The Dual Roles of Human γδ T Cells: Anti-Tumor or Tumor-Promoting

γδ T cells are the unique T cell subgroup with their T cell receptors composed of γ chain and δ chain. Unlike αβ T cells, γδ T cells are non-MHC-restricted in recognizing tumor antigens, and therefore defined as innate immune cells. Activated γδ T cells can promote the anti-tumor function of adaptive immune cells. They are considered as a bridge between adaptive immunity and innate immunity. However, several other studies have shown that γδ T cells can also promote tumor progression by inhibiting anti-tumor response. Therefore, γδ T cells may have both anti-tumor and tumor-promoting effects. In order to clarify this contradiction, in this review, we summarized the functions of the main subsets of human γδ T cells in how they exhibit their respective anti-tumor or pro-tumor effects in cancer. Then, we reviewed recent γδ T cell-based anti-tumor immunotherapy. Finally, we summarized the existing problems and prospect of this immunotherapy.

INTRODUCTION gd T cells are the non-classical cell subgroup characterized by expression of gd heterodimeric T cell receptor (TCRgd) on cell surface. They only account for 1% to 5% of T lymphocytes in peripheral blood circulation and lymphatic circulation, and predominantly reside in the mucosal tissues such as skin, intestine, lung, and uterus (1)(2)(3). gd T cells are the intermediate group of cells between innate and adaptive immune cells, serving as a bridge between innate immunity and adaptive immune response (4,5). They play important roles in tumor immunity. Depending on the microenvironment, different gd T cell subgroups can have anti-tumor or protumor activities.
However, it was reported that gd T cells could also promote cancer development (36). For example, as one of the main sources of interleukin-17 (IL-17), tumor-infiltrating gd T cells were shown to promote tumor development and metastasis by enhancing angiogenesis and recruiting inhibitory cells (37)(38)(39)(40). Tumor-infiltrating gd T cells could also directly induce the apoptosis of anti-tumor immune cells (41).
In this review, we introduced the classification of human gd T cells and summarized how gd T cell subsets play different roles in tumorigenesis. We further discussed the gd T cell-based antitumor immunotherapy which has been widely used in clinic. Finally, we briefly summarized the current limitation and caveats associated with such therapy, and proposed new approach for optimization. We believe that the summary of biological functions of different gd T cells can help us improve our understanding of tumor microenvironment, and provide novel insights for anti-tumor immunity.

CLASSIFICATION OF gd T CELLS
Human gd T cells can be classified into different groups based on the expression of TCRg chains or TCRd chains, and they can be further classified by the expression of different CD molecules (42,43).
Different gd T cells have diversified distribution and functions. Vd1 chain can interact with different g chains to form various gd T cells. They are mainly distributed in the skin, intestine, liver, spleen and mucosal tissues. The role of Vd1 T cells is controversial. In certain situations, they have been shown to have strong anti-tumor effects in colorectal cancer, multiple myeloma, chronic lymphocytic leukemia (48)(49)(50). On the other hand, tumor-infiltrating Vd1 T cells often demonstrate strong immunosuppressive effects. They secreted IL-17 and transforming growth factor-b (TGF-b) (51), expressed programmed cell death 1 ligand 1 (PD-L1), and inhibited the activation of other immune cells (41).
The Vd2 chain only combines with the Vg9 chain to form the Vg9Vd2 T cells, which mainly exist in peripheral blood. Given that the Vg9Vd2 T cells have strong anti-tumor effects in various types of tumors, they were widely used in clinics (52)(53)(54)(55). In addition, they have also been shown to kill the cancer stem cells (CSC) in various tumors including colon cancer, ovarian cancer, and neuroblastoma (56,57).
The Vd3 chain mainly interacts with the Vg2 and Vg3 chains. Vd3 T cells mainly exist in the liver, and also in a small amount in the peripheral blood of patients with chronic lymphocytic leukemia. The functions of Vd3 T cells in tumors have not been elucidated in depth (58)(59)(60)(61).
The Vd5 chain usually combines with the Vg4 chain to form the Vg4Vd5 T cells. They mainly exist in peripheral blood. The TCR of Vg4Vd5 T cells could directly bind to the endothelial protein C receptor (EPCR) to recognize epithelial tumor cells. Like Vd3 T cells, they were rarely studied for their tumor-related functions (19, 62) ( Table 1).

Classifications Based on the Phenotype of CD Molecules
Human gd T cells can be classified based on the expression of CD27 and CD45RA. The naive type (T naive , CD27 + CD45RA + ) and the central-memory phenotype (T CM , CD27 + CD45RA -), mainly exist in the secondary lymphoid organs. T CM can maintain immune memory for a long time and quickly mediate immune response after receiving antigen stimulation. The effector-memory type (T EM , CD27 -CD45RA -) and terminally-differentiated type (T EMRA , CD27 -CD45RA + ) mainly exist at the site of inflammation and exert instant effects, namely secreting cytokines and exerting cytotoxicity (63,64).

Classification of gd T Cells According to Their Cellular Function
Based on their varied functions, gd T cells can be divided into several subtypes. Similar to ab T cells, effector gd T cells can exert an anti-tumor effect through various pathways. Regulatory gd T cells (CD4 + CD25 + Foxp3 + ) or inhibitory gd T cells can regulate the immune balance and maintain immune tolerance (17, 51). In addition, gd T17 cells can produce IL-17 to promote tumor development (6,36,41). Vg9Vd2 T cells recognizes TAA through TCRgd and NKR and Vg9Vd2TCR can recognize PAg. This type of antigen was a product of isoprenoid biosynthesis pathway in eukaryotic cells, such as IPP and the adenylated, thymidylated, and uridylated triphosphate derivatives. In tumor cells, the isoprenoid biosynthetic pathway is enhanced to ensure energy supply and PAg accumulation, prompting recognition by the Vg9Vd2 T cells (65)(66)(67)(68). Vg9Vd2TCR requires the help of Butyrophilin (BTN) 3A1 to recognize tumor cells. BTN3A1 is an immunoglobulin-like molecule with immunomodulatory function, which could mediate the interaction between gd T cells and PAg, and could also be directly recognized by Vg9Vd2TCR (69)(70)(71). There were two theories on how BTN3A1 helps Vg9Vd2TCR recognize PAg. The first proposed mechanism was that BTN3A1 is a sensor that senses the level of PAg inside the cell. The intracellular B30.2 domain of the BTN3A1 molecule is a positively charged pocket that could directly bind to PAg, lead to changes in the structure of the extracellular dimer of BTN3A1 that can be recognized by Vg9Vd2TCR, and then activate the gd T cells (72)(73)(74)(75)(76)(77). The second proposed mechanism was that BTN3A1 formed a BTN3A1-PAg complex with PAg, presented PAg to the outside of the cell, and directly bound to Vg9Vd2TCR to activate gd T cells (78). The latest study found that BTN2A1, which was in the same family as BTN3A1, was also a ligand for Vg9Vd2TCR and necessary for Vg9Vd2 T cells to recognize PAg. BTN2A1 and BTN3A1 can be found on the surface of tumor cells and recognized by two sites of Vg9Vd2TCR. BTN2A1 is recognized by the Vg9 area, and BTN3A1 is recognized by the Vd2 area (79,80). In addition, Vg9Vd2TCR could recognize the F1-ATPaseApoA1 complex. This complex are normally expressed in the inner membrane of mitochondria, but some tumor cells, such as human leukemia (K562) cells and lymphoma (Raji) cells, could ectopic express it on the cell membrane. ApoA1 in the complex could not directly activate Vg9Vd2 T cells, instead it plays a function in stabilizing the interaction between Vg9Vd2TCR and F1-ATPase (13,81). Vg9Vd2 T cells could also recognize TAA through NKR, such as the natural killer 2D receptor (NKG2D) and DNAX accessory molecule 1 (DNAM-1). NKG2D is a lectin-type activation receptor, expressed on most natural killer cells (NK) and natural killer T (NKT) cells and partly expressed on gd T cells and antigen-activated CD8 + T cells (82). When gd T cells contacted by the tumor cells, Vg9 + subpopulations rapidly proliferated, and gd T cells upregulated their NKG2D expression (83). NKG2D ligands on tumor cells include MICA, MICB and ULBP1~4 (84,85). They could be recognized by NKG2D and enable gd T cells to exert antitumor function (82). DNAM-1 is expressed on the gd T cells and believed to promote the secretion of cytokines and enhance the cytotoxicity of immune cells. Vg9Vd2 T cells used DNAM-1 to recognize Nectin-2 and PVR, which were widely expressed on the tumor cells (86)(87)(88). Shielding DNAM-1 from the surface of gd T cells could significantly inhibit its ability to kill tumor cells (89). It was shown that DNAM-1 is one of the important factors mediating gd T cells to recognize tumor cells.
Vd1 T cells also recognize tumor cells through TCRgd and NKR. Vd1TCR could recognize MHC-like molecule CD1d and the lipid antigen presented by it (90,91). CD1d is expressed on a variety of cancers, such as myeloma, breast cancer and prostate cancer (92)(93)(94). The decrease of CD1d molecules on the primitive neuroectodermal tumor cells would cause these cells to evade immune recognition (95). In addition, Vd1TCR could recognize tumor cells through MICA, but the MICA bindings by Vd1TCR and NKG2D were mutually exclusive (96). Vd1 T cells also express NKR. These cells recognize ULBP3 which is expressed on chronic lymphocytic leukemia of B-cell type (B-CLL) through NKR (97). They recognize human breast cancer cells through NKG2D, significantly preventing the disease progression (35). In addition to NKG2D and DNAM-1, Vd1 T cells stimulated by IL-2 or IL-15 also express NKp30, NKp44 and NKp46 (48,98), and have strong IFN-g secretion ability (99,100). Moreover, it has been confirmed that in acute myeloid leukemia, the ligand of NKp30 is B7-H6, a member of the B7 family (101).
Other studies have also confirmed that Vg4Vd5TCR can recognize EPCR, which is expressed on the epithelial tumor cells (19, 20) ( Figure 1).

Anti-Tumor Mechanism of gd T Cells
First, gd T cells could kill tumor cells directly through secreting perforin and granzyme B (82). gd T cells recognize tumor cells and release perforin and granzyme B into the synaptic space. They could further activate caspases to break DNA of tumor cells and lead to tumor cell death (102)(103)(104)(105). gd T cells could kill the human squamous cell carcinoma through perforin and granzyme B (106). Perforin and granzyme B inhibitor significantly reduce the ability of Vg9Vd2 T cells to lyse breast cancer cells in vitro (107). Moreover, in patients with renal carcinoma, activated Vg9Vd2 T cells showed a strong cytotoxicity to autologous tumor cells through perforin and granzyme B (108).
Second, gd T cells kill tumor cells through ADCC. The Fab and Fc segment of antibody could bind to the TAA and gd T cells, respectively. Then gd T cells are activated to kill the tumor cells. Upon interaction with tumor cells, the expression of CD16 (FcgRIIIA) could be up-regulated on gd T cells to induce tumor death through ADCC (82,109,110). In chronic lymphocytic leukemia and breast cancer patients, the cytotoxicity of Vg9Vd2 T cells is significantly enhanced after treatment with monoclonal antibodies including rituximab, trastuzumab and alemtuzumab (111)(112)(113).
Third, gd T cells kill tumors through the Fas/FasL pathway and TRAIL (106). FasL expressed on gd T cells could bind to Fas, and formed Fas trimer, which lead to the binding of the death effector domain (DED) to Fas-associated death domaincontaining protein (FADD), and then activate caspases to induced the apoptosis of target cells (114)(115)(116). Similar to Fas/ FasL, TRAIL also activates caspases through FADD, and then leads to apoptosis of tumor cells (117)(118)(119)(120)(121)(122)(123)(124). In addition, IFN-g could enhance the cytotoxicity of gd T cells by up-regulating the expression of Fas on osteosarcoma cells (125,126). Similar to Vg9Vd2 T cells, Vd1 T cells could kill tumor cells through the perforin-granzyme B, Fas/FasL and TRAIL pathway (49,50,98,101). For example, human skin Vd1 T cells could secrete perforin to kill melanoma cells (127). Granzyme B + Vd1 T cells and TRAIL + Vd1 T cells showed strong cytotoxicity to lymphoma cells and chronic lymphocytic leukemia (128)(129)(130). Beyond that, ex vivo expanded Vd1 T cells highly express FasL, and have strong cytotoxicity on colon cancer cells (131).

gd T CELLS ENHANCE THE ANTI-TUMOR ABILITY OF OTHER IMMUNE CELLS
gd T cells share similar functions as antigen presenting cells (APC), which could activate CD8 + T cells (132,133). When co-cultured with chronic myeloid leukemia (CML) cell lysates, the expression of co-stimulatory molecules (CD40, CD80 and CD86) and antigen-presenting molecule HLA-DR on Vg9Vd2 T cells could be strongly up-regulated. When these gd T cells were co-cultured with CD8 + T cells, the proliferation rate of CD8 + T cells became 3 times faster than that of the control group (134,135). Tumor cell fragments activate MAPK signaling pathways through Vg9Vd2TCR, up-regulate the expression of scavenger receptor CD36, enhance antigen uptake and processing of Vg9Vd2 T cells, and then induced tumor antigen-specific CD8 + T cell response (136). Furthermore, gd T cells toned to interact with cell surfacebound antibodies to acquire the ability of APC (137).
In addition, activated gd T cells could secrete IFN-g, which stimulates CSC to up-regulate the expression of MHC I molecules and CD54, and enhance the killing effect of CD8 + T cells on tumor cells (138). Activated gd T cells could also express CD137L to stimulate NK cells that upon proliferation exhibit strong anti-tumor activity through cell-to-cell contact (31).

The interaction between gd T cells and dendritic cells (DC) is
mutual. gd T cells promote the maturation of DC, and mature DC induces the activation and proliferation of gd T cells, which yield enhanced anti-tumor effect (139,140). For example, activated Vg9Vd2 T cells could secrete IFN-g and TNF-a to promote DC maturation and increase the expression of CD86 and MHC-I molecules on DC (141,142). Mature DC could activate gd T cells through presenting IPP, which synergizes with ATP-binding cassette transporter A1 (ABCA1), ApoA1 and BTN3A1 (143) (Figure 2).

TUMOR-INFILTRATING gd T CELLS PROMOTE TUMOR DEVELOPMENT BY SECRETING IL-17
Interestingly, patients with increased number of tumorinfiltrating gd T cells have higher recurrence rates and likelihood of metastasis (144)(145)(146). Among the tumorinfiltrating gd T cells, Vd1 T cells are present as the main population and secrete IL-17 to promote tumor development. IL-17 can promote the proliferation of tumor cells by activating IL-6/STAT3 and NF-kB pathways. In addition, it can also stimulate tumor cells to secrete vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP) to further help tumor metastasis. High levels of IL-17 have been found in patients with advanced tumor or metastasized tumors (64,147,148). For example, in patients with solid tumors, Vd1 T cells account for a large proportion of tumor-infiltrating gd T cells; unlike Vd1 T cells in adjacent non-tumor tissues, tumorinfiltrating gd T cells do not express granzyme B, perforin, IFN-g, FasL, TRAIL and NKR, but secrete IL-17 (149)(150)(151)(152)(153)(154). Majority of the tumor-infiltrating Vd1 T cells were T EM phenotype, while most of the Vd1 T cells in healthy subjects were T CM phenotype

TUMOR-INFILTRATING gd T CELLS INHIBIT THE ANTI-TUMOR FUNCTION OF OTHER IMMUNE CELLS
IL-17, secreted by tumor-infiltrating Vd1 T cells not only acts on tumor cells directly, but can also recruit myeloid-derived suppressor cells (MDSC) to tumor (147,148,150). MDSC inhibits the activation of CD8 + T cells by expressing high levels of ARG1, which decomposes arginine in the tumor microenvironment (157)(158)(159)(160).
In addition, tumor-infiltrating gd T cells could significantly inhibit the maturation of CD4 + T cells (155). Studies in breast cancer settings showed that tumor-infiltrating gd T cells could inhibit the maturation of CD4 + or CD8 + T cells and change their functions by arresting cell cycle in G0/G1 phase and increasing the expression of p53, P21, and P16. Through secreting IL-10 and TGF-b1, these suppressed T cells further amplified the inhibitory effect (161,162). Beyond that, these cells express high levels of PD-L1 to promote the apoptosis of CD8 + T cells in cancer patients (41).
Vd1 T cells could also inhibit the maturation of DC, reduce the expression of CD80/86 and HLA-DR on DC, attenuate the secretion of pro-inflammatory cytokines TNF-a, and up-regulate the expression of PD-L1 on the surface of DC (161, 163) (Figure 3).

ANTI-TUMOR IMMUNOTHERAPY WITH gd T CELLS
The unique antigen recognition mechanism of gd T cells renders them the ability to kill various types of tumors. Therefore, gd T cell-based therapies have been widely used in clinical as antitumor immunotherapies and achieved good results ( Table 2). At present, the most routine method in these therapies is to activate the anti-tumor activity of natural gd T cells and the Vg9Vd2 T cells, which as the most abundant subtype in peripheral blood are often selected and utilized through transferring back to cancer patient after stimulation in vitro or direct activation in vivo.
The most widely used stimulants for expanding Vg9Vd2 T cells in vitro are zoledronic acid (ZOL) and IL-2. As a kind of bisphosphate, ZOL could specifically inhibit farnesyl pyrophosphate synthase (FPPS) in isoprene biosynthesis pathway, thus causing the accumulation of endogenous PAg in cells and promoting the activation of Vg9Vd2 T cells (65). This method could effectively expand and activate Vg9Vd2 T cells from patients or healthy people in vitro (52). In addition, another kind of PAg, 2methyl-3-butenyl-1-pyrophosphate(2M3B1-PP) could also effectively stimulate and expand Vg9Vd2 T cells (164,165). The activated immune cells are transferred back into the patients to produce anti-tumor effects. In order to track the activated Vg9Vd2 T cells, they are typically labeled with indium 111 . Studies have confirmed that these cells mainly metastasize to the lung, liver and spleen, as well as to the tumor sites (166). In the treatment of patients with multiple myeloma, the stimulated Vg9Vd2 T cells expressed high levels of NKG2D and IFN-g, but not IL-17. After treatment, the number of Vg9Vd2 T cells in the tumor microenvironment increased, lasting as long as 4 weeks (167). In patients with renal cell carcinoma and non-small cell lung cancer, repeated injections of IL-2 has demonstrated good safety (168), enhanced the cytotoxicity of Vg9Vd2 T cells. As results, the deterioration of tumor was alleviated with patients' condition stabilized, and the survival time was pro-longed (164, 165, 169,   170). In the clinical study of malignant ascites, after transferring back the activated Vg9Vd2 T cells, the number of tumor cells in ascites decreased significantly and the level of IFN-g in ascites increased. During the course of treatment, there were no significant adverse effects and the amounts of ascites decreased significantly (171). In addition to the direct anti-tumor effect of Vg9Vd2 T cells, the numbers of CD4 + T and CD8 + T cells could also get increased after the allogeneic Vg9Vd2 T cells were transferred into the patients, as shown in another study of cholangio-carcinoma (52). Vg9Vd2 T cells could also be activated in vivo. Injection of ZOL and IL-2 could directly activate these cells in cancer patients. Serval clinical trials have demonstrated that, upon injection and activation of Vg9Vd2 T cells in vivo, IFN-g was strongly induced and the deterioration of the tumor was controlled (53,(172)(173)(174)(175). Besides ZOL, Vg9Vd2 T cells could also be activated by synthetic PAg or bromohydrin pyrophosphate (BrHPP, IPH1101) with good safety tolerability in patients (176).

OPTIMIZATION OF gd T CELL-BASED IMMUNOTHERAPY
In clinic, repeated use of ZOL and IL-2 carry the liability in inhibiting the proliferation of Vg9Vd2 T cells (172), which could be alleviated by vitamin C (VC) and L-ascorbic acid 2-phosphate (pVC). VC has the ability to reduce the apoptosis of gd T cells during stimulation, and pVC may enhance the expansion of gd T cells. Therefore, VC and pVC have been utilized to improve the efficacy of the gd T cells in anti-tumor immunotherapy (177). In addition, cytotoxicity of Vg9Vd2 T cells could be improved by adding IL-21, IL-15, or IL-33 in vitro (55,(178)(179)(180)(181)(182). Anti-cancer drug Gemcitabine or antiepileptic drug Valproic acid (VPA) in combination with ZOL could also enhance the anti-tumor ability of gd T cells (183,184).
In recent years, chitosan nanoparticles (CSNPs) and antibodies have been developed as potential anti-tumor therapies. CSNPs have been shown to regulate gd T cells by up-regulating the expression of NKG2D, CD56 and FasL, and enhancing their anti-tumor functions (185). TIM-3 could also inhibit the killing effect of Vg9Vd2 T cells on tumor by reducing the expression of perforin and granzyme B. PD-1 and TIM-3 antibodies could protect anti-tumor activity of Vg9Vd2 T cells (186)(187)(188). Beyond these, the application of bispecific antibodies can also promote gd T cells to inhibit tumor development. For example, in the study of hepatoblastoma and pediatric hepatocellular carcinoma, the application of EpCAM/CD3bispecific BiTE antibody (MT110) enhanced the anti-tumor ability of gd T cells; similarly, in epithelial ovarian cancer and pancreatic ductal adenocarcinoma, bispecific antibody [HER2xCD3] and [(HER2)2xVg9] (tribody format) could also effectively enhance the cytotoxicity of gd T cells (189)(190)(191)(192)(193).
Finally, chimeric antigen receptor engineered gd T (CAR-gd T) technology is another new direction in immunotherapy. CAR-gd T cells could target GD2, a TAA on the surface of neuroblastoma cells, and effectively kill tumors. This kind of CAR-gd T cells need to recognize GD2 to become activated. Such mechanism ensures the specificity of these cells in killing tumor cells and offering lower toxicities and side effects (194,195). On the hand, Vg9Vd2TCR could also be transduced into ab T cells. These CAR-T cells are called TEGs, which carry not only the extensive recognition ability of gd T cells but also and the memory ability of ab T cells (196)(197)(198)(199).
SUMMARY Taken together, we described in this review that Vd1 T cells and Vg9Vd2 T cells are the two most important subgroups of human gd T cells. Peripheral Vd1 T cells and Vg9Vd2 T cells could recognize tumor cells through TCRgd and NKR, and kill them through perforin-granzyme B, Fas/FasL and TRAIL. Activated Vg9Vd2 T cells could perform the function of APC, and furthermore, they could activate NK cells and DC directly. On the contrary, tumorinfiltrating Vd1 T cells promoted tumor development by secreting IL-17 and inhibiting the maturation of CD4 + /CD8 + T cells and DC. In immunotherapy, ZOL, 2M3B1-PP or IPH1101 has been commonly used to activate Vg9Vd2 T cells to achieve anti-tumor effect. The failure caused by repeated application of this method can be solved by adding VC or replacing cytokines. In addition, new classes of drugs such as CSNPs, were also applied to gd T cell-based anti-tumor immunotherapy.
It is noteworthy to mention that although Vd1 T cells account for the majority of tumor-infiltrating gd T cells, the definition of gd T cell subsets still rely on their profile in cytokine production (32, 64,149). Secondly, the interaction mechanism between gd T cells and the environment or other immune cells remains to be further elucidated. For example, Vg9Vd2 T cells could ingest LDLcholesterol upon activation and lead to reduced function, suggesting that obesity may inhibit the anti-tumor activity of gd T cells (200). Another myth exists in how exactly soluble molecules mediate the inhibition of gd T cells in tumor microenvironment (161,163). In addition, gd T cell-based immunotherapy needs to be further optimized, with emphasis on how to carry out personalized therapy according to the actual situation of individual patient.
In summary, a more comprehensive understanding of the biological characteristics of gd T cells, an important group of lymphocytes, will guide the improvement of their clinical application methods and provide new strategies to fight against human cancers.

AUTHOR CONTRIBUTIONS
JZ completed the writing of the classification of gd T cells. XC and XW completed the writing of introduction and summary. YL and GL completed the writing of the rest of this review. All authors contributed to the article and approved the submitted version.