γδ T cells in cancer: a small population of lymphocytes with big implications

Abstract γδ T cells are a small population of mostly tissue‐resident lymphocytes, with both innate and adaptive properties. These unique features make them particularly attractive candidates for the development of new cellular therapy targeted against tumor development. Nevertheless, γδ T cells may play dual roles in cancer, promoting cancer development on the one hand, while participating in antitumor immunity on the other hand. In mice, γδ T‐cell subsets preferentially produce IL‐17 or IFN‐γ. While antitumor functions of murine γδ T cells can be attributed to IFN‐γ+ γδ T cells, recent studies have implicated IL‐17+ γδ T cells in tumor growth and metastasis. However, in humans, IL‐17‐producing γδ T cells are rare and most studies have attributed a protective role to γδ T cells against cancer. In this review, we will present the current knowledge and most recent findings on γδ T‐cell functions in mouse models of tumor development and human cancers. We will also discuss their potential as cellular immunotherapy against cancer.


INTRODUCTION
cd T cells constitute non-MHC-restricted innatelike T-cell populations, poised to be activated rapidly within seconds to minutes, rather than days, and bridge the innate and adaptive immune systems. 1 Although cd T cells make up only a minor proportion of the CD3 + compartment in the circulation and most tissues, because of their rapid cytokine production following activation, they constitute an important first line of defence against infections and are important players in antitumor defence. 2,3 Innate recognition of tumor cells and subsequent activation of cd T cells are mediated by a range of cellular and molecular determinants, including tumor-derived stress ligands and cytokine signals ( Figure 1). Despite their well-documented innate properties, the adaptive features of cd T cells are also essential in their development and function. [4][5][6] Unlike ab T cells, activation of cd T cells through their TCR is generally thought not to be restricted to presentation of peptide by MHC molecules, although a human cd T-cell clone capable of recognising melanoma tumor antigens MART-1 and gp100 in a MHC I-restricted fashion was recently generated in an artificial experimental system. 7 The identification of cd TCR ligands and the antigen-presenting molecules they recognise remains a long-standing quest, although several candidates, linked to specific cd T-cell subsets, have been identified. Among these are nonpeptidic phosphorylated metabolites, or phosphoantigens (PAgs), recognised by human Vc9Vd2 T cells and expressed not only by pathogens but also by tumor cells (Figure 1). 8 In addition, human and murine cd T cells are thought to be capable of activation by cytokines, independent of TCR-cognate antigen recognition.
cd T cells can be found in the circulation and in secondary lymphoid organs, but they are mainly resident in barrier tissues, such as the mucosae and the skin, and in adipose tissue. cd T cells expressing specific Vc and Vd chains are enriched in particular locations within the body as illustrated in Table 1. This suggests that tissue-specific factors trigger clonal selection, possibly as a result of infection, cytokine milieu or endogenous antigens, highlighting how little is known about factors controlling activation and expansion of human cd T-cell subsets. In mice, it is clear that two functional subsets of cd T cells can be found, one producing IL-17 and one producing IFN-c. These two subsets can be functionally defined based on differential surface expression of CD27. CD27 is a member of the TNF receptor family and binds to CD70. CD27 is expressed not only on activated lymphocytes but also on tumor cells. While CD27 is present on murine IFN-c + cd T cells, it is absent on the surface of the IL-17 + cd T cells. 9 On the contrary, IL-17 cd T cells preferentially express CCR6 and the transcription factor PLZF, which is considered to confer innate-like properties to lymphocytes. [10][11][12][13] Whereas murine cd T cells acquire TCR-dependent functional maturity during thymic ontogeny, human cd thymocytes are functionally immature and instead acquire their effector functions in response to peripheral cytokine signals. [14][15][16] Nevertheless, human thymic cd T cells exhibit de novo expression of type 1 transcription factors T-bet and eomesodermin, reflecting their capacity to rapidly differentiate into cytotoxic effectors producing IFN-c in response to cytokines IL-2 and IL-15. 15 Unlike in mice, the cd T-cell compartment in humans cannot be functionally defined based on differential ULBPs. cd T cells also display an NK-like phenotype in their expression of NCRs (NKp30, NKp44 and NKp46), particularly following activation. LFA-1, lymphocyte function-associated antigen 1; NKG2D, natural killer group 2 member D; PLZF, promyelocytic leukaemia zinc finger protein; Rae1, retinoic acid early inducible-1; TCR, T-cell receptor; TRAIL, TNF-related apoptosis-inducing ligand; ULBP, UL16-binding proteins. * denotes expression on some clones only. expression of CD27 and the functional distinction among the different subsets is less clear. 9 Human cd T cells can be divided into 3 main subsets based on TCRd-chain usage, Vd1, Vd2 and Vd3, which does not allow for clear discrimination of their different effector functions. Interestingly, Vd4 + , Vd5 + and Vd6 + populations of cd T cells have also been found in patients with diverse infections, but they remain rare and no commercially available antibodies exist for these subsets. 17 Thus, most of the studies of human cd T cells have focused on the Vd1, Vd2 and Vd3 subsets. While tissue-resident cd T cells are mostly Vd1 + (and probably Vd3 + , as they are sometimes described as Vd1 À Vd2 À ), the majority of our current knowledge on the biology of human cd T cells comes from blood-circulating cells, which are mainly Vd2 + (Table 1). Recent studies concerning the human cd TCR repertoire have revealed distinct innate and adaptive roles for cd T-cell subsets, depending on TCRc-and TCRdchain usage. In cord blood, the Vd1 + TCR repertoire is highly diverse and private, but undergoes postnatal clonotypic focusing throughout adulthood, 18 as evidenced by the enrichment of discrete Vd1 + clonotypes during cytomegalovirus (CMV) and human immunodeficiency virus (HIV) 19 infection. Within the Vd2 + subset exist highly clonal adaptive populations expressing a Vc9 À Vd2 + TCR, which undergo differentiation and clonal expansion during acute CMV infection, in contrast to the innate-like Vc9 + Vd2 + TCR with limited recognition kinetics and CDR3 diversity. 20 The Vd2 + subset constitutes an heterogeneous population of cells, producing a range of pro-inflammatory cytokines including IFN-c, IL-17, TNF-a, IL-9, but also IL-10 depending on the setting. [21][22][23][24] While IFN-c-producing cd T cells are abundant in peripheral blood, IL-17 production by human cd T cells is rare at homeostasis. However, significant inflammatory insult such as that seen in some cancers and infections can polarise cd T cells towards a type 17 phenotype. 24,37,39,40 A recent extensive study sequencing bulk transcriptomes of 18 000 human tumors revealed that, among all leucocytes present in the tumors, cd T cells were most strongly associated with good prognosis. 41 However, the computational approach used to characterise these cells has since been disputed. 40 There have also been reports of cd T cells having a potential tumor-promoting role in various human malignancies, 37,40,42 likely attributable to their functional plasticity in various inflammatory microenvironments, although determination of a direct immunosuppressive role for human cd T cells in situ is difficult. Thus, although cd T cells may still provide good prognostic and therapeutic value in human cancers, more research is required into understanding the balance between pro-and antitumor effector functions, and how this is regulated in the tumor microenvironment.

cd T CELLS IN TUMOR IMMUNE SURVEILLANCE AND ANTITUMOR IMMUNITY
Antitumor functions of murine cd T cells Initial studies performed in murine models of cancer have found protective roles for cd T cells against tumor growth. 43,44 Several mechanisms, through which they mediate their antitumor effects, have been described, including not only direct killing of tumor cells mediated by cytolytic proteins or NKG2D-dependent mechanisms, but also indirect effects mediated by their production of IFN-c, as illustrated in Figure 2. In this section, we summarise the current knowledge on the different antitumor functions attributed to murine cd T cells.
Early studies on the protective role of cd T cells in mice have been conducted in murine models of skin cancers, induced chemically or by subcutaneous transfer of melanoma or carcinoma cell lines. In all models, crucial roles for cd T cells in antitumor immunity have been described, and studies have shown a NKG2D-mediated mechanism by tissue-resident Vc5 + dendritic epidermal T cells (DETCs) as a main player in cd T-cell antitumor function. [43][44][45][46] DETCs are dendriticshaped cd T cells, which express a largely invariant Vc5 + Vd1 + TCR, and are considered to be a unique and unusual subset of cd T cells, which restricts the extent of findings on these cells to other populations of cd T cells. DETCs constitute the majority of T cells in the murine epidermis, but cd T cells are far less abundant in human skin, and DETC equivalents are not present in humans, although evidence for antitumor function of skin cd T cells also exists in humans. 47 Interestingly, in vivo studies of skin cancer performed in cd Tcell-deficient mice (TCRd À/À mice) did not allow for discrimination between DETCs and other populations of cd T cells, and might have underestimated the role of dermal Vc4 + cd T cells or other subsets infiltrating the skin. 43,44 Indeed, TCRd À/À mice reconstituted with Vc4 + cd T cells had a restored antitumor response against B16 melanoma cells, which relied on IFN-c and perforin production, two important mediators in antitumor immunity by cd T cells and other lymphocytes ( Figure 2). 45,48,49 Importantly, a protective role for cd T cells in antitumor response in mice has been described in other models of cancer and notably in a spontaneous model of Bcell lymphoma. 49  macrophages and enhances the antigen presentation capacities of professional APCs. 50 Interestingly, IFN-c production by cd T cells also enhances MHC I molecule expression at the surface of B16 melanoma cells, thereby promoting their recognition by CTLs ( Figure 2). 51 A recent study in a mouse model of gastrointestinal stromal tumor describes a protective role for cd T cells mediated through the secretion of GM-CSF. This cytokine promoted the maturation of CD103 + CD11b À dendritic cells, which were associated with infiltration of effector CD8 T cells within the tumor. 52 Tumor immune surveillance by activated murine cd T cells has been linked to their surface expression of the C-type lectin receptor NKG2D, ligands for which Rae-1 and H-60 (MICA and MICB in humans) are expressed at the surface of stressed cells. 43,46 In mice, DETCs together with Langerhans cells (epidermal dendritic cells) and tissue-resident CD8 + memory T cells form a network integrated within the epidermis. 53 Upregulation of Rae-1 by epidermal cells induces activation and remodelling of DETCs from dendritic-to round-shaped cells, leading to a reorganisation of the epidermal architecture. Rae-1 upregulation also promotes expression of the activation marker CD69 on the DETCs within the epidermis and the killing of tumor cells through a NKG2D-mediated pathway. 43,46 While expression of NKG2D ligands in humans is associated with better outcome in several types of cancers, NKG2D ligands are often internalised by tumor cells or secreted as soluble forms during immune evasion, but are promoted following exposure to different factors including chemotherapy. 54 Interestingly, a recent study by Sheppard and colleagues has identified an unexpected tumor-promoting role for NKG2D in a model of hepatocellular carcinoma. The authors proposed that, while NKG2D has evident antitumor function in early stages of cancer, it could exacerbate the proinflammatory microenvironment of the tumor at later stages, leading to tissue damage and enhanced cell proliferation, which promoted tumor progression in the liver environment. 55 While the authors did not look at the implication of cd T cells in this process, these cells could nevertheless play a role, given their enrichment in the liver and their robust cytokine expression in response to many inflammatory signals. 56 The engagement of the cd TCR in tumor recognition and elimination by murine cd T cells is also likely, and this is also true in humans. 57 Indeed, Girardi et al. 43 showed that incubation of murine DETCs with a cd TCR-blocking antibody resulted in impaired lysis of the PDV tumorigenic keratinocyte cell line. However, Dutta et al. 58 have recently shown that blockade of cd TCR with antibodies can induce apoptosis in those cells, which could account for the decrease in killing capacity observed. Recently, Crawford et al. 59 showed that skin-resident intraepithelial cd T cells also induced a rapid adaptive immune response to chemically induced skin carcinogenesis by promoting class switching and secretion of high levels of protective IgE by B cells, indicating that the impact of cd T cells on other cell types might be broader than expected (Figure 2).

Role of human cd T cells in antitumor immunity
The Vd2 + subset Vd2 + cd T cells are the predominant subtype in the blood, accounting for 2-5% of circulating CD3 + lymphocytes. 60 These cells express a TCR with preferential pairing of Vd2 and Vc9 chains, and mediate effective antitumor immunity directly through cytotoxicity via perforin and granzymes, or indirectly through IFN-c and TNF production ( Figure 2). 3 Recognition of tumor cells by Vc9 + Vd2 + T cells can occur through a host of cell surface receptors for self and non-self ligands, including TCR recognition of tumor antigen and stress ligand receptors. These include NKG2D, FCcIII (CD16), FasL, TRAIL and DNAM-1 (CD226). [61][62][63][64][65] Vc9 + Vd2 + T cells recognise tumor-derived phosphorylated prenyl metabolites in a TCRdependent manner, which may accumulate intracellularly as a by-product of dysregulated tumor metabolism (Figure 1). One well-studied PAg, isopentenyl pyrophosphate (IPP), can accumulate in cancer cells as a result of the elevated metabolic flux through the mevalonate pathway of cholesterol biosynthesis. 21,28,66 These non-peptidic antigens are not presented in the context of classical MHC and are instead presented through a non-polymorphic type I transmembrane protein called butyrophilin 3A1 (BTN3A1). BTN proteins of the immunoglobulin (Ig) superfamily consist of a B30.2 intracellular domain and two extracellular Ig domains. 67 The mechanism of activation of Vc9 + Vd2 + T cells by BTN3A1-bound PAg remains controversial, although it is thought to be triggered by initial intracellular binding of PAg to a positively charged surface pocket within the intracellular B30.2 domain. [68][69][70][71] The resultant conformational change within BTN3A1 has been proposed to confer recognition by the Vc9Vd2 TCR in an 'inside out' signalling mechanism whereby surface BTN3A1 is sensitive to the intracellular concentration of prenyl pyrophosphate metabolites. 72,73 These non-MHC-restricted, innate-like recognition kinetics of Vc9 + Vd2 + T cells are an attractive candidate for cancer immunotherapy and have been targeted in clinical settings using aminobisphosphonate drugs.
Aminobisphosphonates are clinically approved potent inhibitors of the mevalonate pathway, thereby not only promoting direct antitumor effects but also leading to a build-up in endogenous isoprenoid metabolites. Zoledronate is an aminobisphosphonate drug that directly inhibits farnesyl pyrophosphate synthase (FPPS), an enzymatic mediator of the mevalonate pathway, leading to a build-up in endogenous IPP. 74 This, in combination with mitogenic IL-2, induces activation and proliferation of type 1 cytotoxic effector cd T cells with antitumor potential producing IFN-c, TNF, perforin and granzymes. 75 Thus far, all clinical trials using cd T cells as an autologous cellular therapy for cancer have focused on ex vivo or in vivo activation and expansion of Vc9 + Vd2 + T cells with aminobisphosphonates, with satisfactory safety profiles observed. 76,77 This highlights the need for a better understanding of how Vc9 + Vd2 + T cells become activated in cancer, how their effector functions are regulated and how this may be exploited for therapeutic gain in cancer immunotherapy.
The Vd1 + subset Vd1 + T cells are a minor population in the blood but represent the predominant tissue-resident population of cd T cells. 57 These cells are mainly found at mucosal sites such as the dermis and intestinal epithelia where they can comprise 20-50% of the tissue-resident lymphoid compartment. 3 Unlike their Vd2 + counterparts, Vd1 + cd T cells do not often preferentially pair with a specific Vc chain (although clonal expansion can be seen in some organs, which can be different among individuals). Vd1 + cd T are not activated by PAgs, but can display an NK-like phenotype in their expression of natural cytotoxicity receptors, (NCRs) NKp30, NKp44 and NKp46, depending on the protocol used to expand them. 62 Although a unique ligand for the Vd1 + TCR has yet to be identified, recent studies have elucidated some cognate TCR recognition properties of Vd1 + T cells. A crystallographic study revealed sequential recognition kinetics of the MHC class I homologue MICA by NKG2D and Vd1 + TCR, thereby providing both TCR and costimulatory signals from the same ligand. 65 Some Vd1 + cell lines have been reported to recognise the lipid antigen a-galactosylceramide (a-GalCer) presented by CD1d. [78][79][80] Furthermore, Vd1 + TCR-mediated recognition of glycolipids presented in the context of CD1c facilitates target cell lysis, Th1 cytokine production and dendritic cell maturation by Vd1 + T cells (Figure 1). Indeed, as with ab T cells, TCR-mediated recognition of host stress ligands by cd T cells may require costimulatory signals. This is exemplified by the TCR-mediated recognition of endothelial protein C receptor (EPCR) expressed on CMV-infected endothelial cells by a cd T-cell clone bearing a Vc4Vd5 TCR, which required CMV-induced upregulation of ICAM-1 by target cells for an optimal response. 81 Similar to Vd2 + cells, Vd1 + cd T cells induce tumor cell death through soluble cytotoxic machinery (perforin, granzymes and granulysin) and cytokine secretion (IFN-c and TNF). The cytolytic function of Vd1 + T cells has been shown for a range of haematological and solid malignancies, including acute lymphoblastic leukaemia (ALL), acute myelogenous leukaemia (AML), B-cell chronic lymphocytic leukaemia (B-CLL), neuroblastoma, melanoma and pancreatic, lung and colorectal cancers (CRC). 36,57,60,[82][83][84] Vd1 + T cells seem to outclass Vd2 + cells in most in vitro and in several in vivo pre-clinical cancer models in terms of cytotoxicity and durability, 57,85 which may have important implications in the development of next-generation cd-based immunotherapies. One advantage Vd1 + cells may have for use in immunotherapy is their resistance to activationinduced cell death (AICD), 86 which has posed significant problems in clinical trials following chronic stimulation of Vc9 + Vd2 + T cells with aminobisphosphonate drugs. 77 Although the cytotoxic capacity of both Vd1 + and Vc9 + Vd2 + T cells makes them attractive targets for the development of next-generation immunotherapies, a broader understanding of how these effector functions are regulated and how they may be polarised towards a pro-tumor phenotype, and whether, like conventional T cells, they become inhibited and exhausted in the tumor microenvironment, is required.

cd T CELLS AS DRIVERS OF TUMOR GROWTH
Pro-tumor function of murine cd T cells Studies performed in mouse models indicate that pro-tumor functions of cd T cells can be largely attributed to the IL-17 + cells (Figure 2). This is in line with the majority of reports on IL-17 production by other innate and adaptive immune cells, although the impact of IL-17 on tumor growth might depend on the type of cancer studied. 87 We and others have found that IL-17 + cd T cells are enriched not only in a variety of murine solid tumor models induced by implantation of tumorigenic cells, but also in spontaneous models of HPV-related carcinogenesis and breast cancer models, for which they are associated with metastasis. 34,[88][89][90][91][92] In murine models of ovarian, pancreatic and lung cancers, IL-17-producing cd T cells in tumors were highly proliferative and displayed an activated phenotype. 34,89,93 They induced angiogenesis and the recruitment of neutrophils, generally associated with poor prognosis in cancer. 92,93 Indeed, neutrophils secrete different tumorpromoting agents, such as growth factors, metalloproteinases (MMPs), neutrophil elastase (NE) and reactive oxygen species (ROS), which directly enhance tumor growth and invasion, promote angiogenesis and suppress antitumor immune cells. Nevertheless, neutrophils display phenotypical and functional plasticity depending on the tumor microenvironment, and have been found to also contribute to antitumor immune response, notably through antibody-dependent cellular cytotoxicity (ADCC) and recruitment of other immune cells. 94 IL-17-producing cd T cells also promoted the recruitment of immunosuppressive neutrophils and small peritoneal macrophages, which inhibit CTL response and enhance tumor growth. 34,88,90,93 In a model of pancreatic ductal adenocarcinoma (PDA), IL-17 + IL-10 + cd T cells were also directly suppressive of T-cell responses. Here, IL-17 + cd T cells expressed the checkpoint inhibitors PD-L1 and Galectin-9, both of which prevented the activation of ab CD4 + and CD8 + T cells, indicating that cd T cells can directly inhibit adaptive antitumor immunity (Figure 2). 89 A direct role of IL-17 + cd T cells on tumor cell proliferation is also possible as IL-17 + cd T cells from lung tumors expressed IL-22 and amphiregulin, both of which can directly promote tumor cell proliferation ( Figure 2). 93 Nevertheless, as IL-17-producing cd T cells are rarely found in humans at steady state, further studies are needed to fully grasp the relevance of these findings for human cancers.
The tumor microenvironment provides favorable conditions for the enrichment of IL-17-producing cd T cells, notably through enhanced levels of the cytokines IL-1b, IL-6, IL-23 and IL-7, which favor CD27 À cd T-cell survival and promote IL-17 expression. 34,88,92 Jin et al. 93 showed that in a spontaneous model of lung adenocarcinoma, tumor development alters the local microbiota, which induces the production of IL-1b and IL-23 by myeloid cells resulting in highly proliferative tissue-resident IL-17 + Vc6 + Vd1 + cd T cells. Interestingly, the IL-17 production in cd T cells via IL-1b axis is also described in promoting tumor metastasis in a spontaneous model of breast cancer metastasis. 92 Interestingly, IL-1b and IL-6 additionally drive the expression of NOS2, associated with tumor evasion, in pro-tumorigenic cd T cells. 95 IL-17 + cd T-cell recruitment is supported by tumor chemokine secretion, such as CCL2/MCP-1, a molecular target for anticancer therapy and ligand for CCR2, which is highly expressed on tumor-infiltrating cd T cells. 89,91,[96][97][98] The chemokine receptor CCR6, involved in the trafficking of IL-17 + cells to tissues at steady state, is also expressed by IL-17 + cd T cells in the tumor bed of PDA and hepatocellular carcinoma. 89,90 Indeed, CCR6 and its ligand CCL20 are associated with tumor progression in models of CRC and pancreatic cancer. 99,100 Interestingly, in other models, recruitment of IL-17 + cd T cells to the subcutaneous B16 melanoma tumors and HPVinduced skin lesions, respectively, is associated with a downregulation of CCR6 expression, 91,96 indicating that the environmental setting in which the tumor develops might influence the phenotype of the immune cells recruited.
Intrinsic metabolic pathways are another parameter that may influence the recruitment and survival of cd T cells within the tumor bed. In fact, as cancer progresses, tumor cells override lymphocytes in competition for nutrients, especially glucose, which is essential for T-cell effector functions. Thus, nutrient availability might favor or limit the survival of particular immune cells. Our unpublished work suggests that CD27 À and CD27 + cd T cells have different metabolic requirements, which might partially explain the enrichment of the IL-17 + subset over the IFN-c + one in the tumor, observed in a number of cancer models. In addition, tumor cells and other cells infiltrating the tumor niche express enzymes and excrete products, which can inhibit normal T-cell metabolism. For instance, in models of hepatocellular carcinoma and peritoneal B16 tumor, tumor-infiltrating IL-17 + Vc6 + cd T cells express low amounts of the antioxidant glutathione, which make them highly susceptible to ROS produced by tumor-associated neutrophils. 101 These recent insights of the effect of metabolic state of the tumor microenvironment on the promotion of pro-or antitumor immune cells require further investigation.

Pro-tumor function of human cd T cells
One potential caveat of the functional plasticity and innate response kinetics of cd T cells is their susceptibility to polarisation by a particular inflammatory milieu. Although human cd T cells rarely produce IL-17, several groups have reported an elevated frequency of IL-17 + cd T (cdT17) cells in response to a combination of Th17-polarising cytokines IL-1b, IL-6, IL-23 and TGF-b in some disease settings. 24,39 Many of these cytokines are elevated in the tumor microenvironment of certain cancers, and indeed, there have been some reports of IL-17-producing cd T cells having a pro-tumor role in various human malignancies.
The first report of IL-17-producing cd T cells having a pro-tumorigenic role in humans was reported by Wu et al. 37 in patients with CRC. They showed that breach of the gut epithelial barrier by tumor dysplasia induced an influx of commensal microbial products, resulting in the accumulation and activation of IL-23-producing inflammatory dendritic cells. This was sufficient to induce cdT17 polarisation of Vd1 + intraepithelial lymphocytes, with cd T cells identified as being the main cellular source of IL-17 in human CRC. Production of IL-8 and GM-CSF by cd T cells resulted in an influx of immunosuppressive neutrophils, which have well-established protumor roles in an array of cancer types in both humans and mice.
McAllister et al. 102 used a murine model of pancreatic intraepithelial neoplasia (PanIN), a histological precursor of PDA, to show that the oncogene Kras can induce the expression of IL-17 receptors on PanIN cells and infiltration of IL-17 + lymphocytes into pancreatic stroma. Within the pancreatic tumor microenvironment exists an abundance of type 17-polarising cytokines such as IL-6 and TGF-b. 103 They showed an increase in the frequency of RORct + cells in PanIN lesions, primarily produced by Th17 (10% IL-17 + ) and cd (50% IL-17 + ) T cells. It has later shown that human PDA consists of a unique inflammatory infiltrate, with cd T cells making up to 75% of infiltrating T cells, 89 although Gunderson et al. 104 have reported a much lower proportion of cd T cells in the PDA inflammatory infiltrate (< 5%). Using a transgenic murine model of PDA, they identified a substantial population of cd T cells, which produced IL-10 and IL-17 and restrained ab T-cell activation through expression of immune checkpoint ligand PD-L1. Although the role of IL-17 in human pancreatic tumorigenesis remains uncharacterised, ablation of cd T cells resulted in enhanced ab T-cell tumor infiltration with superior antitumor effector function in TCRd À/À mice, perhaps highlighting the need for a better understanding of how cd T-cell anticancer function is regulated in the tumor microenvironment.
A pathological role has also been described for cdT17 cells in human gallbladder cancer (GBC), with an increased frequency of cd TCR + cells in the blood and TIL of patients with GBC. 40 Here, cdT17-derived IL-17 induced expression of vascular endothelial growth factor (VEGF) and other proangiogenic factors by GBC cells, facilitating tumor growth and survival. A common feature of the cancer types for which a pro-tumorigenic role of cd T cells has been described is their resistance to conventional chemotherapeutic treatments and poor 5-year survival rates, exemplifying the need for alternative therapies. With the recent clinical success of immunotherapies such as checkpoint blockade and chimeric antigen receptor (CAR) T cells, an argumentative case can be made for targeting cd T cells; however, the factors that govern the pro-versus anticancer phenotype in the tumor microenvironment must first be further explored.

APPLICATIONS OF cd T CELLS IN IMMUNOTHERAPY
Immunotherapy is a rapidly expanding and diversifying field of clinical oncology, which has shown unprecedented success in the clinic. The emergence of immune checkpoint inhibitors and CAR T-cell technology has revolutionised the treatment of malignancy. However, the efficacy of these treatments is limited, for the most part, to haematological neoplasms and solid tumors with high mutational burdens (e.g. metastatic melanoma and MSI-high colon cancer). 105 While current T-cell-based therapies have shown great success in the clinic, several pitfalls in their use still persist. Checkpoint inhibitors are only effective in a minority of patients, acquired resistance and tumor relapse with resistant clones is an increasingly worrying problem. The time and expense involved in the expansion and conversion of patient cells to CAR T-cell products means there is a limited treatment window available to patients with advanced disease. In some cases, this expansion protocol fails completely, leaving few options for further treatment. Similar to checkpoint therapy, the use of CAR T-cells in solid tumors has proven disappointing. This is believed to be in large part by difficulty in drawing them to the affected tissues. Homing to effected tissues requires the expression of a range of chemokine receptors and adhesion molecules, which are not normally expressed by peripheral blood T cells. 106 Successful elimination of tumors is dependent on the persistence of transferred T cells, which can become exhausted. Conversely, some patients suffer detrimental side effects, such as autoimmune colitis and cytokine release syndrome. These side effects can even result in increased morbidity and mortality. 107 Therefore, an off-the-shelf cellular immunotherapy is an attractive proposition. Innate immune cells, such as cd T cells and NK cells, appear to have an improved safety profile with minimal off-target effects. 108 Furthermore, since these cells are not MHC-restricted cell products can be prepared from a pool of healthy donors and expanded, reducing the costs and unpredictability associated with rapid expansion of patient-derived products. The innate nature of cd T cells and their ability to recognise a wide range of tumors makes them potentially excellent candidates for cellular therapy.
A pan-cancer analysis of the TCGA database identified cd T cells as the strongest immune prognostic available in solid tumors. 41 However, the analysis showed wide variability in the infiltration of tumors by cd T cells. In addition, the computational algorithm used to deconvolute these tumor microarrays, CIBERSORT, has then shown to inaccurately distinguish cd T cells from other lymphoid populations. 109 This computational-based identification was later optimised by Tosolini et al., 109 allowing for more accurate assessment of cd TILs from bulk tumor transcriptomes. Moreover, cd T cells are diverse and often plastic so identifying the most suitable subset and maintaining this phenotype in vivo remains a challenge to be addressed in coming years. For example, the presence of IL-17producing cd T cells in colon cancer has been associated with poor prognosis. 37,110 Interestingly, though a pan-cancer analysis of the TCGA database identified a combined Th1/Th17 immune signature as the most beneficial for patient survival, this group showed the most pronounced Th17 gene signature but appeared balanced by the presence of a Th1 response. 111 This study requires further dissection to determine the relative contribution of Th1 and Th17 genes to this signature. IL-17 has previously been considered pro-tumorigenic, with many IL-17mediated diseases eventually leading to malignancy. However, this study indicates that IL-17 in context of a Th1 response may be beneficial, but the source and localisation of IL-17 production cannot be identified in current transcriptomic data sets with reasonable certainty. Therefore, this question may benefit from a new approach, and using single-cell transcriptomic analysis to identify the source of this potentially beneficial IL-17 is worth investigation. Homology between murine and human cd T-cell subsets is poor and makes translation of murine studies to humans a difficult proposition.
Vd1 + T cells make up a small proportion of the circulating cd T-cell population. However, they are highly enriched in mucosal tissues including the skin, gut, lung and liver (Table 1). Residing in tissues, Vd1 + T cells adapt to lower nutrient availability and decreased oxygen levels, which is similar to the tumor microenvironment. Incubation in hypoxia ex vivo has been shown to enhance cd T-cell cytotoxicity. However, tumors in hypoxic environments begin to secrete soluble NKG2D ligands, rendering cd T cells incapable of killing these cells. 112 Having previously homed to target organs, adoptively transferred Vd1 + T cells should be capable of homing again to a target organ containing a tumor. Furthermore, protocols have been developed that allow the rapid expansion of highly cytotoxic donor Vd1 + T cells (DOT cells), which are able to control leukaemic cell growth. 57 These cells acted against a broad range of tumor clones and did not select for resistant strains. 113 It is thought that this is mediated through innate NK receptors in addition to TCR recognition of tumor cells. Vd1 + cd T cells express a range of germ-line-encoded receptors, which recognise cellular stress (NKG2D) as well as tumor-and viral-associated antigens (NKp44 and NKp46; Figure 1). This is consistent with previous reports, showing that expanded Vd1 + T cells possess broad cytotoxic potential in solid tumors, including colon cancer. 114 This provides a unique advantage for cd T cells over conventional ab T cells. Their ability to recognise a broad range of tumor signals through NK receptors and their TCR allows them to avoid some of the most potent immune evasion mechanisms available to tumors. However, Vd1 + T cells have been poorly characterised in solid tumors. Despite their enrichment in specific organs (Table 1), tumors nonetheless develop in these tissues, indicating many tumors are capable of evading recognition by Vd1 + T cells. cd T cells may also succumb to inhibition through checkpoint molecules. cd T cells have been shown to express PD-1 transiently after activation; however, the expression of PD-1 and other immune checkpoints such as CTLA-4, TIM3, LAG3 and TIGIT has been poorly characterised on cd T cells in human tumors and a combination of these molecules may inhibit TCR and NK-receptor recognition of tumors. 115 Vd2 + cells are the majority of circulating cd T cells in humans, and the vast majority of literature surrounding Vd2 + T cells has focused on a subset expressing the TCR Vc9 chain. The transcriptional profile of Vc9 + Vd2 + T cells appears to be an amalgamation of ab T cells and NK cells, giving them aspects of both cells' functions. Vc9 + Vd2 + T cells have adaptive features such as a somatic recombination of receptors, memory formation and professional antigen presentation, alongside innate features such as an absence of MHC restriction, recognition of conserved microbial and self-antigens and ability to perform ADCC. [116][117][118] A wide range of germ-line-encoded activating receptors are also expressed by Vc9 + Vd2 + T cells, which are essential for their antitumor function, including NKG2D, which recognise MICA/B. 61,113 Vc9 + Vd2 + T cells have been detected in over 30 solid and haematological malignancies. 109 In this study, Vc9 + Vd2 + T cells were associated with prolonged overall survival in CLL, AML, colon and prostate cancers. Interestingly, Vc9 + Vd2 + T-cell infiltration was independent of ab T-cell accumulation, indicating that infiltration by cd T cells is via a different mechanism to conventional ab T cells. Vc9 + Vd2 + T cells account for about 5% of peripheral blood T cells, so are readily available for in vivo and ex vivo expansion. The drug zoledronate has been used in several clinical trials to promote the in vivo expansion of Vc9 + Vd2 + T cells. While this proved a safe treatment, the efficacy was disappointing and failed to prevent progression in most patients. 119 Ex vivo expansion of cd T cells using zoledronate and IL-2 has also been trialled in a number of studies, improving disease progression but failing to achieve improved overall survival in a number of solid tumor types (renal cell carcinoma, lung cancer, hepatocellular carcinoma). 119,120 These early trials should be interpreted with caution as they were designed for assessing safety of cd T-cell products and not their efficacy. As of March 2019, there are currently 13 active clinical trials (clinicaltrials.gov) involving the use of cd T cells to treat a broad range of cancers including leukaemia and breast, pancreatic, ovarian, liver, kidney, lung and brain cancers. These trials involve combinations of in vivo expansion using drugs such as zoledronate and alendronate, infusions of ex vivo-expanded cd T cells and surgical interventions such as cryosurgery or irreversible electroporation (NanoKnife; Table 2). However, these trials utilise techniques used in previous trials with low rates of success. Perhaps then, new approaches to cd T-cell-based immunotherapy are required.
While the current trend in immunotherapy involves the use of checkpoint inhibitors to release the suppression of T cells, this therapy may not drive antitumor responses in innate T cells, such as cd T cells. As many cd T cells are not MHCrestricted, the co-inhibitory pathways associated with antigen presentation, such as PD-1 and CTLA-4, may be redundant in their tumor recognition. Therefore, cd T cells may require release of additional immune checkpoints such as TIGIT, a potent inhibitor of NK cells. [121][122][123] The expression of immune checkpoints such as TIGIT, TIM3, LAG3 and NKG2A remains poorly characterised in tumor-infiltrating cd T cells and may provide synergistic targets to combine with conventional T-cell targets such as PD-1.
Recently, cd T cells have been incorporated into CAR therapy, producing sufficient cells from Vd1 + and Vd2 + subsets for clinical studies. 124,125 An additional perquisite of using cd T cells for immunotherapy lies in their ability to crosspresent processed tumor antigen to ab T cells, and this process is retained in CAR-cd T cells, further enhancing their antitumor effects. 124

FUTURE DIRECTIONS
With the advancement of chimeric antigen receptor (CAR) engineering, interest in cellular therapies has increased dramatically. Furthermore, robust expansion protocols for the production of cd T cells en masse have made their use in the clinic feasible. 32,57,114 The safety profile of innate lymphocytes compared to conventional T cells and their lack of MHC restriction makes them an attractive target for off-the-shelf cell therapy. However, further fundamental research is needed to grasp fully the pleiotropic roles of cd T cells in cancer. In addition, inhibitory pathways used by tumors to evade recognition by cd T cells have been poorly characterised and warrant further investigation. Additional and more advancedphase clinical trials are required to determine the efficacy of cd T-cell-based therapies. cd T cells are a strong positive prognostic in most cancers. They naturally infiltrate tissues throughout the body, including lung, liver and the gut, some of the most difficult organs in which to treat malignancies. They recognise a broad range of tumors, not only through their TCR but also through NK receptors. Furthermore, they fail to induce graft-versus-host disease and autoimmune complications. This potent effector function, broad range of activity and safety profile make them an ideal potential cellular therapy to enhance current immunotherapy strategies and improve the treatment of solid malignancies.