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γδ T Cells in the Tumor Microenvironment—Interactions With Other Immune Cells

A growing number of studies have shown that γδ T cells play a pivotal role in mediating the clearance of tumors and pathogen-infected cells with their potent cytotoxic, cytolytic, and unique immune-modulating functions. Unlike the more abundant αβ T cells, γδ T cells can recognize a broad range of tumors and infected cells without the requirement of antigen presentation via major histocompatibility complex (MHC) molecules. Our group has recently demonstrated parts of the mechanisms of T-cell receptor (TCR)-dependent activation of Vγ9Vδ2+ T cells by tumors following the presentation of phosphoantigens, intermediates of the mevalonate pathway. This process is mediated through the B7 immunoglobulin family-like butyrophilin 2A1 (BTN2A1) and BTN3A1 complexes. Such recognition results in activation, a robust immunosurveillance process, and elicits rapid γδ T-cell immune responses. These include targeted cell killing, and the ability to produce copious quantities of cytokines and chemokines to exert immune-modulating properties and to interact with other immune cells. This immune cell network includes αβ T cells, B cells, dendritic cells, macrophages, monocytes, natural killer cells, and neutrophils, hence heavily influencing the outcome of immune responses. This key role in orchestrating immune cells and their natural tropism for tumor microenvironment makes γδ T cells an attractive target for cancer immunotherapy. Here, we review the current understanding of these important interactions and highlight the implications of the crosstalk between γδ T cells and other immune cells in the context of anti-tumor immunity.


INTRODUCTION
For the past 37 years, since the first isolation of the TCR g gene segment (1,2), the knowledge accumulated about the gd T-cell lineage has grown exponentially and received strong clinical interest, especially for cancer immunotherapy development (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Similar to the other two lineages of lymphocytes in the jawed vertebrates that utilize somatically recombined receptors for immunosurveillance (B cells and ab T cells) (16), TCR heterodimers of gd T cells are generated through somatic rearrangements of genes encoding for TCR d chain variable (V), diversity (D), joining (J), and constant (C) gene segments, and TCR g chain V, J, and C gene segments at the thymus (17,18). Hypothetically, such diverse gene rearrangements can result in a total of 10 17 possible distinct gd TCRs (19). Despite the diverse theoretical gd TCR repertoire, human gd T cells can be classified into two major subsets according to their TCR Vd chain usage: Vd2 + populations that are usually paired with Vg9 chain, and Vd2 − populations with diversified Vg chain usage (6,20). Among all 8 TCR Vd gene segments, Vd1, Vd2, and Vd3 are three commonly used segments for d chain rearrangement (21,22).
Importantly, the presence of tumor-infiltrating gd T cells was shown to be the most favorable prognostic marker for overall cancer patients survival in 25 different cancer types and solid tumors (non-brain tumor) (4). Their role in cancer immunosurveillance was clearly evidenced and validated in many tumor models and clinical studies including cutaneous carcinoma (154), melanoma (119,155,156), lymphoma (157)(158)(159), leukemia (44,117,160,161), gastric (162), colorectal (43,163,164), kidney (41), prostate (165,166), and pancreatic (143) cancers. The ability of gd T cells to produce large quantities of cytokines and chemokines rapidly and their tendency to reside in blood circulation or in non-lymphoid tissues (e.g., skin, intestines, and lungs) (8,16,17), helps to provide the first line of immunosurveillance against aberrant cell growth and infectious diseases, and bridges the innate and adaptive immune responses. Thus, it is important to understand the crosstalk between gd T cells and other immune cells in the TME and to harness this knowledge for effective cancer immunotherapy development.

CROSSTALK BETWEEN gd T CELLS AND ab T CELLS
The role of antigen processing and presentation to ab T cells is mostly associated with the classical professional antigenpresenting cells (APCs) like DCs, macrophages, and B cells (167,168). However, with the unexpected discovery by Brandes et al., it was shown that activated but not resting human Vg9Vd2 + T cells were also capable of acquiring professional APC functions (56). Indeed, activated Vg9Vd2 + T cells isolated from both healthy individuals and cancer patients' peripheral blood mononuclear cell (PBMC) exhibited potent APC functions to stimulate robust antigen-specific ab T-cell responses (169).
During the activation process, human Vg9Vd2 + T cells can rapidly gain APC functions by upregulating co-stimulatory (CD40, CD80, and CD86), MHC class I and II molecules (56,57,61,62,97,108,169), and transiently expressed lymph node-homing markers, chemokine receptor CCR4 and CCR7 (62,68,97). This allows recruitment of activated gd T cells from the peripheral sites to secondary lymphoid tissues for antigen presentation and bridges the early phase of rapid innate-like gd T-cell response to microbial or tumor antigens with the later phase of adaptive immune response involving the antigen-specific CD4 + and CD8 + ab T cells (14,15,17,168,170). In a study by Himoudi et al., it was shown that activated human Vg9Vd2 + T cells were "licensed" to acquire their APC functions through recognition of antibodyopsonized tumor cells, mediated targeted cell killing by their innate cytotoxicity, and subsequently helped to release tumor-associ ate d antig ens (TAAs) into the surrounding microenvironment (60). These TAAs can be taken up by activated gd T cells via phagocytosis (62,97,98,108), trogocytosis (171), or pinocytosis (57,58), processed and presented on the cell surface for priming and induction of naïve ab T cells (59, 60) ( Figure 1). Furthermore, it was shown that Vg9Vd2 + gd T cells can uptake microbes and soluble antigens via CD16-mediated phagocytosis, a process that can lead to functional antigen processing and presentation on MHC class II (98), and cross-presentation of immunodominant MHC class I peptides to antigen-specific CD8 + ab T cells (58,60,61). This notion was further supported by the identification of Vg9Vd2 + T cells in malaria patients that readily acquired APC functions upon infection and induced CD4 + and CD8 + ab T-cell activation (61). Interestingly, it was also demonstrated that activated Vg9Vd2 + T cells can uptake CD1d-containing membrane fragments from phosphoantigen expressing Cd1d + target cells via trogocytosis, leading to the presentation of CD1drestricted antigen and the activation of Va24Vb11 + invariant natural killer T cells (iNKT) (172).
When compared to activated ab T cells and monocytes, activated Vg9Vd2 + T cells were shown to be more efficient in presenting antigens and induced 100-fold higher proliferative , and GM-CSF for contact-dependent and independent crosstalk with tumor cells, CD4 + and CD8 + ab T cells, NK cells, DCs, macrophages, and neutrophils. Activated gd T cells cross-present antigens to CD4 + and CD8 + ab T cells; induce B-cell immunoglobulin class switching; co-stimulate NK cells via CD137/CD137L and ICOS/ICOS-L interactions; induce upregulation of CD40, CD86, and HLA-DR expression on monocyte; promote DC maturation via CD40/CD40L and Fas/FasL interactions; and inhibit the immunosuppression function of CD4 + CD25 + FoxP3 + Treg cells on CD4 + ab T-cell activity. In contrast, activated gd T cells can also suppress DC function (downregulation of CD80, CD83, CD86, HLA-DR, IL-1b, IL-6, and IL-12) and mediate DC killing via perforin release. Butyrophilin 2A1 and 3A1 (BTN2A1 and BTN3A1); cyclooxygenase-2 (COX2); granulocyte-macrophage colony stimulating factor (GM-CSF); granzyme B (GzmB); human leukocyte antigen-DR (HLA-DR); immunoglobulin A, E, or G (IgA, IgE, or IgG); inducible T-cell co-stimulator (ICOS); ICOS ligand (ICOS-L); interferon-g (IFN-g); major histocompatibility complex class I and II (MHC-I and -II); MHC class I chain-related antigens A and B (MICA and MICB); natural killer group 2D (NKG2D); programmed cell death 1 (PD-1); PD-1 ligand 1 (PD-L1); prostaglandin E2 (PGE2); reactive oxygen species (ROS); T-cell receptor (TCR); tumor necrosis factor-a (TNF-a); UL16-binding protein (ULBP). responses in naïve CD4 + ab T cells (56). Activated Vg9Vd2 + T cells were also able to cross-present antigens to CD8 + ab T cells with a higher efficiency and reproducibility (57), and induced less CD4 + CD25 hi FoxP3 + Treg cell expansion than moDCs (59). Similar results were seen under pathological condition, when it was shown that gd T cells isolated from gastric cancer patients can acquire APC functions upon activation with cells derived from autologous tumor tissues (99). These clinically relevant tumor-activated gd T cells induced strong antigenspecific CD4 + and CD8 + ab T-cell responses and prevented immunosuppression mediated by CD4 + CD25 + Treg cells (99) (Figure 2). Of note, Muto et al. showed that resting Vg9Vd2 + T cells can significantly upregulate the expression of scavenger receptor CD36 during activation and that this was mediated by a key transcription factor, CCAAT/enhancer-binding protein a (C/EBPa), that supports acquisition of APC functions in activated Vg9Vd2 + T cells (108). In contrast, resting ab T cells expressed a low level of CD36 and did not upregulate it upon activation (108). In DCs and macrophages, the CD36 receptor was shown to facilitate the uptake of apoptotic cells and crosspresentation (173,174), potentially explaining the induction of a stronger antigen-specific ab T-cell response by activated Vg9Vd2 + T-cell APC.
The ability to migrate to the tumor site and cross-present TAAs to ab T cells was also retained when Vd1 + and Vd2 + gd T cells were engineered to express tumor-specific chimeric antigen receptors (CARs) and resulted in an increased cytotoxic level against tumor cells (175). Hence, activated Vg9Vd2 + T cells can process and present antigens and provide critical co-stimulatory signals to prime and induce naïve CD4 + (56) and CD8 + (57) ab T cells for proliferation, differentiation, and cytokine production and to mediate cytotoxic responses against tumors and pathogen-infected cells (176)(177)(178)(179). This remarkable ability of gd T cells to uptake and present antigens and prime ab T cells has been highlighted by Vantourout et al. (168), and the accumulated data so far have illustrated the potential of harnessing the APC functions of gd T cells to crosstalk with ab T cells for immunotherapy development.
Given their natural tropism for TME (14,119,175,(180)(181)(182), activated gd T cells could hence be utilized to prolong the intratumoral immune response by cross-presenting TAAs to other tumor-infiltrating lymphocytes and provide an early source of IFN-g to expand and increase immunogenicity of TAA-specific ab T cells within the TME (155,183,184), and to upregulate expression of MHC class I and II on tumor cells (185,186) for ab T cell-mediated killing ( Figure 2). The presence of tumor-infiltrating gd T cells within the TME as revealed by genomic data analysis in over 18,000 human tumors has uncovered a strong correlation to good prognosis (4). In the context of cancer immunotherapy, the capability of activated gd T cells to cross-present TAAs to ab T cells could be further boosted through the "licensing" pathway (60, 187) by using therapeutic monoclonal antibodies against tumor cells, e.g., rituximab (anti-CD20) and trastuzumab (anti-HER2/neu) (145,188,189). Such combination treatment could greatly improve the outcome of gd T-cell cancer immunotherapy. Activated Vg9Vd2 + T cells can also modulate ab T-cell activity indirectly by co-stimulating NK cells via inducible Tcell co-stimulator (ICOS)/ICOS-L and CD137/CD137L engagements to enhance IFN-g and TNF-a production (100,101), which, in turn, helps to support ab T-cell activation (190). Another study has shown that activated Vg9Vd2 + T cells can induce B-cell and DC maturation and subsequently leads to alloreactive stimulation of ab T-cell proliferation and IFN-g production by mature B cells and DCs (72). The interactions between gd T cells and other immune cells (B cells, DCs, and NK cells) will be discussed later in this review.
Despite their ability to exert positive immune modulation functions on ab T cells, activated gd T cells can also negatively regulate ab T-cell response by upregulating an immune checkpoint inhibitory ligand, programmed cell death 1 ligand 1 (PD-L1) (11, 151,191). The suppressive phenotype of activated Vd2 + gd T cells on autologous ab T cells was shown to be mediated by the PD-1/PD-L1 interactions and correlated well with the strength of Vd2 + gd TCR signaling during the activation process but was independent of TGF-b and FoxP3 expression (192) (Figure 2). Daley et al. showed that tumor-infiltrating gd T cells with high expression levels of checkpoint inhibitory ligands PD-L1 and Galectin-9 could inhibit ab T-cell activation through checkpoint receptor ligation (193). The immunosuppressive effect can also be mediated by the interaction between CD86 on activated Vd2 + gd T cell and cytotoxic T lymphocyteassociated antigen 4 (CTLA-4) on activated ab T cells (191). Such gd T cell-mediated immunosuppression of ab T cells, however, can be significantly reduced by disrupting PD-1/PD-L1 and CTLA-4/CD86 interactions with blocking antibodies (191,192). Furthermore, Peng et al. identified tumorinfiltrating Vd1 + gd T cells that could suppress naïve/effector ab T-cell proliferation and IL-2 production through the Toll-like receptor (TLR) 8 signaling pathway and may lead to tumor immune escape (102). The immunosuppressive activity of Vd1 + gd T cells can be reversed using TLR8 ligands, and this signaling involved the myeloid differentiation primary response 88 (MyD88), TNFR-associated factor 6 (TRAF6), IKB kinase a (IKKa), IKKb, and mitogen-activated protein kinase 14 (MAPK14), but not transforming growth factor-b-activated kinase 1 (TAK1), Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) molecules in Vd1 + gd T cells (102). It was also reported that gd + NKG2A + intraepithelial lymphocytes (IELs) can mediate suppression of CD8 + ab + IEL cytotoxic responses (IFN-g and granzyme B) in patients with celiac disease through TGF-b secretion (194). The immunosuppressive effect on CD8 + ab + IELs can be further enhanced upon gd + IELs NKG2A receptor ligation with the cognate ligand, human leukocyte antigen-E (HLA-E) (194). This immunosuppressive effect can be reduced by blocking NKG2A/HLA-E interaction and TGF-b with blocking antibodies (194). Therefore, it is important to consider these negative immunomodulatory roles of gd T cells when designing novel immunotherapeutics.
Apart from the PD-1/PD-L1 and CTLA-4/CD86 immune checkpoint axes, other non-conventional checkpoint receptors [killer Ig-like inhibitory receptors (KIRs), Ig-like transcript 2 (ILT-2), and NKG2A] can be expressed on Vg9Vd2 + T cells, inhibit their cytotoxic function, and prevent tumor cell lysis upon recognition of specific HLA class I ligands on tumor cells (195)(196)(197)(198)(199)(200)(201)(202)(203). In this context, the presentation of HLA class I molecules on tumor cells can be a double-edged sword. On one hand, it facilitates the presentation of antigenic peptides to activate CD8 + ab T cells, but at the same time, it can also inhibit the activation of Vg9Vd2 + T cells. Such inhibitory signals on immune cells mediated by KIRs, ILT-2, or NKG2A can be blocked using monoclonal antibodies targeting KIRs (lirilumab and IPH4102), ILT-2 (anti-ILT-2, anti-HLA-G1, anti-FasL), or NKG2A (monalizumab) (204,205). In a study by Andreé t al., treatment with monalizumab indeed led to enhanced anti-tumor immune responses elicited by T and NK cells (206). As a type 2 inhibitory membrane receptor, NKG2A carries cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and forms heterodimers with CD94 to recognize non-classical HLA-E molecule (207). Many human tumors have been shown to express HLA-E including in the colon, cervical, endometrial, head and neck, liver, lung, pancreas, ovarian, and stomach (206). Moreover, a majority of Vg9Vd2 + T cells in healthy individuals express NKG2A/CD94 (197,198,200,208), and the expression levels can be induced by IL-15 and TGF-b (209,210). Therefore, treatments targeting these non-conventional checkpoint receptors on Vg9Vd2 + T cells (KIRs, ILT-2, and NKG2A) to disrupt the interactions with their respective HLA class I ligands on tumor cells (HLA-C, HLA-G, and HLA-E) may help to enhance the effectiveness of Vg9Vd2 + T cell-based tumor immunotherapy. Recent work by Payne et al. suggests that BTN3A, itself part of the molecular complex required for phosphoantigen-mediated activation of Vg9Vd2 + T cells, can also inhibit tumor-reactive CD8 + ab T cells when bound to N-mannosylated residues of CD45 by preventing its segregation from the immunological synapse (52). In this study, the suppression of ab T-cell activation was shown to involve BTN3A1 but not BTN2A1, and the immunosuppressive effect could be blocked by BTN3A1specific monoclonal antibodies such as clone 20.1, 103.2, and CTX-2026 (52). Targeting BTN3A1 with the agonistic antibody CTX-2026 induced BTN3A1 switching from immunosuppressive to immunostimulatory conformations and promoted coordinated Vg9Vd2 + and CD8 + ab T-cell anti-tumor responses against BTN3A1 + tumors (52). Hence, BTN3A1 may be an attractive immune target for intervention to orchestrate effective and coordinated gd and ab T-cell anti-tumor responses.

CROSSTALK BETWEEN gd T CELLS AND B CELLS
gd T cells have been previously reported to interact with B cells and modulate their immune functions (5,8,168,211,212).
Vg9Vd2 + T cells can adopt a role similar to T follicular helper (Tfh) cells and provide B-cell help, thereby regulating B-cell maturation. Specifically, a subset of CXCR5 + Vg9Vd2 + T cells present in circulation and in tonsil tissue expresses costimulatory molecules (ICOS and CD40L) upon antigen stimulation and secrete cytokines (IL-2, IL-4, and IL-10), which can promote the development of antibody-producing B cells via immunoglobulin class switching [including immunoglobulin A (IgA), IgE, IgG1, IgG2, IgG3, and IgG4] (8,213,214) in the extra-follicular or within germinal centers (65-67, 69) ( Figure 2). Furthermore, upon stimulation with IL-21 and HMBPP, activated tonsillar Vg9Vd2 + T cells can express CXCL13 receptor, CXCR5, induce lymphoid-homing phenotype and clustering in germinal centers, and sustain the production of germinal centers (70,71). Similarly, IPP-stimulated Vd2 + gd T cells with functional CCR7 expression can also induce transient lymph node-homing, migration, and clustering of Vd2 + gd T cells within B-cell zones of germinal centers in lymphoid tissues (64,68).
Phosphoantigen-activated Vd2 + gd T cells can additionally induce the expression of B-cell co-stimulatory molecules (CD40L, OX40, CD70, and ICOS) and affect the downstream production of circulating IgA, IgG, and IgM antibodies by B cells (68). In patients with specific mutations (RAG1 and CD3D) that impair ab T-cell function, gd T cells are responsible for hyper-IgE syndromes or the elevated production of circulating IgA, IgG, and IgM (215,216). gd T cells can also suppress antibody responses via the induction of CD4 + Foxp3 + Treg cells (217). Conversely, some B cells can express BTN2A1 and BTN3A1, required for Vg9Vd2 + T-cell activation (33-35), thereby directly influencing Vd2 + gd T-cell activation (218,219) as shown by early studies using Daudi cells, a B-cell malignancy cell line (Burkitt's lymphoma) (220)(221)(222)(223)(224)(225)(226). Vg9Vd2 + T cells can directly engage BTN2A1 expressed on B cells via the TCR Vg9 chain (3,(32)(33)(34)(35)(36)227), and in concert with BTN3A1, this results in Vg9Vd2 + T-cell activation and expansion (101,212). Hebbeler et al. showed that the Vg9Vd2 + T cells activated and expanded by phosphoantigen or Daudi B lymphoma cells use public TCR Vg9 clonotypes, and elicit comparable cytotoxic responses against tumor cells (228). Further investigations revealed that the germline-encoded region between TCR Vg9 CDR2 and CDR3 is responsible for contacting BTN2A1 on target cells (33, 34). Such findings indicate the inherent property of TCR Vg9 to recognize diverse range of cell types that express BTN2A1 including B cells (212,(227)(228)(229). In addition to BTN2A1 and BTN3A1, B cells also express other closely related BTN molecules such as BTN3A2 (in naïve or germinal center B cells), BTN3A3 (in memory B cells), BTN1A1, and BTN2A2 (3,50). The contribution of these other BTN molecules in B cells for gd T-cell activation remains elusive. Similarly, circulating activated B7 + CD39 + B cells can stimulate Vd1 + gd T-cell proliferation (133,134). The Vd1 + gd T-cell stimulatory ligand is upregulated in B cells upon activation and can induce polyclonal Vd1 + gd T-cell responses (133). This B cellmediated immunostimulatory effect on Vd1 + gd T cells can be blocked with antibodies against B7 and CD39 (133,212).
In summary, Vg9Vd2 + T cells can regulate B-cell maturation during development or initiation of an immune response, sustain the production of germinal centers within secondary and possibly tertiary lymphoid structures, and affect the production of circulating (auto)antibodies for humoral immunity (168,211,212), while B cells can activate Vd1 + and Vg9Vd2 + T cells (230).

CROSSTALK BETWEEN gd T CELLS AND NK CELLS
Human NK cells are important innate immune subset for controlling early tumor growth and metastasis through cellmediated cytotoxicity and show broad reactivity to tumors that escaped immunosurveillance by loss or aberrant MHC class I expression (14,231,232). Being a specialized group of innate lymphoid cells (ILCs), NK cell functions are closely regulated by a range of cytokines such as IFN-g, TNF-a, IL-2, IL-12, IL-15, IL-18, and IL-21 (233,234). These effector molecules are important for the initiation of anti-viral and anti-tumor immune responses (235)(236)(237)(238). However, more established tumors can evade NK cell surveillance by developing resistance to NK cell-mediated cytotoxicity, leading to tumor immune escape (239).
In order to overcome NK-resistant tumors, Maniar et al. showed that activated human NK cells (CD25 hi , CD54 hi , CD69 hi , and CD137 hi ) increased surface expression of natural NKG2D receptors to promote tumor cytolysis and death (73). NKG2D is a lectin-like type 2 transmembrane receptor mostly expressed by human NK cells and binds to MHC-related ligands such as ULBPs, MICA, and MICB, which are highly expressed in tumor cells but rarely in healthy cells (231,240). IPP-activated Vd2 + gd T cells upregulate CD137L (4-1BBL), engage with CD137 + NK cells, and can in turn lead to enhanced NKG2D expression and NK cellmediated cytotoxicity against tumors (73) (Figure 2), highlighting a potential key role for gd T cells in this process. CD137 or 4-1BB is a member of the tumor necrosis factor receptor superfamily (TNFRSF) and is expressed by a range of immune cells (190). Expression of CD137 on NK cells is induced by IL-2 and IL-15, and following CD137 signaling, it promotes NK cell proliferation and production of IFN-g, which, in turn, can support NK tumor effector functions (101,190). This finding was further corroborated by Liu et al., and they demonstrated that in the context of liver fibrosis, gd T cells engaged with conventional and liver-resident NK cells through CD137/CD137L interactions to promote NK cell-mediated cytotoxicity against activated hepatic stellate cells and conferred immune protection (75).
Similar to NK and CD8 + ab T cells, human gd T cells also express NKG2D to detect stress-inducible ligands on tumors and pathogen-infected cells (125,(241)(242)(243)(244)(245). Several studies have shown that NKG2D ligation to its cognate ligand can costimulate Vg9Vd2 + T-cell activation (CD25 and CD69 upregulation) and promotes the release of IFN-g, TNF-a, and cytolytic granules to mediate killing of NKG2D ligand-expressing tumors (163,(246)(247)(248)(249)(250)(251). In the context of leukemia and lymphoma cell recognition by Vg9Vd2 + T cells, it was reported that tumor-expressed ULBP1 was a strong marker for tumors susceptible to Vg9Vd2 + T cell-mediated cytotoxicity (252). Similarly, it was shown that ULBP1 overexpression in tumor cells can lead to enhanced killing by Vg9Vd2 + T cells (253). Hence, blocking NKG2D-mediated Vg9Vd2 + T-cell recognition of tumor cells with anti-NKG2D and anti-MICA/B monoclonal antibodies inhibits tumor cell killing to varying degrees (247,249,253). Vd1 + gd T cells can also recognize and kill NKG2D ligand-expressing tumors via NKG2D receptor (8,11,245,254). The number of Vd1 + gd T cells and ULBP3 expression level are negatively correlated with disease progression in chronic lymphocytic leukemia patients (254). A study reported by Kamei et al. demonstrated a longer overall survival in gastric cancer patients with high expression levels of NKG2D and ULBP1 (255). Hence, upregulation of stressinducible NKG2D ligand in tumor cells and NKG2D receptor in tumor-infiltrating immune cells can help to orchestrate concerted NKG2D-mediated NK, CD8 + ab, and gd T-cell antitumor responses within the TME. Of note, several anti-cancer drugs have been found to induce expression of NKG2D ligand in tumor cells, including the proteasome inhibitor bortezomib and the alkylating agent temozolomide, and these can help to promote tumor cell lysis by NK and gd T cells (256,257). Therefore, it is feasible to target NKG2D and its ligands for gd T cell-based immunotherapy development.
It was later shown that IPP-activated Vg9Vd2 + T cells can upregulate ICOS and signal NK cells via ICOS/ICOS-L engagement to promote CD69 and CD137 expression, which then leads to enhanced production of IFN-g, TNF-a, MIP-1b, I-309, RANTES, and soluble Fas ligand by activated NK cells (100). Such ICOS/ICOS-L-mediated crosstalk enables NK cells to acquire the "license" to kill mature DCs that may play a role in inflammation and tumor growth (100). These studies have uncovered the immunomodulatory role of IPP-activated Vg9Vd2 + T cells to circumvent NK-resistant tumors and to promote NK-mediated DC editing function by modulating NK cell cytotoxicity through CD137/CD137L and ICOS/ICOS-L engagements (73,101) (Figure 2). Such findings will provide an alternative strategy for gd T cell-based immunotherapy development against difficult-to-treat solid tumors or to prevent metastasis (239,258,259).
However, NK cell activity can also be negatively regulated by gd T cells. Zoledronate-activated Vd2 + gd T cells not only can co-stimulate early NK cell activation for IFN-g production but also lead to premature ending of the response by inducing cytotoxicity against CD56 + DC-like cells (74). In the absence of activated Vd2 + gd T cells, CD56 + DC-like cells survived (74) and maintained NK cell activity through secretion of NK cellactivating cytokines such as IL-1b and IL-18 (260,261). Therefore, further studies will help to provide a better understanding of the immunosuppressive role of Vd2 + gd T cells on NK cells.

CROSSTALK BETWEEN gd T CELLS AND MONOCYTES/MACROPHAGES
gd T cells share many of their innate functions with other immune cell subsets, including NK cells, monocytes, and macrophages (56,98,262,263). These are integral to the innate inflammatory response against infectious pathogens and tumors, which, in turn, activates a strong and targeted adaptive immune response (170). While the hallmark of Vg9Vd2 + T cell is recognition of phosphoantigens produced by bacteria-infected or tumor cells (25, 264), monocytes are adept at potentiating this process by taking up and accumulating phosphoantigen for subsequent presentation to gd T cells (262,263). Conversely, the prototypical roles of myeloid cells, such as phagocytosis and MHC class II presentation, are also shared by Vg9Vd2 + T cells, which can act as professional APCs (56,98). The close interconnection between these cell types and partial redundancy in functional properties denotes multiple implications for tumor immunity.
Vg9Vd2 + T cells have been shown to activate monocytes, induce adhesion and aggregation, and increase their survival (76,265). This occurs via production of inflammatory molecules including IFN-g, TNF-a, granulocyte-macrophage colony stimulating factor (GM-CSF), lymphocyte function-associated antigen 1 (LFA-1), and CCL2 (76,78). In turn, this leads to changes in monocyte markers such as downregulation of CD14, and upregulation of CD40, CD86, and HLA-DR (76, 77) ( Figure 2). Bidirectionally, zoledronate-or HMBPP-primed monocytes can activate Vg9Vd2 + T cells through phosphoantigen accumulation and presentation, leading to gd T-cell proliferation and bacterial pathogen killing (76,263). However, in vitro, it has also been reported that in the presence of zoledronate, monocytes and Vd2 + gd T cells can negatively regulate each other by inducing apoptosis (266,267). It is interesting to note that the contact-dependent stimulation of Vg9Vd2 + T cells by monocytes via the intercellular adhesion molecule 1 (ICAM-1)/LFA-1 engagement can be disrupted by blocking CD11a with monoclonal antibody (78). In contrast to these in vitro results, in vivo treatment with zoledronate or other aminobisphosphonates has shown varying effects, with some studies reporting an increase in circulating monocyte numbers, while others found no difference (77,268). This suggests that the relationship between these cells may be more nuanced and context-dependent than first thought and will require further investigation.
The crosstalk between gd T cells and macrophages has not yet been thoroughly elucidated; however, the effects are again cell subtype-and context-dependent. Macrophages have been demonstrated to recruit Vg9Vd2 + T cells to the site of infection via IP-10 and CXCR3 receptor-ligand interactions (80). Once this occurs, Vd2 + gd T cells can drive the local cytotoxic response via granzyme and perforin release or Fas ligand binding (79,81,82). Both Vd1 + cells and Vd2 + cells have been shown to produce CCL3, CCL4 (MIP-1a and MIP-1b), and CXCL10, which find their respective cognate receptors expressed by macrophages (70,138). In vitro, the supernatant of cultured gd T cells has been shown to induce macrophage activation via IFN-g, TNF-a, and GM-CSF production, arguing for a tightly regulated and balanced interplay between these immune cell populations (265). This was further demonstrated by studies showing that IFN-g and TNF-a released by activated Vg9Vd2 + T cells can induce cyclooxygenase-2 (COX2) expression and prostaglandin E2 (PGE2) release by both macrophages (Figure 2) and tumor cells, and this downregulates the cytotoxic response of gd T cells (269,270) and plays a major role in tumor immune escape (271,272). Furthermore, galectin-9 on both gd T cells and pancreatic tumor cells has been shown to bind dectin-1 on tumorinfiltrating macrophages, leading to M2 macrophage polarization and subsequent downregulation of IFN-g and TNF-a production by gd T cells (273,274).

CROSSTALK BETWEEN gd T CELLS AND NEUTROPHILS
Neutrophils are another immune cell population with complex interactions with gd T cells at peripheral sites of inflammation and in the TME. Zoledronate-activated Vg9Vd2 + T cells release cytokines and chemokines such as IFN-g, TNF-a, IL-6, and MCP-2, and these have been demonstrated in vitro to induce neutrophil migration, activation, phagocytosis, degranulation, and release of a-defensins (83). In a differing context using a bacterial phosphoantigen, HMBPP-activated Vg9Vd2 + T cells produce CXCL8 and TNF-a, which together mediate neutrophil recruitment, induce CD11b upregulation and prevent apoptosis, and downregulate CD62L, allowing neutrophil adhesion (78). This finding was further corroborated by Sabbione et al., showing that HMBPP-activated Vd2 + gd T cells can stimulate CD11b expression and myeloperoxidase production by neutrophils (86), all of which imply a stimulatory role of gd T cells towards these granulocytes. In another study, tissue-resident Vd1 + gd T cells were shown to regulate the recruitment of neutrophils to the site of bacterial infection via IL-17 secretion (275). In the absence of Vd1 + gd T cells, the production of IL-17 is reduced and leads to lower numbers of neutrophil recruitment to the site of infection (275).
Interestingly, activated neutrophils can inhibit CD25 and CD69 expression, IFN-g production, and cell proliferation of Vd2 + gd T cells either spontaneously or in response to HMBPP (86). This is dependent on initial TNF-a production by gd T cells, which then induces reactive oxygen species (ROS) secretion from neutrophils (86) (Figure 2). These processes can be independent of cell-cell contact; however, the inhibition is more potent if cells are allowed to interact and form conjugates (86). Neutrophils can take up zoledronate, and despite also expressing BTN2A1 and BTN3A1, they do not have the capability of activating Vg9Vd2 + T cells, which may be due to their extremely limited production and accumulation of IPP (276)(277)(278). Rather, these zoledronate-activated neutrophils inhibit TNF-a and IFN-g production and proliferation of Vg9Vd2 + T cells via ROS, arginase-1, and serine protease production. Some serine proteases are also able to downregulate BTN3A1 expression on PBMCs, which has downstream consequences for BTN-mediated activation of Vd2 + gd T cells (84,85). Furthermore, Vd1 + gd T cells have been shown to exhibit reduced proliferation in the presence of hydrogen peroxide as well as decreased glutathione production, which may be indicative of ROS-dependent neutrophil inhibition (139). In some instances, however, neutrophils that have phagocytosed HMBPP-producing bacteria subsequently release HMBPP, which is then able to activate Vg9Vd2 + T cells. This results in CD25, CD69, LFA-1, IFN-g, and TNF-a production and is crucial for initiating an immediate anti-inflammatory response (78).
Functionally, pancreatic tumor cell killing by gd T cells within a PBMC context is decreased in the presence of neutrophils, in both unstimulated and zoledronate-activated conditions (279). However, when pancreatic tumor cells are co-cultured with purified, expanded gd T cells and neutrophils, tumor cell lysis is increased compared to co-culture with gd T cells alone, which can be attributed to elevated granzyme B and IFN-g production. These conflicting observations may be explained by differences in immune cell subpopulation crosstalk within PBMCs, or by differing polarization of neutrophils: N1 neutrophils are tumor suppressive while N2 neutrophils have a pro-tumoral phenotype (280). It is worth noting that a higher neutrophil-to-lymphocyte ratio in a cohort study of 1,714 cancer patients treated with immune checkpoint inhibitors was recently reported to significantly correlate with low progression-free survival, poor response rates, and low clinical benefit (281). Considering the immunosuppressive functions of activated neutrophils on gd Tcell activation as discussed above, this may partly contribute to the poor outcomes in cancer patients with higher neutrophil-tolymphocyte ratios.
In summary, different gd T-cell subsets can either aid and promote or inhibit DC maturation and function (7,13,304,305), while DCs can activate and expand Vg9Vd2 + T cells (7,13,(304)(305)(306)(307). The crosstalk between gd T cells and DCs can thus have downstream anti-or pro-tumoral effects with therapeutic potential, albeit warranting further investigation using DCs that are not monocyte-derived (8,150,308).

OUTLOOK AND FUTURE PERSPECTIVE
Our understanding on gd T cells continues to expand and their contributions in bridging the innate and adaptive anti-tumor immune responses are becoming more evident. Multiple studies are now highlighting their role in interacting with and orchestrating a variety of other immune cell subsets as reviewed here. Traditionally, gd T cell-based cancer immunotherapies have been focused on assessing the efficacy of activated gd T cells alone in mediating tumor clearance (41-46, 145, 157, 163, 165, 309). Although these past clinical trials have shown that gd T cell-based immunotherapies were safe and well tolerated in patients, given the limited success to date (8,10,19,101,(310)(311)(312), more innovative strategies aiming to overcome the challenges and immunosuppression within the TME should be thoroughly explored. Notably, with the ever-increasing numbers of studies demonstrating the intricate network of immune interactions within the TME, it is high time to deeply explore some of these interactions and to gain valuable insights into the unique immunomodulatory functions of gd T cells in the context of cancer immunotherapy. Such acquired knowledge can be fully harnessed to develop a multipronged gd T cell-based immunotherapy focusing on gd T cells' capability to influence the activities of other tumor-infiltrating immune cells via rapid cytokine and chemokine secretion, expression of various co-stimulatory molecules, and the professional APC functions in cross-priming and presenting antigens to ab T cells.
For example, we are now armed with several potent therapeutic agents including the agonist antibodies against BTN3A1 (clone 20.1, CTX-2026, and ICT-01) and BTN2A1 (ICT-0302) that are capable of activating and enhancing the immunomodulatory functions of Vg9Vd2 + T cells (48-53, 227, 313, 314). Treatment targeting BTN3A1 (CTX-2026) can induce coordinated Vg9Vd2 + and ab T-cell responses for tumor cell killing and represents a promising therapeutic approach that could be combined with other immune checkpoint inhibitors targeting PD-1/PD-L1 (nivolumab and p e m b r o l i z u m a b ) , C T L A -4 / C D 8 6 ( i p i l i m u m a b a n d tremelimumab), KIRs (lirilumab and IPH4102), ILT-2 (anti-ILT-2, anti-HLA-G1, anti-FasL), and NKG2A (monalizumab) to circumvent potential immunosuppression in TME (11,204,205). These anti-tumor responses could potentially be further enhanced by inducing the expression of NKG2D ligands in tumor cells using a proteasome inhibitor (bortezomib) and an alkylating agent (temozolomide) to promote orchestrated NKG2D-mediated tumor cell lysis by tumor-infiltrating NK, CD8 + ab, and gd T cells (240,256,257). Moreover, CD137 (4-1BB) co-stimulation with recombinant human CD137L can boost the therapeutic effect of Vg9Vd2 + T cell-based immunotherapy and lead to heightened NK cell-mediated cytotoxicity (73,75,101,315). Taken together, such combined therapeutic treatment will be a powerful approach to elicit concerted anti-tumor responses in different tumor-infiltrating immune cells and help to maximize the efficacy of future gd T cellbased immunotherapy treatments in cancer patients.

AUTHOR CONTRIBUTIONS
KFC, JDGD, SO, and AB wrote and prepared the manuscript draft. KFC prepared the figures. All authors contributed to the article and approved the submitted version.