γδT cells are primarily distributed in subcutaneous tissue, intestinal mucosa, the respiratory tract, and the urogenital canal [1]. Since they can quickly identify infected and mutated cells, they are considered the first line of defense for “dissidents” inside the human body [2]. γδT cells participate in immune regulation in the body, present antigens, induce the maturation of dendritic cells (DCs), and exert cytotoxic effects to kill tumor cells [3, 4]. Activated γδT cells can secrete perforin, granzyme A/B, interferon-γ (IFN-γ) and tumor necrosis factor-alpha (TNF-α), and can express Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) simultaneously [5]. In addition, γδT cells infiltrate a variety of tumor tissues, such as colorectal, breast, prostate, and ovarian carcinomas [6, 7]. The importance of γδT cells in immune surveillance has also been confirmed in multiple mouse models [8]. Mice lacking γδT cells are prone to malignant skin tumors [9]. In humans, the distribution and phenotype of γδT cells in tumors can also serve as prognostic indicators in cancer patients [10]. Some studies have also reported that interleukin-17 (IL-17)—secreting γδT cells exert a tumor-promoting effect, which will not be described in this review [11].

Studies on the genetic engineering technology of tumor immunotherapy based on allogeneic transplantation predominantly focus on αβT cells, but the main limitation of this approach is that these cells can cause severe graft-versus-host disease (GvHD) [12], and inefficient infiltration of solid tumors has led to a limited curative effect in solid tumors [13]. However, γδT cells can recognize various tumor cells through a variety of innate cytotoxic receptors expressed on their surface [14]. This broad response can reduce the opportunity of tumors to escape, and the independent manner by which major histocompatibility complex (MHC) molecules recognize tumor cells [15] to effectively avoid GvHD and cytokine release syndrome (CRS); therefore, this approach allows more opportunities for γδT cells to be designed through genetic engineering.

γδT cells are relatively small in circulating T cells, with a content of about 1–5% in peripheral blood. They express a diversified T-cell receptor (TCR) repertoire composed of a γ chain and a δ chain. Different functional subsets of γδT cells may have a different function in antitumor immunity.

Vγ9Vδ2 T cells are the most abundant subset [16] in peripheral blood, as they account for 50–90% of all of the γδT cells [17]. The main target of this group of cells is the nonprotein pyrophosphate metabolite produced by the host mevalonate pathway [isopentenyl pyrophosphate (IPP)] or the microbial nonmevalonate pathway (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), namely phosphoantigens (pAg) [18]. This process can be blocked by nitrogen-containing bisphosphonates (N-BPs), which leads to the accumulation of pAg in cells, this, in turn, activates Vγ9Vδ2 T cells. Zoledronate (ZOL) as a representative stimulus plays an important role in the activation and expansion of Vγ9Vδ2 T cells [17]. In some studies, the combination of ZOL in the form of liposomes and Vγ9Vδ2 T cells can significantly improve its efficacy [19, 20].

Vδ1⁺ T cells constitute the main population of γδT cells in the mucosa and skin. These cells can be activated by many ligands, including stress-induced autoantigens, glycolipids presented by CD1c/d, and self-lipids, as well as potential ligands that have not yet been determined [21, 22]. It has been reported that Vδ1⁺ T cells have a wide range of tumor cell recognition capabilities [23], which indicates that they are a potential tool in antitumor immunotherapy.

Most non-Vγ9Vδ2 T cells and non-Vδ1⁺ T cells in humans express Vδ3⁺ TCR chain, accounting for about 0.2% of circulating T cells, but they are enriched in the liver and some patients with chronic virus infection and leukemia. Vδ3 T cells are glycolipid reactive T cells, which specifically recognize CD1d, release T-helper 1 (Th1), Th2, and Th17 after activation, and induce DC cells maturation [24]. However, due to their small number, there is no effective large-scale amplification method for immunotherapy, so it will not be introduced in this review.

Different from αβT cells, γδT cells do not rely on MHC molecules for TCR mediated antigen recognition, and can directly mediate cytotoxicity through cytotoxic cytokines, granzyme, and perforin [1]. At least three cytotoxicity pathways of how γδT cells kill tumor cells have been described. One is γδTCR-dependent cytotoxicity, another is cytotoxicity mediated by receptor-ligand binding, such as NK cell receptor (NKG2D)-NKG2D ligands (NKG2DL), Fas-FasL, TRAIL-TRAIL receptor (TRAILR) and so on, and the third is antibody-dependent cytotoxicity (ADCC) via the Fc domain of antibodies that bind to tumor cell antigens recognized by γδT cells [25]. Based on these, γδT cells represent a promising immune population for the next generation of immunotherapy.

Adoptive immunotherapy using γδT cells has produced ideal effects in both solid and hematological tumors in mice and humans. Regardless of whether the cells are used alone or in combination with IL-2 or ZOL, the recipients experience no serious adverse reactions [26–31]. Since γδT cells account for a relatively small proportion of peripheral blood and tumor-infiltrating T lymphocytes, many studies are devoted to obtaining clinical-scale γδT cells. It has been reported that vitamin C helps to increase the expansion of γδT cells [32]; moreover, Vγ9Vδ2 T cells incubated with IL-2 and IL-15 exhibit stronger cytotoxicity [33], and in the absence of IL-2, the delivery of Vγ9Vδ2T cells in vivo requires high doses of IL-15 [34]. Some researchers developed a new formula to improve the expansion of γδT cells in vitro. This formula consists of ZOL, IL-2, IL-15, and vitamin C. Compared with the culture system that only adds ZOL and IL-2, γδT cells expanded according to the new formula can produce higher levels of effector molecules (IFN-γ, TNF-α, and NKG2D), which increases cytotoxicity against a variety of tumor cell lines in vitro and in vivo [35]. IL-21 is a cytokine that has been proved to promote the cytotoxicity of natural killer (NK) cells and CD8⁺ T cells and can enhance cytotoxicity and proliferation [36]. Studies have shown that HMBPP can induce the up-regulation of IL-21 receptors (IL-21Rs) on human γδT cells in the amplification system. γδT cells show stronger antitumor activity [37], such as granzyme and perforin. The signal of the IL-21R can inhibit γδT cells secreting IL-17 [38]. The presence of IL-21 can also affect the differentiation and polarization of γδT cells and have more memory phenotypes [39]. The erythroleukemia cell line K562 was modified, irradiated, and used as a feeder cell line to express costimulatory molecules CD19, CD64, CD86, CD137L, and the membrane-bound protein IL-15 (mIL-15), which was supplemented by the addition of exogenous IL-2 and IL-21. This engineered cell line can substantially expand γδT cells derived from the peripheral blood of healthy donors or tumor patients. γδT cells expanded in vitro are not a specific subtype and these polyclonal γδT cells exhibit a wide range of resistance to tumor cells [7, 40–42]. However, IL-21 also increased the expression of T-cell immunoglobulin and mucin domain 3 (TIM-3), making its efficacy limited.

Bispecific antibodies enhance cytotoxic activity by selectively targeting tumor-associated antigens [such as human epidermal growth factor receptor 2 (HER2), CD19, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM)] recognized by γδT cells, by carrying CD3, CD16 or Vγ9 domains in the recombinant protein which activates γδT cells, CD107a on the surface of γδT cells, intracellular IFN-γ and TNF-α are significantly increased, which can lead to stronger cytotoxic activity in tumor cell lines isolated in vivo and drug-resistant tumor cells [40, 43–46]. In addition to conventional antibodies, nanobodies that lack the CH1 domain of light and heavy chains are produced by camelids and can be used as alternatives to traditional antibodies [47]. Some researches based on the specific heavy chain of HCAbs (VHH) of Vγ9Vδ2 T cells can not only induce activation, degranulation and cytokine production in Vγ9Vδ2 T cells, but it can also be used in flow cytometry analysis, magnetic bead sorting, and immunohistochemistry [48].

Some drugs also affect the cytotoxicity of γδT cells by affecting the signaling pathways involving γδT cells. In mice, rapamycin can inhibit the mechanistic target of rapamycin (mTOR) signaling in Vγ4δ T cells expanded in vitro, which leads to the upregulation of NKG2D and increased TNF-α expression, thereby enhancing their cytotoxic effects against a variety of tumor cell lines [49]. In the treatment of patients with nonmuscular invasive bladder cancer (NMIBC), Bacillus Calmette-Guerin (BCG) is the standard treatment method. When rapamycin was used in combination with BCG, the levels of IL-8 and TNF-α were significantly increased in the patients’ urine, and in a mouse xenograft model, rapamycin promoted the killing of humans squamous cell carcinoma mediated by γδT cells [50]. Some therapeutic monoclonal antibodies, such as rituximab, can activate the phospho-zeta-chain-associated protein kinase-70 (ZAP70) and phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathways in γδT cells, and enhance their ADCC activity [51–53]. The Wnt signaling pathway plays a key role in the differentiation, survival, and immune response of T lymphocytes. As an activator of this pathway, TWS119 can enhance the activity of γδT cells to lyse on tumor cells through the perforin-granzyme pathway [54]. Studies have found that the T cell immunoglobulin domain and TIM-3 are significantly up-regulated during the expansion of γδT cells in vitro, which reduces the production of perforin and granzyme B in γδT cells through the ERK1/2 pathway. The use of TIM-3 inhibitors can prevent apoptosis of γδT cells and promote their accumulation at the tumor site [55, 56]. In one interesting study, acute dynamic exercise was used as an experimental model. Controlled exercise of a certain intensity within 30 minutes could increase the number of γδT cells in the peripheral blood by nearly three-fold; these effects occur through β2 adrenergic receptor signaling, which enhances the expansion and antitumor activity of γδT cells in vitro [57].

Selenium is an essential trace element that helps maintain the health and survival of eukaryotic cells [58, 59]. Selenium nanoparticles (SeNPs) are considered as excellent antitumor active substance [60]. The particles can reorganize the cytoskeleton by upregulating tubulin-α, which increases the rigidity of γδT cells. SeNP-pretreated γδT cells can significantly upregulate the expression of NKG2D, CD16, IFN-γ, and other cytotoxicity-related molecules while downregulating the expression of programmed cell death protein 1 (PD-1), which will prolong the survival time of γδT cells in vitro [61].

Some drugs can affect the susceptibility of tumor cells to toxicity by affecting the NKG2DL-NKG2D axis, Fas-FasL signaling, or TRAIL-TRAILRs signaling between γδT cells and tumor cells; examples of such drugs include, disintegrins and metalloproteinase inhibitors [62], bortezomib [63, 64], doxorubicin [65], decitabine [66], temozolomide [67], 4,5-diazafluorene derivatives (14c) [68], carboplatin [69], valeric acid [70]. Pretreatment of tumor cells with these drugs can increase the expression of NKG2DL, Fas, or TRAILRs on their surface, thereby promoting tumor cell apoptosis. Even bisphosphonates that are used to amplify γδT cells can increase the sensitivity of tumor cells to γδT cells when they are pretreated [71–74]. It had been reported that the replacement of the hydroxyl group in ZOL with fluoride ions, and as a result of this structure, γδT cells became more capable of killing tumor cells [75] (Figure 1).

The complementarity determining region 3 (CDR3) of the γδTCR determines the binding affinity of pAg-expressing tumor cells [76]. Some research identified a specific CDR3 region in tumor infiltrating lymphocytes (TILs) in human ovarian epithelial carcinoma and named it OT3 [77]. Thus, a fusion protein containing OT3 and the human IgG (hIgG) Fc domain was designed. This fusion protein exhibited the ability to bind properties to a variety of human tumor cell lines in vitro and could mediate cell killing through ADCC. It also significantly inhibited tumor progression in human ovarian cancer xenograft models and improved the survival rate of tumor-bearing mice [78].

All the methods described above provide better combination treatment strategies that can improve γδT cell antitumor immunotherapy at a later stage.

It has been reported that Vδ1⁺ T cells are increased in patients with B-cell chronic lymphocytic leukemia (B-CLL) [110, 111], while human CMV infection is a complication after allogeneic stem cell transplantation that activates, Vδ1⁺ T cells in vivo after transplantation. This activation has a positive impact on patients’ prognosis [112], which indicates that the CMV antigen is crucial to the expansion of Vδ1⁺ T cells. The low abundance of Vδ1⁺ T cells in peripheral blood hinders their clinical use, and thus, some studies have used irradiated artificial antigen-presenting (aAPCs) that express CD86, 41BBL, CD40L, and CMV-pp65 as feeder cells to expand γδT cells. The resultant polyclonal cell population showed high expression of activated memory markers without signs of exhaustion, namely, Vδ1⁺ T cells, and could exhibit antitumor activity in vivo in a mouse model of leukemia [113]. The addition of concanavalin A (ConA), phytohemagglutinin (PHA), or OKT monoclonal antibody (mAb) to the culture system results in a high percentage expansion of the Vδ1⁺ cell. These TCR agonists can selectively induce Vδ1⁺ T cells to express NK cell receptors (NCRs) such as NKp30, NKp44, and NKp46, which increases their resistance to activation-induced cell death and exhaustion after continuous stimulation [114–116].

A rapid and reproducible clinical transformation protocol developed by the Almeida et al. [116] termed DOT cells. The program is divided into two phases. The first phase involves a TCR agonist and IL-4 costimulation to greatly expand the number of cells, and during the second phase, IL-15 is added to induce NCR expression. Using this scheme, Vδ1⁺ T cells can be expanded by more than 1,000 times in three weeks. Genetic manipulation and feeder cells are not needed for the production process. In vitro, DOT cells target unspecified leukemia cells and bind to a wide range of recognition domains. DOT cells also exert a cytotoxic effect on drug-resistant acute myeloid leukemia (AML) cells that survive after chemotherapy [116, 117].

TILs are activated by autoantigens of hematological tumors, and the detection of TILs in other solid tumors also supports the response of Vδ1⁺ T cells to malignant tumors. Vδ1⁺ T cells isolated from colorectal cancer TILs mainly express NKp46, not only because autologous or allogeneic epithelial tumor cells are cytotoxic but also because this can delay the risk of metastatic tumors [118, 119]. After Vδ1⁺ T cells and Vδ2⁺ T cells from TILs derived from samples were expanded in vitro, all Vδ1⁺ T cells were able to exert cytotoxic effects on the A375 cell line cultured in vitro, while only two Vδ2⁺ T cell samples were cytotoxic [120].

Vδ1⁺ T cells are not susceptible to apoptosis induction. They have unique innate recognition and regulation abilities, and thus, they exert strong lytic activity on tumor cells. They can persist in circulation for many years and provide lasting immunity against certain cancers [22]. Consequently, Vδ1⁺ T cells are gradually becoming a promising candidate for cancer immunotherapy.

At this stage, γδT cell adoptive immunotherapy has achieved certain successes. So far, there are no serious adverse reactions related to γδT cells therapy, which has satisfactory therapeutic effects on hematologic tumors and solid tumors. γδT cells can also distinguish tumor cells from healthy cells by sensing the stress signals specifically expressed by tumor cells. However, the treatment process is associated with two main problems: on the one hand, γδT cells apoptotic during continuous stimulation in vitro, which results in reduced cytotoxicity; on the other hand, it is difficult for the infused γδT cells to infiltrate or accumulate at the tumor site [55]. Therefore, understanding the mechanism of dysfunction of adoptively transferred γδT cells is essential for their future clinical application. The distribution and abundance of T cells play a key role in tumor immunotherapy. Research has led to the development of a stable and accurate method for evaluating the abundance of immune cells from transcriptome data. This Immune Cell Abundance Identifier (ImmuCellAI) can provide important insights for dynamic immune cell infiltration during immunotherapy, and provide valuable indicators into immunotherapy [121]. Studies have also reported that γδT cells in mice that secrete IFN-γ completely rely on glycolysis for their metabolism, and thus, glucose supplementation could enhance the antitumor function of these cells. However, the level of oxidative metabolism of IL-17 secreting cells is increased, and the mass and activity of mitochondria are increased with the uptake of lipids, which accelerates tumor progression in obese mice. It was found that the metabolic resources needed by different subsets of γδT cells can be controlled to amplify antitumor effects, which provides a metabolism-based plan for tumor intervention treatment [122].