM are strongly reduced in HIV-infected patients and during active pulmonary tuberculosis [32]. Vγ9Vδ2 T cell can be further subdivided according to the pattern of expressed chemokine receptors [36]. The CD45RA (mostly TEM) subset strongly expresses CCR5, a receptor for inflammatory chemokines including RANTES and MIP-1β [34, 37], while the Tnaïve and TCM subsets express the lymph node homing receptor CCR7 together with CD62L [34]. The skin homing chemokine receptor CCR6 is expressed on 20–40% of circulating Vγ9Vδ2 T cells in healthy adults [38]. Interestingly, a selective reduction in CCR6+ Vγ9Vδ2 T cells is observed in the blood of psoriasis patients, seemingly due to the recruitment of these cells from blood to the inflamed skin [38]. Furthermore, CXCR5 has been shown to identify approximately 15% of blood Vγ9Vδ2 T cells which simultaneously express CCR7, CD45RO and CD62L. CXCR5+γδ T cells secrete Th2 cytokines IL-4 and IL-10 and help B-cells in antibody production [39].

Additional classes of surface markers expressed on subpopulations of blood Vγ9Vδ2 T cells include NK-cell-related antigens/NK-receptors, Fc-receptors, co-stimulatory/-inhibitory receptors, Toll-like receptors and others. The activating natural killer group 2 member D (NKG2D) molecule, a lectin-like type II membrane receptor for stress-inducible and tumour-expressed MHC class I related molecules A/B (MICA/MICB), is expressed on essentially all γδ T cells [40] and provides a co-stimulatory signal [41] but may also directly activate Vγ9Vδ2 T cells independently of TCR signalling [42]. Another CD94-associated receptor, NKG2C which recognizes the non-classical MHC class I HLA-E molecules, is absent on γδ T cells from many donors but present on up to 70% of γδ T cells from other apparently healthy donors [43]. In these instances, NKG2C is confined to the cytotoxic CD56+ CD16+ TEMRA subset [43]. Importantly, up to 70% of blood γδ T cells also express the HLA-E-specific inhibitory receptor NKG2A [43, 44], and a regulatory role of NKG2A in the control of Vγ9Vδ2 activation including a cross-talk between NKG2A/NKG2C has been described [43, 44]. Furthermore, the killer cell-lectin-like receptor G1 (KLRG1) is also expressed on many Vγ9Vδ2 T cells, preferentially on the TEM subset but the functional significance has not yet been precisely defined [45]. Further surface markers with functional relevance for the Vγ9Vδ2 T cells are CD56 and CD16. Among the few CD56+ CD3+ T cells in peripheral blood, approximately 15% are γδ T cells [46]. CD56 is up-regulated upon phosphoantigen stimulation, and CD56+ Vγ9Vδ2 T cells display more potent anti-tumour cytotoxicity than the CD56 population [47]. CD16, the low affinity type 3 receptor for Fc portion of IgG (FcγRIIIA), is expressed on the CD45RA+CD27 TEMRA subset of circulating Vγ9Vδ2 T cells [48] and is up-regulated upon phosphoantigen activation [49]. The expression of CD16 confers to Vγ9Vδ2 T cells potent ADCC activity in the presence of tumour-targeting monoclonal antibodies [50].

Like conventional αβ T cells, γδ T cells express several activating and inhibitory co-stimulator receptors. CD27 used to identify Tnaïve and TCM Vγ9Vδ2 T cells, transmits survival and proliferative signals upon encounter of its ligand CD70 [33]. The prototype co-stimulatory receptor CD28 is expressed by 40–60% peripheral blood γδ T cells, but its role in γδ T cell activation is less clear than for αβ T cells [51]. The inducible co-stimulatory receptor ICOS is absent on ex vivo analysed Vγ9Vδ2 T cells but is readily induced upon in vitro activation [39, 51]. Programmed cell death-1 (PD-1) is a negative regulator which is low or absent on circulating Vγ9Vδ2 T cells but again is rapidly induced upon cellular activation [52]. Together, there is strong evidence that the initiation and termination of Vγ9Vδ2 T cell activation is subject to similarly complex regulation by receptor–ligand interactions as in αβ T cells [51].

Finally, it should be mentioned that Vγ9Vδ2 T cells also express innate immune receptors, notably Toll-like receptors (TLR). TLR2 is readily detected on the cell surface, as is the intracellularly located TLR3. The corresponding TLR2 and TLR3 ligands provide potent co-stimulation to TCR-activated Vγ9Vδ2 T cells [53–55]. A summary of the discussed marker profile of ex vivo analyzed Vγ9Vδ2 T cells is presented in Table 1. It should be noted that this is not an exhaustive or complete list.

Table 1.   Subpopulations of Vγ9Vδ2 T cells: ex vivo analysis.
PhenotypeFunctionProportion/expressionReferences
CD45ROMemory markerMajority (>90%)[29, 30]
CD27Co-stimulationMajority (≈80%)[33]
CD45RA+ CD27+Naïve15–20%[34, 35]
CD45RA CD27+Central memory40–60%[34, 35]
CD45RA CD27Effector memory≈30%[34, 35]
CD45RA+CD27Terminally differentiated memory≈7%[35]
CCR5Receptor for inflammatory chemokinesOn Tnaive and TCM[34, 36]
CCR7Lymph node homingOn TEM and TEMRA[34]
CCR6Skin homing20–40%[38]
CXCR5B-cell help, Th2 cytokines≈15%[39]
NKG2D(Co-)activating receptorMajority (>90%)[40, 41]
NKG2CActivating receptor0 to 70%[42]
NKG2AInhibitory receptor≈70%[42, 43]
CD56Cytotoxic activity15–20%[46, 47]
CD16Cytotoxic activity, ADCCOn TEMRA[48]
CD28Co-stimulatory receptor40–60%[51]
ICOSInducible co-stimulatorAbsent[39, 51]
PD-1Inhibitory receptorMinimal[52]
TLR2Peptidoglycan receptorMajority[53]
TLR3dsRNA receptor (intracellular)Majority[53, 54]

Cytokine production

Within a few hours after TCR-dependent activation, Vγ9Vδ2 T cells secrete high amounts of cytokines including TNF-α and IFN-γ and chemokines including RANTES and MIP-1α, which can be further augmented by co-stimulatory signals such as TLR ligands [53, 54, 56]. TNF-α secretion is also triggered through engagement of CD16 [49]. While these data would suggest that Vγ9Vδ2 T cells are primed towards a Th1 phenotype, it is obvious that the cytokine spectrum of Vγ9Vδ2 T cells is subject to regulation by the environmental milieu. Th1-driving conditions (IL-12, anti-IL-4) push Vγ9Vδ2 T cells towards high IFN-γ and low if any IL-4 production, while anti-IL-12 plus IL-4 induce a prominent Th2 phenotype with strong IL-4 production [57]. IL-21 induces a TCM phenotype with low IFN-γ/IL-4 production [58] but high expression of the follicular B-cell attracting chemokine CXCL13 [59]. The cytokine spectrum can be also correlated to the expressed chemokine receptor pattern. On the basis of CXCR5 expression, TNF-α and IFN-γ production could be assigned to the CXCR5 CD27 TEM subset, whereas IL-2, IL-4 and IL-10 secretion was almost exclusively restricted to CXCR5+ CD27+γδ T cells [39]. An in-depth characterization of the gene profile differentially induced in phosphoantigen-activated Vγ9Vδ2 T cells exposed to IL-2 versus IL-4 versus IL-21 revealed the pleiotropy of γδ T cell cytokines and chemokines at the population level but did not allow to dissect the pleiotropy at the level of an individual Vγ9Vδ2 T cell [59].

IL-17 has recently attracted much attention because of its pivotal role in regulating neutrophil migration and inflammation. In the mouse, γδ T cells are a major innate source of IL-17 [60]. A proportion of Vγ9Vδ2 T cells isolated from adult blood can be differentiated in vitro into IL-17 producing cells by cytokine cocktails including IL-1β, IL-6, TGF-β and IL-23 [61, 62], some of which simultaneously produce IFN-γ [61]. IL-23 alone in conjunction with a γδ T cell–specific stimulus seems to be able to induce IL-17 production in neonatal Vγ9Vδ2 T cells [63]. Taken together, the Vγ9Vδ2 T cells have the potential for diverse cytokine production.

Regulatory potential

Regulatory T cells (Treg) are essential for maintaining peripheral immune tolerance and the regulation of immune responses. In addition to the naturally occurring prototype CD4+ CD25highCD127FoxP3+ Treg, several inducible regulatory T cells have been described [64]. The proliferative response of Vγ9Vδ2 T cells to phosphoantigen stimulation is inhibited by CD4+ CD25high Treg [65, 66], and TGF-β has been identified as the inhibitory effector molecule which might derive from Treg or tumour cells [67]. Proliferation and cytokine production of phosphoantigen-reactive γδ T cells is also inhibited by mesenchymal stem cells through a prostaglandin E2-dependent pathway [68]. Quite surprisingly, however, γδ T cells themselves can exert regulatory activity on αβ T cells. Earlier studies reported that peripheral blood γδ T cells inhibited CD4 T cell proliferation even more potently than Treg did and suggested on the basis of differential TGF-β production that Vδ1 cells would be more suppressive than Vδ2 [69]. Notably, FoxP3 was not detected in γδ T cells [69]. Similarly, phosphoantigen-activated purified blood γδ T cells were found to inhibit the proliferative expansion of purified CD4 T cells in response to superantigens or tetanus toxoid protein antigen [70]. Given the known role of TGF-β in the induction of FoxP3 expression in ‘non-natural’ Treg [71], the relationship between inducible FoxP3 expression and regulatory activity was investigated. Kang et al. [72] observed that although freshly isolated γδ T cells were FoxP3-negative, FoxP3 expression was induced upon TCR activation in vitro, even in the absence of TGF-β. However, the FoxP3-expressing γδ T cells did not display regulatory activity. In contrast, Casetti et al. [73] reported the induction of FoxP3 expression and regulatory activity in Vγ9Vδ2 T cells upon antigen stimulation in the presence of TGF-β and IL-15. A note of caution has been raised regarding the intracellular staining pattern of different FoxP3 antibodies, suggesting that some antibodies (e.g. clone PCH101) might be less specific (and in consequence stain ‘activated’ cells more non-specifically) than others (e.g. clone 259D) [74]. Our own experiments indicate that highly purified γδ T cells inhibit CD4 T cell proliferation in an APC-free co-culture system with anti-CD3/CD28-coated beads as a stimulus. Although addition of TGF-β induces some γδ T cell FoxP3 expression (using clone 259D) in these cultures, inhibition also occurs in the absence of added TGF-β (Peters C, unpublished data). Taken together, it seems safe to conclude that human γδ T cells can display regulatory activity, even though the precise role of FoxP3 and additional Treg-associated transcription factors such as Helios [75] is presently unclear. Furthermore, the possible in vivo significance of regulatory Vγ9Vδ2 T cells is uncertain. Available evidence suggests that regulatory γδ T cells might play a role in controlling tumour-infiltrating lymphocytes [76], tolerance in liver graft recipients [77, 78], CD4 T cells in systemic lupus erythematosus [79] and cytotoxic αβ T cells in coeliac disease [80]. In these instances, however, regulatory γδ T cells seem to belong to the Vδ1 rather than the Vδ2 subset [76–80]. This was not specifically addressed in coeliac disease [80] but appears likely in view of the dominance of Vδ1 cells among intestinal intraepithelial T cells [8].

Cytotoxic activity

Vγ9Vδ2 T cells are potent killer cells, making use of both the secretory (perforin/granzyme) and the death receptor/ligand pathways when delivering the lethal kiss to bacteria- or virus-infected cells [81–83] and tumour cells [47, 84]. As discussed above, the Vγ9Vδ2 TCR recognizes microbial and tumour-derived pyrophosphates [20, 26]. In addition, cytotoxic effector activity of Vγ9Vδ2 T cells can be directly triggered by NKG2D signalling, independently of TCR activation [42]. The contribution of TCR- versus NKG2D-dependent triggering of cytotoxicity depends on the tumour target and clonal Vγ9Vδ2 T cell interaction and may vary between tumour cells [85]. Not all Vγ9Vδ2 T cells display similar cytotoxic activity. This seems to be primarily confined to the CD56+ and/or CD16+ subsets [47, 50]. Given the wide-spread interest to develop γδ T cell–based tumour immunotherapies [86, 87], the identification of pathways modulating the cytotoxic activity is an important issue.

Antigen-presenting capacity

Recent studies have attested yet another – quite unexpected – function to the Vγ9Vδ2 T cells, that is, the capacity for ‘professional’ antigen presentation. While activated human T cells can present peptide antigens to other T cells because of their induced expression of HLA class II molecules [88], Moser’s group has shown that phosphoantigen-activated Vγ9Vδ2 T cells can specifically pick up complex protein antigens and virus-loaded cellular debris, and process such molecules for presentation via the MHC class I- or class II pathway to CD8 or CD4 αβ T cells [89–91]. Within 18 h of activation, phosphoantigen-stimulated Vγ9Vδ2 T cells not only up-regulated surface molecules important for antigen presentation such as HLA class II, CD80/CD86 and CD40 but also acquired the capacity to process and present to CD4 T cells PPD and tetanus toxoid protein antigens [89]. More importantly, activated Vγ9Vδ2 T cells were capable of processing exogenous soluble viral and tumour proteins and cross-presenting peptide-HLA class I complexes to antigen-specific CD8 αβ T cells [90]. This capacity for cross-presentation is a specific feature professional APC, for example, dendritic cells (DC). A comparative analysis of the antigen presentation machinery between γδ T cells and DC revealed an even superior ability of activated Vγ9Vδ2 T cells to translocate soluble antigen into the cytosol for processing via the proteasomal degradation pathway [91]. The ability of Vγ9Vδ2 to phagocytose particulate antigens and execute HLA class II-dependent antigen presentation has been confirmed by other groups [92]. It has not yet been determined whether Vγ9Vδ2 T cells can act as professional APC also in vivo. If confirmed, Vγ9Vδ2 T cells might represent an attractive alternative cellular source for antigen-presenting cells in cell-based immunotherapies, because of the ease of their large-scale expansion in vitro [93]. In the context of tumour immunotherapy, the opsonisation of tumour cells by tumour-targeting monoclonal antibodies strongly enhances the γδ T cell–dependent cross-presentation to HLA class I-restricted tumour antigen-specific αβ T cells [94]. Therefore, the combined activation (or adoptive transfer) of Vγ9Vδ2 T cells plus application of tumour-directed mAb might be beneficial through at least two mechanisms, that is by exploiting γδ T cells as ADCC effector cells [50] and as cross-presenting APC for activation of tumour-reactive αβ T cells [94]. Albeit less well studied in the murine system, activated γδ T cells from immunized mice were also shown to up-regulate MHC class II, CD80 and CD40, and to present peptides to antigen-specific αβ T cells [95]. Moreover, a recent study suggested that murine γδ T cells might be capable of cross-presenting skin-derived antigens to CD8 αβ T cells, as evidenced in a skin transplantation model employing TCRγδ-deficient mice [96]. Finally, APC function was also reported for bovine γδ T cells in a virus infection model where the activated γδ T cells produced IFN-γ, exerted NK-like killer activity and presented protein antigens [97].Therefore, sharing the capacity for phagocytosis and antigen presentation with myeloid cells underscores the emerging picture of γδ T cells as innate immune cells bridging innate and adaptive immunity.

Clonal plasticity or multiple subsets?

As summarized here, Vγ9Vδ2 T cells display a surprisingly broad array of functional activities. Is this plasticity an intrinsic feature of the Vγ9Vδ2 T cell population or can it be assigned to distinct subsets? This is an important issue when it comes to the design of γδ T cell–based therapies. While it might be of interest to boost the γδ T cell regulatory activity in some instances, other applications would aim at optimizing for example, the APC or the cytotoxic activities. In terms of cytokine production and cytotoxic activity, Vγ9Vδ2 T cells can be divided into subsets on the basis of surface marker expression. Highest IFN-γ secretion is confined to the CD45RACD27 TCM subset, while strong cytotoxic activity resides within the CD45RA+CD27 TEMRA population, whereas naïve CD45RA+CD27+ display low if any functional activity [34]. Upon in vitro activation and extended culture in IL-2, a sequential differentiation pattern Tnaïve→ TCM→ TEM→ TEMRA has emerged, which is further influenced by homoeostatic cytokines such as IL-15 [35]. Moreover, additional cytokines including IL-4 and IL-21 together with a TCR stimulus drive differential differentiation programs, as shown by gene expression profiling [50]. In this study, however, positively selected total γδ T cells were subjected to distinct cytokine exposure. Therefore, the observed differences in induced gene profiles could not be discerned at the level of e.g. Tnaïve, TCM, TEM or TEMRA subpopulations [50]. As to the capacity for IL-17 production, studies with cell sorter-purified subsets indicated that only the CD45RA+CD27+ Tnaïve subset differentiates into IL-17 producing cells when exposed to Il-1β, IL-6, IL-23 plus TGF-β [62]. IL-17+ Vγ9Vδ2 T cells displaying a TEMRA phenotype induce neutrophil migration through production of CXCL8 and up-regulate β-defensin production in epithelial cells [62]. The stability of the Th17-like phenotype is unclear, as is the stability of the cytokine profile of other Vγ9Vδ2 T cell subsets such as the Il-4 plus IL-10-secreting CXCR5+ subset [39]. Although the expression of lineage-associated transcription has been addressed in some studies, no systematic approach to correlate the expression of transcription factors with the cytokine profile in Vγ9Vδ2 T cells has so far been reported. Similarly, the cytotoxic activity can be assigned to specific subsets at the population level, notably the CD45RA+CD27 TEMRA and the CD56+/CD16+ cells [34, 47, 48], but the plasticity at the clonal level is uncertain. Whether the additionally described functions such as APC and regulatory activity are confined to specific subsets is much less clear. Up-regulation of APC-associated surface molecules such as HLA class II, CD40, CD80, CD86 occurs rapidly on the majority of phosphoantigen-stimulated Vγ9Vδ2 T cells [89], but it is currently unknown which proportion of those cells actually displays APC activity. Interestingly, the transcriptome analysis of ‘total’ Vγ9Vδ2 T cells did not indicate up-regulation of APC-associated genes suggesting that only a subset might be endowed with this activity [98]. Similarly, the precise characterization of regulatory Vγ9Vδ2 T cells is presently unknown. Although a sizeable proportion of per se FoxP3-negative Vγ9Vδ2 T cells can be induced to express FoxP3 in the presence of appropriate cytokines [73], it remains unclear whether the regulatory activity resides within the FoxP3+ cells. In fact, we observe regulatory potential of Vγ9Vδ2 T cells under conditions of little FoxP3 expression (Peters C. unpublished data). Several models can thus be envisaged to account for the multifunctionality of Vγ9Vδ2 T cells, as illustrated in Fig. 1. The most simple model would postulate that Vγ9Vδ2 T cells differentiate in a milieu-dependent fashion into subpopulations stably maintaining mutually exclusive functions. At least in the ‘extreme version’ depicted in Fig. 1A, this seems rather unlikely as in vitro expanded cytotoxic Vγ9Vδ2 T cells usually also produce cytokines. Alternative models displayed in Fig. 1B,C appear more likely, that is, a stimulus/milieu-dependent ‘primary functional phenotype’ which can then acquire additional functions in a milieu- and time-dependent fashion (Fig. 1B), or the existence of clonally separate subsets destined to exert different function which, however, might degenerate to display additional functions, again depending on activation signals and the surrounding milieu (Fig. 1C). Culture systems based on single cell deposit FACS sorting of Vγ9Vδ2 T cells according to differentially expressed surface markers (e.g. CD45RA/CD27 subsets) and exposure of growing clones to well-controlled differential cytokine conditions (thus mimicking the micromilieu) are required to experimentally differentiate between these models.

Figure 1.

 Different models to explain the multifunctionality of Vγ9Vδ2 T cells. (A) A given γδ T cell is initially activated by TCR stimulation; different co-stimulatory signals, TLR signals etc. determine a stable functional phenotype (cytotoxicity, APC function, regulatory activity). (B) The initial stimulus in combination with milieu-dependent signals determines the functional phenotype (cytokine production, APC function, cytotoxicity, regulatory activity) which however can be modulated to adapt alternative (or additional) functions upon additional signals. (C) Distinct subsets of Vγ9Vδ2 T cells acquire initially clonally distinct functions (cytokine production, APC function, cytotoxicity, regulatory activity); broadening of the functional repertoire might occur upon additional environmental signals.

Conclusions

At the population level, human Vγ9Vδ2 T cells combine features of innate immune cells (rapid cytokine production), myeloid cells (APC function) and adaptive immune cells (antigen-specific TCR expression). A more detailed understanding of the molecular mechanisms that govern the functional plasticity or the lineage commitment of individual Vγ9Vδ2 T cells will be required to design efficient γδ T cell–based immunotherapy for certain tumours and possibly certain infectious diseases. As an example, a precise knowledge of the pathways that enable Vγ9Vδ2 T cells to present and cross-present exogenous antigens to conventional αβ T cells might help to develop more efficient cell-based tumour vaccines. On the other hand, the anti-infective capacity of Vγ9Vδ2 T cells might be significantly enhanced once the pathways maintaining stable cytokine and for example, anti-viral chemokine production in these cells have been fully deciphered.

Acknowledgment

D.K. is supported by the DFG-funded Cluster of Excellence ‘Inflammation-at-Interfaces’ and the CRC877, project A7.

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