Gut γδ T cells as guardians, disruptors, and instigators of cancer

Colorectal cancer is the third most common cancer worldwide with nearly 2 million cases per year. Immune cells and inflammation are a critical component of colorectal cancer progression, and they are used as reliable prognostic indicators of patient outcome. With the growing appreciation for immunology in colorectal cancer, interest is growing on the role γδ T cells have to play, as they represent one of the most prominent immune cell populations in gut tissue. This group of cells consists of both resident populations—γδ intraepithelial lymphocytes (γδ IELs)—and transient populations that each has unique functions. The homeostatic role of these γδ T cell subsets is to maintain barrier integrity and prevent microorganisms from breaching the mucosal layer, which is accomplished through crosstalk with enterocytes and other immune cells. Recent years have seen a surge in discoveries regarding the regulation of γδ IELs in the intestine and the colon with particular new insights into the butyrophilin family. In this review, we discuss the development, specialities, and functions of γδ T cell subsets during cancer progression. We discuss how these cells may be used to predict patient outcome, as well as how to exploit their behavior for cancer immunotherapy.


| INTRODUC TI ON
One of the most prevalent immune cell populations in gut tissue are γδ T cells. γδ T cells represent a collection of diverse subsets with independent phenotypes and functions-some subsets reside in tissue-resident and other subsets circulate in blood. In humans, these subsets are defined by their expression of the δ chain (Vδ1, Vδ2, and Vδ3), whereas γδ T cell subsets in mice are characterized by the expression of the γ chain (Vγ1, Vγ4, Vγ5, Vγ6, and Vγ7). One interesting aspect of γδ T cells is that the γδTCR often dictates where the cells localize anatomically. Human Vδ1 and Vδ3 cells are frequently found in organs, including the gut, skin, and liver, 1-3 while Vδ2 cells circulate in the peripheral blood. Similarly, mouse Vγ5, Vγ6, and Vγ7 cells are tissue-resident, whereas Vγ1 and Vγ4 cells traffic from tissue to lymph nodes. Human Vγ4Vδ1 cells and mouse Vγ7 cells account for the majority of gut-resident γδ T cells, 1,4 but other subsets can infiltrate diseased, damaged, and dysplastic gut tissue. This review will focus on the development, phenotype, and function of both resident and infiltrating γδ T cells in the gut during cancer progression. We highlight the individual contribution of γδ T cell subsets in colorectal cancer (CRC), as well as strategies to boost their anti-tumor properties or methods to mitigate their pro-tumor functions. The role of γδ T cells in other cancer types has recently been reviewed elsewhere. 5

| DE VELOPMENT OF CIRCUL ATING AND G UT-RE S IDENT γδ T CELL S
γδ T cell generation in humans and mice is highly complex and tightly regulated. Recent years have seen a surge of new findings in this area. Here, we describe the aspects of γδ T cell development related to the subsets found in gut tissue. More detailed information can be found in recent review articles. [6][7][8] In mice, the rearrangement of the Vγ4/5/6/7 locus is evident from embryonic day 12 (E12), 9 preceding the start of αβ T cell development at E17. 10,11 E14 is just after completion of thymus development in the embryo, whereas hematopoiesis in the liver occurs from E12 and then in the bone marrow from E16.5. 12 Beginning at E12-E14, γδ T cells develop in ontogenic waves by functional subtype in the fetal thymus, starting with Vγ5 cells. Generation of Vγ5 cells is completed by E18. 10,13 After Vγ5 cells, Vγ6 cells start to expand from E16 in mice followed by Vγ4 and Vγ1 cells. 10,11,14,15 Recently, a monoclonal antibody specific for Vγ6 chain was developed, which has allowed the acquisition of more specific information about Vγ6 cell development. 15 Vγ6 cells arise in the fetal thymus, like other subpopulations. Their numbers (together with Vγ5 cells) start diminishing after birth, while Vγ1 and Vγ4 cells continue to expand. 15 Vγ5 cells, which express an invariant TCR chain, then migrate to the skin, proliferate within the skin, and exist there for their entire life as dendritic epidermal T cells (DETCs). 16 Vγ6 cells also express a semi-invariant TCR, 17,18 and these cells localize primarily to reproductive organs, tongue, and lung. [19][20][21] In adult mice, Vγ5 and Vγ6 cells (and some IL-17-producing Vγ4 cells in the colon) no longer require the thymus for development. They exist mostly as self-generating, tissue-resident cells. 15,[22][23][24][25][26] The development of Vγ1 and Vγ4 cells in the thymus continues throughout adult life, where these cells emerge in a naive state, similar to αβ T cells. Vγ1 and Vγ4 cells travel throughout the body, and they are enriched in secondary lymphoid organs. 27 Gene expression datasets, including single-cell sequencing, have provided more insight into the hierarchical development of γδ T cells in the thymus. 28,29 The generation of γδ T cells in the fetal or adult thymus of mice results in two main functional populations-not including skin Vγ5 cells or gut Vγ7 cells. These two groups of γδ T cells are defined by the cytokines they secrete, IFNγ and IL-17, rather than the TCR they express. IFNγ-producing γδ T cells are associated with Vγ1 and Vγ4 TCRs, while IL-17-producing γδ T cells are associated with Vγ4 and Vγ6 TCRs. The programming of each effector population undergoes a distinct developmental process. Upon stimulation of γδTCR in thymic development, the common αβ/γδ precursors are committed into γδ T cell progenitors, upregulating CD24 as an immature marker. After this, the fate of both effector types is also regulated by TCR stimulation, lymphotoxin, and IL-7 signaling. [30][31][32][33] Strong signaling on the γδTCR together with CD27 co-stimulation and lymphotoxin signaling directs progenitor cells into IFNγ-producing γδ T cells, leading to downregulation of CD24 and CD25. 30,31 On the other hand, the development of IL-17-producing γδ T cells is dependent on weak TCR signaling, the lack of CD27 signaling, strong IL-7α signaling, and positive cues from the Notch pathway. 30,31,[34][35][36] During this process, IL-17-producing γδ T cells lose CD27 expression and upregulate other receptors, such as CCR6 and CD44. 37 Certain transcription factors are required for the generation of IL-17-producing γδ T cells, such as RORγt, MAF, HES1, and STAT5. 30,[38][39][40] However, there are different developmental requirements of transcription factors between IL-17-producing Vγ4 and Vγ6 cells. For example, SOX4 and SOX13 are essential for the differentiation of Vγ4 cells, whereas they are dispensable for Vγ6 cells. 41 Conversely, PLZF is important for Vγ6 cells, but dispensable for Vγ4 cells. 42 These data imply that IL-17-producing effector subsets are driven by both shared and discretely independent mechanisms. Whether these mechanisms are related to the timing of development-embryonic development (Vγ6) versus postnatal thymic development (Vγ4)-is unknown. In adult mice, three developmental pathways for γδ T cells have been identified that can be defined using CD24, CD25, CD73, CD117, CD200, and CD371. 43 Human γδ T cells also develop in an embryonic wave. 44 The origin of mouse liver-resident and gut-resident γδ T cellswhose functions will obviously be important in primary CRC and liver metastasis-is somewhat unclear. Thus far, at least two subsets of liver-resident γδ T cells have been identified: one CD1d-expressing subset that produces IL-17 governed by lipid antigens from gut commensal bacteria, 58 and another CD8αα + subset regulated by the MHC class I-related molecule, H2-Q10. 59 Similar populations of resident cells are also found in human livers, characterized by the expression of Vδ1 as well as established resident markers, such as CD69, CXCR3, and CXCR6. 3 However, the original source of γδ T liver cells in humans or mice is currently unknown. The origin of gut-resident γδ T cells is controversial. Rearrangement of the Vγ7 locus occurs as early as E11 in the liver and gut before T cell progenitors migrate into developing thymic lobes. 9 69,70 The appearance of human gut Vγ4 cells may also occur independently of the thymus, but the ontogeny of these human cells is still not well addressed. 71 Thus, the contribution of gut-resident γδ T cell development from the thymus seems minimal.

| LO C ATI ON AND FUN C TI ON OF γδ T CELL SUBS E TS IN THE G UT
Covering the surface of the intestinal lumen is a tight layer of epithelia organized as crypts and villi in the small intestine (SI) and crypts in the colon (Figure 1). This single layer of cells separates the gut tissue from digested food, dietary antigens, and microbes. Intestinal stem cells at the base of the crypts, marked by the leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) protein, give rise to all types of intestinal epithelial cells (IECs), including enterocytes (or absorptive epithelial cells), goblet cells, enteroendocrine cells, Paneth cells, and Tuft cells. 72 Lgr5 + stem cells divide about every 24 hours to self-maintain. These cells also exit the cell cycle, move upward toward the villus tip, and differentiate to replenish the various cell types that form the luminal layer of the gut. Once cells reach the top of the villus (except for Paneth cells) in a process that takes about 3-5 days, cells are most exposed to gut luminal contents and the microbiome. Tip cells undergo apoptosis and slough off into the lumen. The rapid turnover of new epithelial cells is necessary to prevent a break in the barrier.
Enterocytes are the most abundant cell type in the epithelium and form tight junctions with their neighboring cells to seal the barrier. Enterocytes and goblet cells synthesize transmembrane mucins to protect apical surfaces and expedite food waste. Cells in the colon form two thick layers of mucus compared with the SI, and the mucus has a high viscosity with a gradient from proximal to distal. The mucus layer in the SI is much thinner but is augmented by anti-microbial peptides (AMPs) synthesized by Paneth cells and enterocytes.
Paneth cells are found only in the SI; the AMPs they produce include defensins, cathelicidin, lysozyme, and regenerating islet-derived protein 3 gamma (REG3G or REGIIIγ). In the colon, Paneth-like cells, also known as deep crypt secretory cells, support Lgr5 + stem cells, but it is unknown whether these cells can secret AMPs. 73,74 The constituents of the microbiome are very different between the SI and colon. There is a gradient of bacterial load from proximal F I G U R E 1 T cell location in the intestine. Stem cells in the crypt receive support and survival cues from Paneth cells. These stem cells give rise to epithelial cells that line the gut and protect internal organs from microorganisms in the gut lumen. During differentiation, epithelial cells express BTNL1 in mice (and BTNL3 in humans), which localizes Vγ7 cells (or Vγ4 cells in humans) adjacent to epithelial cells and maintains their survival. αβ T cells that express CD8αα homodimers or CD8αβ heterodimers also reside next to epithelial cells. In the lamina properia, a variety of other γδ and αβ T cells traffic in and out of the tissue through blood vessels or lymphatic vessels. M cells, specialized epithelial cells, transport antigen to Peyer's patch, which contains mostly B cells and CD4 T cells to distal gastrointestinal tract: ordered by duodenum, jejunum, ileum, cecum, proximal colon, and distal colon. 75 Bacterial load is much higher in the colon than the SI, partly because of the inhospitable environment of the SI, which is more acidic and more abundant in AMPs than the colon. 76 Therefore, it is easily presumed that immunity in the SI and colon is suitably distinct. Coincidentally, human tumors develop mainly in the colon and rarely in the SI. Accumulating evidence suggests that certain species of bacteria are associated with and drive CRC development. 77 γδ T cells in the SI and colon serve as one of the first lines of defense in the immune surveillance program of gut tissue (Figure 1). γδ T cells in this organ consist of both resident and infiltrating populations. The resident subset, called intraepithelial lymphocytes (IELs), roams up and down the villus along the basement membrane behind enterocytes in the lateral intercellular space that separates epithelial cells from the lamina propria. 78 IELs can express either a γδTCR or αβTCR, and these TCRs are oligoclonal in humans and mice. [79][80][81][82] γδ and αβ IELs that express the CD8αα co-receptor but not CD8β or CD4 are the so-called natural or type B IELs. SI enterocytes express the ligand for CD8αα, thymus leukemia antigen (TLA), which is a non-classical MHC class I molecule. 83 A few key molecules control the localization and active migration patterns of IELs. CD103, also known as αE integrin, is one of these molecules expressed by IELs. CD103 dimerizes with β7 integrin, and this complex binds to E-cadherin on epithelial cells. 87,88 In both CD103-and β7-deficient mice, IEL number is reduced with a greater reduction seen in β7-deficient mice. 89,90 The milder effect of CD103 loss compared with β7 loss on IELs might be explained by the fact that β7 can form another heterodimer with α 4 integrin, which is also involved in homing to the gut. 91 Interestingly, αβ IELs are more affected by CD103 loss than γδ IELs. CD103 expression on IELs is stimulated by TGFβ and runt-related transcription factor 3 (RUNX3), 92,93 as well as by the CCL25-CCR9 axis. 94,95 CCL25 is a chemokine highly expressed by SI epithelial cells, 96 and its receptor CCR9 is expressed on IELs. 97 Despite ubiquitous expression of CCR9 by all IELs, CCL25-and CCR9-deficient mice exhibit a specific reduction in γδ IELs to half their number in wildtype mice. 98,99 CCR9 expression and IEL homing to SI seem to be regulated in part by the vitamin D receptor (VDR), as VDR knockout mice exhibit reduced CD8αα-expressing IEL that coincides with decreased IL-10 levels. 100 IL-15 is another key molecule that regulates both γδ and αβ IEL maintenance and localization. [101][102][103][104][105][106] This cytokine and its receptor, IL-15Rα, are expressed by enterocytes and lamina propria DCs, where they form a complex that is transpresented to IELs. The use of sophisticated mouse models and advanced live cell imaging techniques has shown that IL-15 controls Vγ7 cell localization and migratory behavior within the lateral intercellular space, as blocking IL-15 signaling causes their patrolling nature to idle. 104 In addition, the G protein-coupled receptor, GPR18, regulates γδ IEL abundance in the gut and this molecule is important for positioning of IELs next to epithelial cells as opposed to the lamina propria. 107,108 By contrast, GPR55 negatively regulates γδ IELs, as confirmed by intravital imaging in GPR55-deficient mice that not only have more γδ IELs, but these cells also migrate faster and establish greater crosstalk with epithelial cells. Moreover, loss of GPR55 fails to affect αβ IELs. 109 Thus, αβ and γδ IELs share many similarities; however, their homing and motility are differentially regulated.
The phenotype of αβ and γδ IELs is very similar, almost indistinguishable in fact. 110,111 Both αβ and γδ IELs express the semaphorin, CD100 (also called SEMA4D), which controls their proliferation. 112 Compared with other T cells, IELs exist in a constitutively active, cytolytic state with elevated effector potential. IELs constitutively express a number of cytolytic molecules, including granzymes A and B, perforin, and Fas ligand (FasL). Granzymes and perforin work together to penetrate the cell membrane of target cells and induce killing. FasL is a member of the tumor necrosis factor (TNF) ligand superfamily that binds Fas on target cells to trigger apoptosis. IELs also produce molecules normally associated with NK cells, such as 2B4/CD244, NKG2A, NKG2D, NKp46, and NK1.1. IELs uniformly express CD69, an early activation marker of TCR activation as well as a tissue-resident marker. 113,114 On the other hand, another marker of TCR stimulation, CD25, is scarcely expressed on IELs. 110 Taken together, the phenotype of IELs indicates that these cells are always alert and poised for attack.
As suggested by their phenotype, the role of IELs in the gut is to maintain homeostasis and epithelial tight junction integrity [reviewed in Refs 91,115,116]. Crosstalk between the microbiome, epithelial cells, and IELs stimulates epithelial cell proliferation and function to reinforce the barrier. 117 γδ IELs respond to microbiota through the Toll-like receptor (TLR)/MyD88 pathway that induces REGIIIγ to protect against invading bacteria. 118 In chronic inflammatory diseases of the gut, including Crohn's disease and ulcerative colitis, loss of barrier integrity is accompanied by an influx of luminal pathogens, a subsequent massive immune response, and sustained tissue damage, illustrating the importance of intestinal homeostasis for the maintenance of a healthy gut. γδ IELs play a protective role in these pathologies by suppressing Th1 type responses. 119,120 γδ T cell-deficient mice are susceptible to spontaneous colitis with age (5% of mice over 8 months old), as well as experimentally induced colitis. However, transfer of IELs into these γδ T cell-deficient mice reverses colitis, presumably through TGFβ-mediated suppression of IFNγ and TNF. 119 By contrast, overactivation of γδ IEL is thought to perturb homeostasis in the gut, such as in inflammatory bowel disease (IBD) or celiac disease where elevated IL-15 expression is observed. [121][122][123][124][125] In addition to human Vγ4 cells and mouse Vγ7 cells that constitute the γδ IEL population, other γδ T cell subsets infiltrate the gut tissue ( Figure 1). These cells expressing invariant or polyclonal γδTCR repertoires arrive via the blood or lymph system and land in the lamina propria. 91,126 The lamina propria is the core of the villi that contains connective tissue, blood and lymphatic vessels, fibroblasts, extracellular matrix, and a variety of immune cells. Gutinfiltrating γδ T cells may acquire a cytotoxic, effector-like phenotype-like IELs, but other subsets express a completely different range of cytokines, such as IL-17 or IL-22. These cytokines, often in response to IL-23 and/or IL-1β, control the secretion of AMPs and tight junctions between enterocytes to sustain barrier function. 25,127,128 In the mouse, IL-17-and IL-22-producing cells express either the Vγ4 or Vγ6 TCR. The abundance of these subsets changes between different regions of the gut with the highest enrichment of Vγ4 cells in the distal colon. 25  A few studies have reported on the structure of various γδT-CRs. [132][133][134][135][136][137][138][139] In comparison with the αβTCR, the V regions of the γδTCR are similar, whereas the C regions are substantially different. 134 The antigen-binging domain of γδTCR has more protrusions and clefts, exhibiting more similarity to the surface of immunoglobulin heavy chain (V H ) than to the αβTCR, which has a flat surface. The determinant for antigen binding lies within amino acid sequences separated into 3 regions called complementarity-determining regions (CDRs). CDR1 and CDR2 are germline-encoded loops derived from the V segment, and CDR3 forms loops from the recombined region around the junction of the V, D, and J segments. CDR3 forms the center of the antigen-binding site. Interestingly, the length of the CDR3 of TCRδ is as very variable and long as that of V H , 140 indicating that the γδTCR is closer related to immunoglobulins than TCRα and thus can recognize a variety of antigen types. The more striking difference between γδTCRs and αβTCRs is observed in the C domain, where angles, glycosylation sites, and charges of residues are distinct. Given that the C domain binds CD3 subunits, these differences suggest that the association with CD3 subunits and subsequent intracellular signaling may be different to that of αβTCR. This view is supported by biochemical assays and knockout mice, which have demonstrated that most mouse γδTCRs do not associate with the CD3δε heterodimer, but with two CD3γε heterodimers. [141][142][143] However, thymic γδ T cells from Cd3d +/− Cd3g +/− mice exhibit reduced γδTCR expression with dysfunctional differentiation into IFNγ-producing γδ T cells, 144 indicating that the CD3δε heterodimer is important for some γδTCRs. Human γδTCRs are entirely reliant on CD3δε and CD3γε heterodimers. 143 Antigens and ligands for γδTCRs constitute a wide variety of unconventional molecules. An important distinction between γδT-CRs and αβTCRs is that γδTCRs do not bind major histocompatibility complex (MHC) molecules, and γδ T cells rarely express CD4 or CD8 γδTCR antigens fall into two main categories: adaptive-like molecules and B7-like molecules with similarity to the CD28 co-stimulatory receptor. Circulating γδ T cells in both humans and mice can bind the self-stress molecule endothelial protein C receptor (EPCR), 146,147 phycoerythrin, 148  Vγ9Vδ2 cells recognize the BTN3A1-BTN2A1 heterodimer, a receptor complex that acts as a sensor for a group of pyrophosphate-containing metabolites called phosphoantigens that may be endogenous or exogenous (ie, non-self, microbial-derived) products. 160,161 Some of these phosphoantigens include isopentenyl pyrophosphate (IPP) generated from the mevalonate pathway important for cholesterol biosynthesis 162,163 or the microbial hydroxyl-methyl-butyl-pyrophosphate (HMBPP) metabolite produced from the isoprenoid pathway. 164 BTN3A1 and BTN2A1 sense the accumulation of intracellular phosphoantigens such as IPP and HMBPP, and binding of these phosphoantigens to intracellular domains of BTN3A1 causes a conformational change in the protein, promoting recruitment of BTN2A1 that directly binds the Vγ9Vδ2 TCR. 161,[165][166][167] The buildup of IPP is a common occurrence in many types of cancers with dysregulated metabolism, making cancer cells amenable to Vγ9Vδ2 cell killing. The role of BTN/BTNL family members in regulating resident γδ T cell subsets is discussed below.
Pioneering work on thymic development and maturation of mouse skin-resident Vγ5 cells led to the first link between BTN/ BTNL family members and γδ T cells. This discovery identified SKINT1, which is a BTN/BTNL family member specifically expressed in the skin and thymus, as a crucial protein for Vγ5 cell expansion during development and survival in skin. [168][169][170] More recently, mouse BTNL1 and human BTNL3 were demonstrated to be the equivalent of SKINT1 for gut Vγ7 and Vγ4Vδ1 IELs, respectively. 1 These BTNL family members are expressed solely in the SI and colon. 171 Btnl1deficient mice display a marked reduction in Vγ7 cells in gut tissue.
BTNL1 dimerizes with BTNL6, and BTNL3 dimerizes with BTNL8 to maintain γδ IEL survival and function in both humans and mice, indicating a highly conversed network between species. In subsequent studies, mutagenesis, computational modeling, and functional assays showed that the Ig-V regions of BTNL3 directly interact with the germline-encoded non-variable regions of the Vγ4 TCR. 172,173 These data uncover a unique ability of γδTCRs to recognize and respond to ligands via variable and non-variable sequences. Given these sophisticated properties of combining innate and adaptive traits at the cell-intrinsic level, the term "adaptate" was coined to better represent γδ T cell biology. 174,175

| COLOREC TAL C AN CER E TI OLOGY AND MODELING IN MICE
Colorectal cancer is the third most common cancer in men and second most common in women worldwide, 176 rising from fourth and third most common in 2002, 177 respectively. CRC is estimated to kill over 881 000 people worldwide. 176 Incidence and mortality are likely to increase as life expectancy rises and developing countries become increasingly westernized. Key risk factors for CRC include increased red meat consumption, low fiber intake, and a low level of physical activity. [178][179][180] The progression from adenoma to CRC is driven by the acquisition of multiple genetic aberrations. There are two types of adenomas whose genetic mutations differ that correspond with two postulated avenues to metastatic CRC. In the case of sessile serrated polyps (SSPs), mutational drivers consist of BRAF mutations, CpG island methylator phenotype (CIMP), and microsatellite instability (MSI). 181,182 In contrast, traditional serrated adenoma conversion to carcinoma is driven by mutation of the tumor suppressor protein, APC, the inactivation of which allows for stabilization and translocation of β-catenin to the nucleus where it participates in upregulation of WNT target genes. 183,184 Further mutations occur in KRAS, TGFβ signaling, and p53, leading to progression of the tumor, [185][186][187][188] and may be influenced by MSI. 189 Although significant hereditary factors are present in approximately 35% of CRC incidence, 190 driver mutations in genes causing established hereditary syndromes account for approximately 5% of CRC incidence. 191,192 The most prevalent of these hereditary syndromes is Lynch syndrome,193,194  ganoid technology, which can be transplanted into syngeneic or immunodeficient mice, and new genetically engineered mouse models that fully recapitulate CRC progression from adenoma to metastasis. [212][213][214][215] However, the use of these models either older or newer in γδ T cell biology is limited. The importance of γδ T cells in CRC is discussed next.

cer-associated fibroblasts (CAFs), the proliferation of γδ T cells and
IFNγ expression is reduced, while IL-17 expression is increased. 222 These observations indicate that the tumor microenvironment may influence the ability of γδ T cells to recognize cancer cells. In support of this notion, the characterization of Vγ9Vδ2 cells in CRC patients with liver metastasis treated with standard of care, 5-fluorouracil/ oxaliplatin (FOLFOX), has shown that absolute numbers of circulating Vγ9Vδ2 cells are reduced. 223,224 Vγ9Vδ2 cells in these patients exhibit a higher frequency of terminally differentiated and senescent phenotype with impaired effector function, when compared to Vγ9Vδ2 cells from healthy donors. In addition, the number of chemotherapy cycles is correlated with a decrease in Vγ9Vδ2 cells expressing central memory markers, and chemotherapy also skews Vγ9Vδ2 cells toward terminal differentiation. 223 However, whether this change in Vγ9Vδ2 cell effector function adversely impacts prognosis or patient survival is unknown.
As discussed above, metabolites of the mevalonate pathway such as isopentenyl pyrophosphate (IPP) activate Vγ9Vδ2 cells via recognition of the BTN3A1-BTN2A1 heterodimer on cancer cells. 160,161,[165][166][167] Blocking the mevalonate pathway to inhibit IPP accumulation in cancer cell lines reduces Vγ9Vδ2-mediated cell lysis. 221 Conversely, the use of nitrogen-containing bisphosphonates, such as zoledronate, to increase IPP accumulation in CRC stem cells, sensitizes cancer cells to Vγ9Vδ2-induced cytotoxicity. 225,226 These data Interestingly, one study reported that the cytotoxic ability of Vδ1 cells from the primary tumor of three CRC patients (one metastatic) is markedly higher against epithelial tissues than alternative tissues such as hematopoietic cancer cell lines, as quantified by percentage cell lysis and IFNγ release. 227 Thus, Vδ1 cells may be reacting to a ligand which is native to epithelial tissues by a receptor that is constitutively expressed on these cells, such as NKG2D. 114,133,[231][232][233][234] In comparisons between Vδ1 cells and Vδ2 cells isolated from peripheral blood of healthy donors and CRC patients, Vδ1 express higher levels of activation markers, cytotoxicity markers, and terminal differentiation markers. This apparent difference in phenotype is also observed functionally, as Vδ1 T cells induce greater lysis of CRC cell lines than did Vδ2 T cells. 228,229 As interest in Vδ1 cells continues to grow, research is beginning to elucidate more specific details about these cells, how they differ from Vδ2 cells, and ultimately how they function. An NKp46 + subpopulation of IELs that mostly consist of Vγ4Vδ1-expressing cells has recently been characterized. 114 The abundance of these cells is less in stage III/IV than stage I/II. After coculture with the myelogenous leukemia cell line K562, the NKp46 + γδ T cells produce more IFNγ, granzyme B, and CD107a than the NKp46population and kill K562 cells more efficiently.
Blocking NKp46 also reduces K562 killing in these cocultures. 114 Collectively, Vδ1 appear to demonstrate a stronger anti-tumorigenic potency than their Vδ2 counterparts. The anti-tumorigenic roles of γδ T cells are summarized in Figure 2.
The majority of cancer deaths are a consequence of metastasis so understanding how γδ T cells may counter the metastatic process is of great interest. In an orthotopic mouse xenograft model of luciferase-expressing HT29 cells injected into the cecum of immunodeficient mice, the administration of Vδ1 T cells reduces primary tumor growth as well as the formation of spontaneous liver and lung metastases. Similarly, Vδ1 cell immunotherapy decreases HT29 cell growth in the lung after intravenous injection. 235 Thus, Vδ1 cells counteract CRC growth regardless of a tumor's anatomical location, indicating that Vδ1 cells not only exhibit anti-tumor potential but also anti-metastatic potential.

| THE ROLE OF PRO -TUMORI G ENI C γδ T CELL S IN CRC
The knowledge of how γδ T cells may promote CRC is still limited, but what is known is largely centered on IL-17-producing γδ T cell subsets ( Figure 3). This is also true for other tumor types, where we and others showed that IL-17-producing γδ T cells drive cancer progression and metastasis. [236][237][238][239][240] In mouse models driven by mutant Apc or loss F I G U R E 2 Anti-tumorigenic functions of γδ T cells in colorectal cancer. In humans, two major subsets of γδ T cells can recognize and kill cancer cells: One is the gut-resident Vδ1 cell subset and the other is the Vγ9Vδ2 cell subset that enters the gut from the circulation. Both subsets express cytotoxic molecules, such as granzyme, perforin, FasL, IFNγ, and TNF. During immunosurveillance, γδ T cells may sense abnormalities through the NKG2D receptor by stress ligands expressed on cancer cells. Vδ1 cells that express the Vγ4 chain (or Vγ7 chain in mice) and NKp46 may bind cancer cells through BTNL3 (or BTNL1 in mice). By contrast, Vγ9Vδ2 cells recognize cancer cells through BTN3A1/BTN2A1 heterodimers, which bind to the γδ T cell receptor (TCR) after activation by the IPP metabolite, a product of the mevalonate pathway of Apc, inflammation activated through the Toll-like receptor (TLR) pathway plays a central role in tumorigenesis. 241,242 When MyD88, an adapter molecule through which TLR signaling is mediated, is deleted in these Apc models, tumor formation and pro-inflammatory molecules including COX-2, IL-6, IL-23, and IL-1β are reduced. Microbial products exposed to myeloid cells via breakdown of the epithelial barrier trigger these pro-inflammatory molecules and potentiate can- To prevent tumor formation in the ETBF model, both Th17 cells and γδ T cells must be ablated. 247 This redundancy is not observed in F I G U R E 3 Pro-tumorigenic functions of γδ T cells in colorectal cancer. Breakdown of the epithelial barrier by the disorganization of cancer cells allows bacteria to penetrate gut tissue. These microorganisms activate dendritic cells (DC) and macrophages to secrete the cytokines, IL-1β and IL-23, which are received by γδ T cells expressing Vγ6 or Vγ4 cells (in mice or Vδ1 cells in humans). In response to this stimulus, these γδ T cell subsets release IL-17A, and IL-17A can induce proliferation of cancer cells or induce G-CSF expression by other cells. G-CSF mediates neutrophil expansion; neutrophils are drawn into the tumor microenvironment by the chemokines, CXCL1, CXCL2, or CXCL5. Neutrophils and γδ T cells can suppress the anti-tumor activity of CD8 T cells to promote cancer progression every mouse model of cancer. In the K14-Cre;Cdh1 F/F ;Trp53 F/F model of breast cancer, we found that both CD4 T cells and γδ T cells increase expression of IL-17A in response to tumor-associated macrophage-derived IL-1β; however, the depletion of Th17 cells failed to reduce pro-metastatic neutrophils. 236,249 These data indicate that Whether the importance of IL-17-producing γδ T cells is conserved in human cancer is controversial. Circulating Vγ9Vδ2 cells from healthy donors do not readily produce IL-17A, although they can be enticed to secrete this cytokine in vitro when given IL-23, IL-1β, TGFβ, and/or IL-7. 34,253,254 Nevertheless, IL-17-producing γδ T cells are found in human CRC tissue, and these cells are more prevalent in tumors than in normal tissue. 222,[255][256][257] Their frequency positively correlates with tumor stage, tumor size, invasion, lymph node metastasis, vascular and lymphatic invasion, and immunosuppressive neutrophils. 255 Interestingly, IL-17-producing γδ T cells outnumber Th17 cells in at least one cohort of CRC patients. 255 Some IL-17-producing γδ T cells may have regulatory functions that suppress anti-tumor T cells and express CD39. 257 The lack of knowledge surrounding the role of γδ T cells in CRC is partly related to the paucity of more sophisticated mouse models that incorporate other common CRC genetic mutations, such as KRAS, p53, and TGFβ signaling. New CRC models have recently been developed that should provide further insight into the pro-tumorigenic role of γδ T cells. 210 Some of these new models fully progress to the carcinoma stage and metastasize to distant organs, so they should allow researchers to dissect γδ T cell function at different stages of disease progression. A transplantation model with organoids carrying mutations in Apc, Kras, Tgfbr2, and Trp53 genes demonstrated that TGFβ signaling plays a critical role in colorectal cancer metastasis. 213 In this study, combination treatment of TGFβ inhibitor and PD-L1 inhibitor improves survival of tumor-bearing mice and reduces metastasis formation. As γδ T cells can produce TGFβ and suppress cytotoxic CD8 T cells through PD-L1 expression, 258 γδ T cells might be involved in metastasis via immunosuppression in this CRC model. We reported on the ability of IL-17-producing γδ T cells to control immunosuppressive neutrophils in breast cancer and potentiate metastasis to the lung. 236,249 Therefore, γδ T cells may also control pro-metastatic neutrophils in CRC. Recently, neutrophils were shown to promote liver metastasis in a new genetically engineered mouse model whose tumors are driven by mutations in KRAS, loss of p53, and overexpression of NOTCH1. 212 From these new models, we may also learn additional mechanisms of tumor promotion independent of IL-17 expression. γδ T cells can express galectin-1 to suppress anti-tumor T cells in a Kras/p53 sarcoma model, 259 as well as IL-22 and amphiregulin (AREG) to stimulate epithelial cells in a Kras/p53 lung cancer model. 250

| THE PROG NOS TIC P OTENTIAL OF γδ T CELL S IN CRC
Prognostic indicators of disease progression are of key interest specifically in CRC, as the gold standard tumor burden/nodal status/ metastasis or TNM staging system is not sufficiently accurate prognostic markers for stage II and stage III patients. In addition, approximately 25% of stage II and stage III patients relapse, despite the lack of evidence for residual cancer cells or distant metastasis following surgical resection. 260,261 Moreover, TMN cannot predict response to chemotherapy. Attempts to refine or replace the TNM staging system have given rise to the Immunoscore, 262 which is based on the observation that T cells have a strong, favorable prognostic role in CRC. This originated from a study in 415 CRC patients showing that increased infiltration of CD3, CD8, or CD45RO (effector/memory) T cells at the tumor center or invasive margin, but particularly when high in both, is associated with greater disease-free survival. 263 Importantly, this analysis outperformed the TNM staging system. In follow-up studies using two independent cohorts of 602 combined patients, only 4.8% of the high Immunoscore group exhibited relapse after 5 years. 264 Immunoscore can also be applied to lung and liver metastatic lesions to predict patient outcome. 265 The Immunoscore encompasses tumor-infiltrating γδ T cells due to their expression of CD3 molecules. However, these cells represent less than 5% of the total CD3 population, 222,266 so it is unclear whether their abundance actually contributes to the overall predictive power of Immunoscore in CRC. To date, a comprehensive histological analysis of the prognostic power of γδ T cells in CRC has not been reported. Given the association of high Immunoscore with favorable outcome, one may speculate that γδ T cells will correlate with good prognosis, although increased intratumoral γδ T cells in breast and gallbladder correlate with poor prognosis. 267,268 In a study from 2015 that is often quoted by the γδ T cell community to justify the use of anti-tumorigenic γδ T cells in cancer immunotherapy, γδ T cells were the best indicator of good prognosis, among every immune cell population in multiple tumor types. 269 To arrive at this conclusion, about 18,000 human tumors including CRC were analyzed for their composition of immune cells using a computational method called CIBERSORT that infers individual immune cell populations from bulk transcriptomic datasets. After pooling cancer types to determine global leukocyte prognostic patterns based on overall survival, this study found that myeloid cell populations were generally unfavorably prognostic, while lymphoid populations were more positive markers of patient outcome. Interestingly, γδ T cells scored highest in the favorable prognosis group across more than 20 cancer types. 269 However, the delineation of a γδ T cell gene signature from transcriptomic data by the CIBERSORT method has been challenged, 270 because there is a significant overlap between the γδ T cell gene signature and other lymphocyte subsets. CIBERSORT relies on a series of reference gene signatures derived from peripheral blood immune cells. 271 When CIBERSORT was applied to Vγ9Vδ2 cells purified from the peripheral blood of 12 healthy donors, there was significant overlap between CD8 T cells, CD4 T cells, and NK cells. 270 Therefore, the CIBERSORT γδ T cell gene signature was refined to include 375 genes, which significantly improved specific detection of Vγ9Vδ2 cells. Further analysis showed that αβ T cell abundance in CRC correlates with TCR signaling, TLR signaling, antigen processing, cytolytic activity, and interferon response pathways, whereas Vγ9Vδ2 abundance did not correlate with any of these pathways. Regardless, tumors with high infiltration of Vγ9Vδ2 cells or αβ T cells associated with good outcome of CRC patients. 270 This observation was validated in a separate cohort of CRC patients using the same methodology. 222 It would be interesting to learn the prognostic value of Vδ2cells, particularly the gut-resident Vδ1 subset, and the IL-17-producing subsets in those patient cohorts.
Independent of γδ T cells, IL17A expression is associated with poor survival in CRC, 222,272 but IL-17-producing γδ T cells are also associated with poor outcome in CRC. 255,257 The simple presence of γδ T cells in CRC is not what dictates their contribution to tumorigenesis of course, but rather their functional capabilities, such as cytolytic activity. A quantitative measure of immune cell cytolytic activity (CYT) has been developed using mRNA expression levels of two genes, granzyme A and perforin. 273 In TCGA datasets, CYT is higher in normal colorectal tissue than colorectal tumor tissue with MSI expectantly exhibiting the highest CYT among all CRC subtypes. [273][274][275] The CYT score is a favorable prognostic factor for both overall and disease-free survival. Interestingly, γδ T cells are more abundant in the CYT high colorectal tumors together with CD4 T cells, NK cells, and anti-tumorigenic macrophages, when compared to CYT low colorectal tumors. 274,275 These γδ T cells are presumably Vγ9Vδ2 cells, since the original CIBERSORT method was used to identify them. Their association with CYT is perhaps not surprising given that Vγ9Vδ2 cells are enriched in MSI tumors such as αβ T cells. 222,276 Whether γδ T cells contribute to CYT score by directly producing granzyme A and perforin in CRC is unclear.
One area of CRC-specific oncology that needs further development is the ability of γδ T cell subsets to predict response to T cell checkpoint inhibitor immunotherapy, such as anti-CTLA4 and anti-PD-1. This is because γδ T cell subsets are biomarkers of immunotherapy response in other cancer types. In patients with melanoma that received ipilimumab (anti-CTLA4), the discernment between circulating levels of Vδ1 and Vδ2 cells is imperative, as the frequency of γδ T cells in peripheral blood of cancer patients versus healthy donors is similar. However, when melanoma patients have low levels of Vδ1 cells or high levels of Vδ2 cells, overall survival-assessed from the first dose of ipilimumab-is positive. 277 Another consideration for immunotherapy is whether γδ T cells either play a role in the immunotherapy-induced inflammatory side effects, such as colitis, or could serve as biomarkers for inflammation. A recent investigation into the mechanisms responsible for immunotherapy-driven colitis in melanoma patients showed that gut-resident γδ IELs are reduced in colitis patients when compared to patients on immunotherapy without colitis or healthy tissue from patients undergoing colonoscopies.
By contrast, infiltrating γδ T cells were unaffected in immunotherapy-induced colitis. 278

| THE THER APEUTI C P OTENTIAL OF γδ T CELL S
γδ T cells are of increasing interest in cancer immunotherapy due to their potent cytotoxicity and ability to recognize MHC-unrestricted antigens. For solid tumors, case studies, and phase I trials in renal cell carcinoma, metastatic breast cancer and lung cancer suggest that Vγ9Vδ2 cell immunotherapy can significantly impact cancer progression in patients. [279][280][281][282] This type of immunotherapy is also welltolerated. 283,284 Therefore, considerable effort has been focused on preconditioning γδ T cells ex vivo to bolster their anti-tumorigenic functions before adoptive cell transfer into cancer patients. In many studies, IL-2 and zoledronate are used to expand Vγ9Vδ2 cells in vitro; however, zoledronate must be slowly diluted during expansion because continuous exposure is toxic. 280,281,285,286 Autologous Vγ9Vδ2 cells expanded in vitro with zoledronate have been given to CRC patients following surgery to remove pulmonary metastasis.
These cells maintain their effector functions as determined by IFNγ production and CD107a expression during culture conditions, 287 224 suggesting that interfering with B7-H3 signaling may further improve cytotoxicity by these cells. Thus, Vγ9Vδ2 cells may provide a safe and effective form of immunotherapy for CRC patients. Given that Vδ1 cells are consistently shown to be more potent responders to CRC than Vγ9Vδ2 cells, harnessing Vδ1 cells for immunotherapy in CRC and utilizing different γδ T cell subsets may also prove beneficial in patients.
Another strategy to boost Vγ9Vδ2 cell killing capacity is to increase their ability to recognize cancer cells. Given that the human Vγ9Vδ2 subset shows cytotoxicity against CRC cell lines via IPP-stimulated activation of BTN3A1, 160,221,226,[292][293][294] inducing IPP accumulation will position BTN3A1 and BTN2A1 in the right conformational position for Vγ9Vδ2 cell recognition. Zoledronate sensitizes CRC cells to Vγ9Vδ2 cell killing. 226,295 Zoledronate allows the accumulation of IPP by blocking farnesyl pyrophosphate synthase so that IPP is not converted into cholesterol or ubiquinones. However, in order for Vγ9Vδ2 cell immunotherapy to be effective in CRC patients, the right patient population must first be selected. There are clues from the literature that p53 status may be the key to patient selection. In CRC, breast and liver cancer cells, p53 suppresses mevalonate pathway-related enzymes, whereas p53-mutant or p53-deficient cells show elevated expression of mevalonate pathway-related enzymes. 296  and discover new biology, more sophisticated CRC mouse models that capture CRC development from tumor initiation to metastasis formation should be employed. Along these lines, the existing mouse models for in vivo imaging should be applied to the cancer context to determine how γδ T cells move in and around gut tumors. Organoids or tissue explants derived from human tumors that are cocultured with human γδ T cell subsets will also be valuable. Overall, the tools currently available to manipulate γδ T cell subsets are limited, but they will be necessary to fully understand their pro-and anti-tumorigenic functions. These models may generate additional data in lessstudied areas of γδ T cell biology including immunometabolism. In regard to cancer immunotherapy, we must learn which γδ T cell strategy is most efficacious and which patient population to treat with γδ T cell therapy. Together, these new methodologies and insights will uncover various ways to exploit γδ T cell biology for CRC treatment.

CO N FLI C T O F I NTE R E S T
The authors have no conficts of interest to declare.