The capability of heterogeneous γδ T cells in cancer treatment

γδ T cells, a specialized subset of T lymphocytes, have garnered significant attention within the realm of cancer immunotherapy. Operating at the nexus between adaptive and innate immunological paradigms, these cells showcase a profound tumor discernment repertoire, hinting at novel immunotherapeutic strategies. Significantly, these cells possess the capability to directly identify and eliminate tumor cells without reliance on HLA-antigen presentation. Furthermore, γδ T cells have the faculty to present tumor antigens to αβ T cells, amplifying their anti-tumoral efficacy.Within the diverse and heterogeneous subpopulations of γδ T cells, distinct immune functionalities emerge, manifesting either anti-tumor or pro-tumor roles within the tumor microenvironment. Grasping and strategically harnessing these heterogeneous γδ T cell cohorts is pivotal to their integration in tumor-specific immunotherapeutic modalities. The aim of this review is to describe the heterogeneity of the γδ T cell lineage and the functional plasticity it generates in the treatment of malignant tumors. This review endeavors to elucidate the intricate heterogeneity inherent to the γδ T cell lineage, the consequential functional dynamics in combating malignancies, the latest advancements from clinical trials, and the evolving landscape of γδ T cell-based oncological interventions, while addressing the challenges impeding the field.


Introduction
There is a distinct and conserved population of T lymphocytes called gd T cells, named for the g and d chains making up the T cell receptor (TCR) that sets them apart from the classical T cells (CD4 + and CD8 + ) that contain ab TCRs.They represent a distinctive subset of T cells that exist in a transitional state between the adaptive and innate immune systems (1)(2)(3).Functionally, there is compelling evidence to suggest that their antigen receptors exhibit greater specificity and diversity compared to the surface antigen receptors found in ab T cells or B cells (4).gd T cells play an important role as the first line of defense of the immune system while also participating in the adaptive immune response.Serving as a conduit between innate and adaptive immunity to elicit potent reactions (5), gd T cells are viewed as promising immunotherapeutic agents within the realm of cancer treatment, offering a fresh perspective in the field of anti-tumor immunity (6, 7).

Development and differentiation of human gd T cells
In many mammalian species, gd T cells emerge as the primary lymphocyte subset during fetal development (8,9).Their receptor, composed of a g and a d chain, is formed through somatic variablediversity-joining (V(D)J) recombination, similar to the segments of aand b-chains in ab TCRs (10).There are numerous configurations for the T cell receptor's gd variable region (Vg) and delta chain variable region (Vd), and the fusion of these two regions allows for the formation of a sizable collection of roughly 10 20 TCR clonotypes (11), providing significant diversity to gd T cell subsets.Human gd T cells are traditionally classified into three primary subgroups: Vd1, Vd2, and Vd3, determined by the Vd chain usage (12-14).Among the three main gd T cell subsets in humans, Vd1 T cells predominantly pair with the Vg I family, which includes (Vg2/3/4/5/8), and the Vd2 subset predominantly binds Vg II (Vg9), typically Vg9Vd2 T cells (15).Unique tissue localization, activation, and function are displayed by various gd T cell subsets and their distribution within the human body can be distinguished clearly (16)(17)(18)(19)).Vd1 T cells are predominantly located in epithelial tissues, including the intestines and skin, as well as organs like the spleen and liver.These cells have a vital function in safeguarding the preservation of epithelial tissue integrity (20).Vd2 T cells, mainly the Vg9Vd2T cell subset, account for approximately 60-95% of peripheral gd T cells in the circulation (21).These cells make up around 2-5% of the circulating CD3 + T cell population and play a dual role as both effector cells and antigen-presenting cells (APCs) (22).Vd3 T cells, which are infrequently observed in circulatory systems, are notably prevalent in hepatic tissues, particularly in individuals with infections or malignancies.

gd T cell in complex tumor microenvironments 3.1 Recruitment of gd T cells to the tumor microenvironment
The tumor microenvironment (TME) significantly influences the activity of gd T cells across various cancers.In the complex TME, gd T cells are recruited or activated toward the tumor site.However, there also exists a synergistic or pleiotropic effect of tumor cells and multiple factors in the TME, where infiltrating gd T cells are activated or depleted, or polarized to a tumor-promoting phenotype, thus supporting cancer progression (18).Some investigators have analyzed the effect of the TME on gd T cell recruitment in a preclinical transplantable B16 melanoma model, where human Vd1 T cells use the CCR2/CCL2 pathway to migrate toward the tumor, where they exert critical non-redundant anti-tumor functions (23).Consistent with this study, Vd1 T cell infiltration was abundant in breast and primary prostate cancers with significantly upregulated CCL2 expression (24,25).Furthermore, in cases of hepatocellular carcinoma (HCC), tumor cells harness the CCL4/CCL5 chemokine pathway, interacting with the CCR1/CCR5 receptors, thereby orchestrating the mobilization of gd T cells either from the peripheral blood or peritumor region to the tumor region (26).In the TME of breast cancer, breast cancer cells secrete IP-10, which mediates the transport and migration of gd1 T cells to the tumor site via IP-10/CXCR3 (27,28).It has also been claimed that the CCR4/CCR8-CCL17/CCL22 pathway also significantly induces Vd1 T cell migration.Meanwhile, high levels of CCL17 and CCL22 were detected in a variety of tumors, such as lung cancer, gastric cancer, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, and peripheral T-cell lymphoma.In lymphomas, CCL17 was specifically expressed in classical Hodgkin's lymphoma, whereas CCL22 was expressed in nodular lymphocyte-predominant Hodgkin's lymphoma and B-cell non-Hodgkin's lymphoma (29).

Heterogeneity of gd T cells in the tumor microenvironment
Both in vivo and in vitro studies have revealed the multifaceted roles of various gd T cell subtypes in modulating tumor cell proliferation, underscoring their intricate contribution to the dynamics of cancer progression.Flow cytometry and Frontiers in Immunology frontiersin.orgtranscriptome analyses revealed that tumor-infiltrating lymphocytes contained an average of 4% gd T cells, most of which expressed Vd1.Among gd T cells in the TME, the Vd1 T cell subset highly expresses CXCR1 and weakly expresses CCR5, whereas Vg9Vd2 T cells show only strong expression of CCR5 (30).Moreover, Vg9Vd2 T cells concurrently expressed CCR3 and CXCR3, enabling them to initiate anti-tumoral responses in peripheral tissues, especially during the metastatic processes (18).Vg9Vd2 T lymphocytes have been identified to demonstrate cytotoxic properties against breast cancer cells, enhancing apoptotic pathways and attenuating angiogenic signaling processes (31).Accumulated gd1 T cells in the breast TME are termed gd1 Tregs (32,33), and these breast tumor-derived gd Tregs suppress innate and adaptive immunity by inducing immune senescence and preventing dendritic cell maturation and activity (24,34).
In the study of gd T cells in the TME of colorectal cancer (CRC), the results showed that gd T cells were mainly detected in paracancerous tissues but rarely in intra-tumoral tissues, and there was no significant increase in the number of T cell subpopulations of Vd1 and Vd2 in the CRC-infiltrating gd T cells, but the main subpopulation was Vd1 T cells (35,36).The shifted balance between these subpopulations might hold implications for the progression of colon cancer (37).
Transcriptomic analysis of the peripheral blood of leukemia patients showed the presence of many tumor-infiltrating Vg9Vd2 cells, which positively correlated with the survival of these patients (18,38).But then a new finding emerged that patients with chronic lymphocytic leukemia (CLL) had an increased percentage of Vd1 cells, which replaced Vg9Vd2 cells as the predominant gd T-cell subtype in the peripheral blood (39).And the study noted that a higher percentage of Vg9Vd2 cells was associated with a poor prognosis in patients with untreated CLL, as these lymphocytes exhibited signs of functional failure with reduced NKG2D expression (40,41).
Infusion of large numbers of gd T cells (Vd1 and Vd2 T cells) into high-risk leukemia patients by allogeneic hematopoietic stem cell transplantation (HSCT) contributes to the rapid control of infections and leukemia relapse.In HSCT recipients, Vd2 and Vd1 T cells were found to be cytotoxic to primary acute leukemia cells, whereas newly generated Vd1 and Vd3 cells in the TME underwent an adaptive response driven by cytomegalovirus (CMV) reactivation (42).

gd T cells funtional flexibility
Despite accounting for a relatively small proportion of total T cells, gd T cells have a complex and crucial role in the onset and progression of cancer.The function of gd T cells in the TME can be altered by several circumstances to become either support tumor growth or combat it.Subsets of gd T cells indirectly achieve antitumor immunity by producing specific factors to promote Th1, Th2, or Th17 differentiation (43)(44)(45) or cross-transmitting signals with B cells (46,47), natural killer (NK) cells (45), and dendritic cells (48) in TME (49).There are also specific subpopulations of gd T cells secrete a quantity of IL-17, which can directly act on epithelial cells to promote the progression of cancer, and gd T can affect ab T cells through immune checkpoints, supporting the creation of an immunosuppressive microenvironment that promotes tumorigenesis (2, 50).This dual role may be attributed to the inherent plasticity of gd T cells, which includes the recruitment or residence of specific gd T cell subsets at the tumor site and the ability to differentiate into different functional cell subsets based on the TME (51, 52).

Anti-tumor function
In the realm of oncology, gd T cells serve as a robustly positive prognostic indicator in most malignancies (47,53,54).Pan-cancer analysis based on the TCGA database in 2015 showed that gd T cells were the best predictor of the prognosis within a range of solid tumors (50).gd T cells are crucial for cancer immune surveillance and indeed studies have found that the incidence of cancers in mice lacking gd T cells increases (55).Notably, gd T cells accumulate in tumor-associated lymphoid tissues (38,56) and can penetrate solid tumor tissues (57, 58).They can naturally infiltrate into the tissues of the whole body, including the lung, liver, and intestinal tract, which can be difficult malignancies to penetrate therapeutically.
gd T cells have characteristics of both the innate and adaptive immune systems and can act directly or indirectly on tumor cells (Figure 1; Table 1).To directly attack cells, gd T cells rapidly migrate into the local tumor microenvironment by recognizing NK cell receptors on the cell surface.gd 1T cells and gd 2 T cells are both capable of ex vivo lysing of tumor cells and express chemokine receptors that enhance tumor homing (4).Activated gd T cells can release granzyme and perforin to kill tumor cells directly (78).In addition, different gd T cell subsets attach to tumor cells through the death receptors TNF-related apoptosis-inducing ligand receptor (TRAILR), CD95 (also known as FAS), and TRAIL and lyse cancer cells (65,79,80).The cell surface receptors NKG2D (81) and CD16 (48) also mediate the direct killing of gd T cells based on antibody-dependent cytotoxicity and effector responses (48,82).Complementing their cytotoxic capabilities, gd T cells can also secrete cytokines IFN-g and TNF-a, jointly suppressing tumorassociated angiogenesis (83).In some hematologic tumors, gd T cells have been found to be capable of immunosurveillance by NK-like mechanisms (81,84).Remarkably, around 80% of quiescent circulating gd T cells express NK receptors.Most of these cytotoxic Vg9Vd2 T cell clones express HLA class I inhibitory NK cell receptors, such as CD94/NKG2A, KIR2DL1, KIR2DL2, KIR3DL1, or KIR3DL2 (85).Intriguingly, the majority of the

Pro-tumor function
Specific gd T cells within the tumor microenvironment are known to secrete IL-17 (92), which promotes the emergence of autoimmune and inflammatory disorders (93,94).At the same time, IL-17-producing gd T cells promote the growth of tumors in a variety of ways.Recent studies have elucidated five key characteristics that underscore the tumor-promoting roles of these gd T cells (Figure 2; Table 1).Firstly, gd T cells have been shown to have a pro-angiogenic effect (95).Vascular endothelial growth factor (VEGF) and angiopoietin-2 (ANG-2) are angiogenic factors that gd T cells can produce to promote angiogenesis (92,96).Moreover, Margarida Rei et al. discovered that small peritoneal macrophages (SPM) were activated by IL-17-secreting Vg6 gd T cells, which accelerated the progression of ovarian cancer.Migration inhibitory factor (MIF) and IL-6 are two of the many tumor-promoting mediators that SPM can generate.They may promote the development of a variety of pro-inflammatory and pro-angiogenic molecules, while also protecting tumor cells from death (96).Secondly, these cells can prevent immune cells from performing their anti-tumor immunological functions.Specifically, IL-17 production from gd T cells can directly suppress the anti-tumor activities of CTL and Th1 cells.Additionally, a significant proportion of gd1 Treg cells can be found in the human breast tumor microenvironment, and they exert potent inhibitory effects on the proliferation of CD4 + , CD8 + , and Vg9Vd2 T cells by inducing senescence in responding immune cells and impairing the maturation and function of DCs (24,27,32).Elevated BMP2 in Acute Myeloid Leukemia (AML) patients induces the production of CD25 + CD127 low Vd2 + T cells (named Reg-Vd2).Reg-Vd2 cells produce a number of regulatory cytokines rather than inflammatory cytokines, and the anti-AML activity of effector Vd2 cells is significantly inhibited by Reg-Vd2 cells (97).Furthermore, Vg1 gd T cells secrete IL-4 and decrease the NKG2D, perforin, and interferon expression levels in Vg4 gd T cells (76).
Thirdly, gd T cells can directly construct a tumor immunosuppressive microenvironment.In human colorectal cancer, gd T cells are polarized by microorganisms present due to disruption of the tumor epithelial barrier and inflammatory dendritic cells (Inf-DCs) in the TME to produce cytokines such as TNF-a, GM-CSF, IL-17 and IL-8.These cytokines recruit myeloid derived suppressor cells (MDSC) into the TME, regulate the development of tumor cells as well as induce Treg differentiation (73).In addition, these cells encourage G-CSF-mediated tumorassociated neutrophils (TAN) proliferation and accumulation in the TME.These TAN, in turn, release a variety of cancer-promoting factors, such as growth factors, neutrophil elastase (NE) and metalloproteinases (MMPs), and produce reactive oxygen species ( R O S ) .T h e s e a c t i o n s p r o m o t e d e v e l o p m e n t o f a n immunosuppressive tumor microenvironment, inducing the depletion of CD8 + T cells and supporting tumor metastasis, tumor growth and invasion (31).Zhang et al. demonstrated that the "gdT cell-IL17A-Neutrophil" axis in the breast cancer tumor microenvironment promotes immunosuppression as well as enhancing the breast cancer's tolerance to high-dose anti-VEGFR2 therapy (98).
In addition to this, there are two important protumor mechanisms, IL-17 secreted by gd T cells modulates adhesion molecules and upregulates endothelial cell permeability to promote tumor metastasis (99).gd T cells also produce IL-22 and amphiregulin (AREG), which directly induced tumor cell proliferation (100).

gd T cell-based cancer therapy
gd T cells can directly identify and kill tumor cells therefore, adoptive and in vivo-induced gd T cell expansion therapies are promising avenues to explore for anti-cancer immunotherapy purposes (101,102).gd T cells may be more favourable for use in adoptive cell immunotherapy compared to ab T cells as they react more quickly to targets to produce effector factors, and they are found in a range of organs (103).In the hypoxic tumor microenvironment, gd T cells, particularly the Vd 1 subset, exhibit greater tissue tendency and greater invasiveness compared to ab T cells (104).Moreover, graft versus host disease (GvHD) and allogeneic response risks can be decreased by using gd T cells' MHC-independent identification of target cells (105).Currently, various strategies are being used to activate and target gd T cells, including drugs, antibodies, and genetic engineering.These strategies aim to enhance the anti-tumor response of gd T cells and use them to combat hematological or solid tumors, such as B-cell malignancies (106).

CAR-gd T
CAR-T cell therapy is a type of immunotherapy that uses the patient's own immune cells to fight cancer.In this approach,T cells are collected from the patient's blood and genetically modified in the laboratory to express chimeric antigen receptors (CARs) on their surface.These CARs are designed to recognize specific proteins, called antigens, on the surface of cancer cells.Once the T cells have been modified, they are grown in large numbers and infused back into the patient's body.The CAR-T cells can then seek out and destroy cancer cells that express the target antigen (107).
In the context of CAR-gd T cells, diverse extracellular and intracellular domains can be fashioned based on the target antigen, the required co-stimulatory signal, and the signaling partner (108).Some examples of CAR designs for gd T cells are: CD19-CAR, GD2-CAR (109), CD20-CAR (110), NKG2D-CAR (111), CCR (chimeric co-stimulatory receptor) (112), and NSCAR (non-signaling CAR) (113).To generate CAR gd T cells, different methods of delivering genes can be used, for example, retrovirus (114), lentivirus (115), transposon (116), or mRNA electroporation (117).Traditional CARab T cell therapy has produced good clinical data in leukemia and other hematological malignancies, but it has not achieved the same success in solid cancer.In this regard, CAR-gd T cells might offer a more promising avenue, as they have innate cytotoxicity capabilities, can recognize multiple antigens (118) and acquire the phenotypic and functional properties of antigen-presenting cells (APCs) (59,119).In preclinical studies, CAR gd T cells have exhibited potential against a diverse range of hematological and solid tumors, including B-cell lymphoma (110), glioblastoma (120), melanoma (121), colorectal cancer, and ovarian cancer (111).Nevertheless, several challenges persist in the development and application of CAR gd T cell therapy (122), such as reduced tumor-toxicity, homing, in vivo persistence, heterogeneity, inter-donor variability, tumor microenvironment adaptation, etc.

Adoptive transfer and In vivo expansion of gd T cells
Adoptive transfer of T cells is a form of cancer treatment that involves the infusion of a patient's own gd T cells that have been expanded and activated outside the body.Nevertheless, previous clinical trials utilizing autologous gd T cells sourced from cancer patients have only demonstrated limited clinical efficacy (123).Hence, current research is increasingly focusing on adoptive transfer therapies with allogeneic Vg9Vd2 T cells (124), which have been shown to enhance immune function, including CD4 + T cell, CD8 + T cell, and NK cell counts in cancer patients, even leading to total remission of recurrent hepatocellular carcinoma in one notable case (125).Another study investigated the use of adoptive cell therapy with IL-15-induced gdT cells in a patient-derived renal cell carcinoma xenograft model.The study concluded that IL-15induced gd T cells effectively suppressed tumor growth in vivo and prolonged the survival time of RCC-bearing patient−derived xenograft (PDX) mice (126).
In vivo expansion of gd T-cells stands out as a unique approach to cancer immunotherapy.Unlike the ex vivo expansion seen in adoptive transfer, this method seeks to stimulate gd T-cells directly within the patient's body.This approach aims to enhance the antitumor activity of gd T cells by using agents such as zoledronate, phosphoantigens, or specific cytokines such as IL-15 or IL-2 (124).In vivo expansion of gd T cells has been shown to induce tumor regression and prolong survival in some animal models and clinical trials.A pilot study evaluated the adoptive transfer and in vivo expansion of haploidentical gd T cells in patients with advanced hematological malignancies ineligible for allogeneic transplantation.Patients received peripheral blood mononuclear cells from half-matched family donors, followed by zoledronate and IL-2 to stimulate donor gd T cells in vivo.This resulted in significant expansion of donor gd T cells, NK cells, and double-negative ab T cells.Impressively, three out of four patients achieved complete remission despite prior refractoriness (127).

Clinical trials: current state of the art
At present, many preclinical studies have been conducted by researchers that suggest that gd T cell therapy works well in multiple tumor models.While treating advanced ovarian cancer, gd T cells function in the patient's ascites and tumor by innate and adaptive immunological methods, respectively.This may make gd T cells a viable treatment option for advanced ovarian cancer (128).For hematological malignancies, researchers have explored various ways to treat tumors using gd T therapy.Ganesan et al. created a Vg9/CD123 bispecific antibody that specifically triggers Vg9 + gd T cells and causes cytotoxicity to the tumor in vitro.This antibody efficiently induces Vg9 + gd T cells to engage with tumor cells.In patients with AML, these cells possess a variety of strategies for mounting an efficient immune response against overloaded tumor cells ( 129).gd T cells can identify cancer antigens other than peptides, so extending the pool of possible targets for tumor cell eradication.Combining this feature, Xu et al. proposed a new TCR-T platform.They designed the AbTCR with non-MHC-restricted targets like CD19, which allows for the management of cytokinerelated toxicity beyond existing anti-CD19 CAR-T therapies and provides comparable tumor suppression (115).Contrary to conventional CD19 CAR-ab T, CAR-gd T cells may still be able to target leukemia cells that lack the CD19 antigen and as such are useful for cases in which the antigen has been lost (130).
Early clinical results have established the promising vista of gd T cells therapies in leukemia and other hematological tumors and solid tumors such as lung, gastric, and liver cancers.A landmark study led by Zhinan Yin's team monitored patients with advanced liver and lung cancers over three years post-reception of allogeneic gd T cell therapy.The team used allogeneic Vg9Vd2 gd T cells from healthy human sources.By treating 132 patients with advanced lung and liver cancer tumors with a total of 414 cell transfusions, their study found that there was not a single case of serious side effects from the allogeneic gd T cell transfusions and only some patients developed transient, mild clinical reactions (125,131).Furthermore, the results highlighted a significant extension in survival among eight liver cancer patients and ten lung cancer patients who received ≥5 cell infusions (130).During this decade, dozens of clinical trials have been approved and several products have emerged as well (132).Numerous biotechnological enterprises are channeling significant investments into this burgeoning domain (Table 2).The gd T treatment proposed by French biologics company ImCheck Therapeutics comprises a novel humanderived anti-BTN3A antibody, ICT01.ICT01 is a monoclonal antibody that specifically promotes Vg9Vd2 T cells targeting of BTN3A, which is extensively expressed in diverse solid and hematologic malignancies.ICT01 has been shown to have antitumor activity in vitro and in vivo tumor models against a The Dutch biotech startup Lava Therapeutics have described a humanized bispecific gd T cell binding antibody (gd bsTCE).gd bsTCE directly induces the effective killing of tumor cells through its unique targeting of Vg9Vd2 T cells and tumor-associated antigens (TAA).Two of its company's projects, LAVA-051, and LAVA-1207, have entered clinical phase 2 trials.Multiple myeloma, chronic lymphocytic leukemia, and acute myeloid leukemia all include the antigen CD1d, which is recruited by LAVA-051 to gd T cells (137).LAVA-051 has been given orphan drug status by the FDA for the treatment of (CLL) based on preliminary data from the Phase 1/2a clinical study and has a satisfactory safety and tolerability profile.Meanwhile, LAVA-1207 was designed to be a gd bsTCE targeting prostate-specific membrane antigen (PSMA), with its clinical study focusing on metastatic castration-resistant prostate cancer.US-based biotech company IN8bio has also updated positive data from its ongoing phase 1 clinical trial of the allogeneic gd T cell therapy INB-100 in high-risk AML patients who have previously undergone haploidentical hematopoietic stem cell transplantation (HSCT).From the data, all three patients treated with INB-100 received at least 12 months of follow-up which showed all three were in complete remission (CR).Remarkably, 100% of evaluable-dose patients remained on study and were in CR, with one patient having a progression-free disease course of more than 3 years (NCT03533816).Another ongoing project, INB-200, uses genetically modified autologous gd T cell immunotherapy for the treatment of glioblastoma (GBM).Data according to the Phase 1 clinical trial of INB-200 for GBM showed that 100% of the six treated patients exceeded the median and expected progression-free survival (PFS).Two of the patients had exceeded the expected overall survival (OS), and the medication was generally well-tolerated and robust.
Innovative developments in CAR-gd T-cell therapy is also advancing at a rapid pace.The UK company TC Biopharm is developing a new CAR-T therapy that takes advantage of the inherent specificity of gdT cells for phosphorylated antigens expressed only by cancerous and infected cells to develop the ImmuniCAR.OmnImmune, is being tested in a Phase 2b/3 clinical trial, following a 50% CR in Phase 1b/2a clinical data for this therapeutic candidate for AML.Concurrently, Adicet Bio announced clinical data for its allogeneic CAR-gd T cell therapy ADI-001 for relapsed or refractory B-cell non-Hodgkin lymphoma (NHL).Data from the study showed ADI-001 demonstrated a 75% overall remission rate (ORR) and CR in eight patients who had received multiple prior therapies, including those who relapsed after using CAR-ab T therapy.Gadeta, a Dutch company, has also innovated in CAR-gd T-cell therapy, designed to use ab T cells to carry the T-cell receptor for gd T cells.The company's TEGs technology enables the efficient expression of gd TCR in ab T cells, mediates tumor-specific proliferation of ab T cells, and extensively infiltrates CD8 + effector T cells and CD4 + helper ab T cells into tumors while not affecting normal organs.
Beyond the aforementioned therapeutic strategies, innovative gd T cell-based treatments for diverse cancers are continually emerging.

Limitations and potential of gd T-cell therapy
It should be emphasized that gd T-cell therapy still has some issues that need to be addressed.Firstly, the scarcity and low efficiency of in vitro expansion remains a serious limitation to entry of gd T cells into the clinical pipeline.Expanding a considerable number of cell products through in vitro methods is crucial for the success of gd T cell adoptive cell therapy.However, the effectiveness of this approach is limited by the inherent differences between donors (140).Recent research has shown that the level of physical activity in a donor can be used as a gauge for determining the in vitro expansion potential of their gd T cells (124).The dominant subtype of gd T cells in the peripheral blood of humans and other primates is Vg9Vd2 T cells, which account for only 1-10% of circulating lymphocytes in peripheral blood (141, 142).Currently, gd T cells are largely obtained from peripheral blood mononuclear cells (PBMC) or umbilical cord blood isolated from healthy donors, followed by in vitro stimulation and expansion using synthetic PAgs or bisphosphonates (143)(144)(145)(146)(147)(148).
Gene modification and iPSCs techniques to produce specific gd T cells in large quantities are major approaches of pharmaceutical companies to improve production and create a more clinically viable option (149).Efforts are underway to identify strategies that amplify the potency of gd T cells in antitumor activities.For instance, IL-15 which can render a more active phenotype, greater proliferative capacity, and greater cytotoxicity in gd T cells, is being investigated Combining IL-15 and gd T cell immunotherapy may be able to enhance antitumor immunotherapy (150).In this regard, more research is warranted to examine the impact of diverse settings on the expansion of gd T cells in vitro and to identify measures to promote the toxicity of Vg9Vd2 T cells, including candidates IL-2, IL-15, vitamin C, and TGF-b (126,151,152).
Another significant hurdle in advancing gd T cell therapies pertains to the engineering of gd T cells (153).For immune cell engineering, the most common method is to use lentivirus or retrovirus transfection.However, compared with ordinary ab T cells, due to the natural antiviral properties of gd T cells, viral transfection of gd T cells is extremely difficult.It is also prone to the loss of CAR genes in cells during culture (115).
The broad spectrum of gd T cells has to be taken into account when talking about the potential of gdT cell therapy.The heterogeneity of gd T cells we described previously includes different subpopulations that mediate opposite immune responses to tumors.These subgroups are widely distributed throughout the body (12, 16).In addition to the Vd2 T cell subpopulation, which is primarily present in peripheral blood and has been developed for antitumor therapy, the Vd1 T cell subpopulation, which is present in tissues, has demonstrated strong cytotoxic potential against tumors when isolated from a variety of human solid tumors, which may partially address the limitations of current CAR-T therapies against solid tumors (37,122).Combining contemporary high-throughput technologies to grasp the different subsets of gd T cells at the single-cell level, such as Vd1T cells (16), with manipulations such as gene editing techniques to enhance the immunological anti-tumor function of gd T cells, may increase their potential application.It's imperative to recognize that the tumor microenvironment is replete with various inhibitory immune cell populations.Immunosuppressive cytokines released by these cells can cause gd T cells to become pro-tumor oriented and secrete IL-17, which drives cancer progression.In certain instances, the leukemic microenvironment adopts strategies to evade the anti-tumor response of these lymphocytes, leading to their exhaustion or polarization into a tumor-promoting phenotype (18).Confronted with these challenges, targeted screening of anti-tumor subsets, exclusion of pro-tumor subsets, determination of how to prevent the initial tumor killer cells from metamorphosis to promote tumor progression cells, or effective depletion of specific pro-tumor gd T cell subsets, will be the focus of future research.Much work remains, particularly with regards to dissecting the multitude of subsets present in the body and determining how best to promote their anti-tumor activity.Current production bottlenecks further restrict their clinical application.Nevertheless, with ongoing research, it is anticipated that gd T cells will cement their place as a cornerstone of cancer immunotherapy in the coming years.

FIGURE 1
FIGURE 1 The anti-tumor function of gd T cells.gd T cells elicit antitumor immune responses through multiple pathways (1) Direct killing effect; (2) Secretion of IFN-g and TNF-a; (3) Induced B cell transformation to secrete large amounts of Ig E and produce adaptive immune responses; (4) Eliciting CD8 + T cell responses.TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; ADCC, antibody-dependent cell-mediated cytotoxicity; CTL, cytotoxic T lymphocyte; NK, natural killer cell; DC, dendritic cells.
Induced pluripotent stem cells (iPSCs) termed T-iPSCs were formed by Watanabe et al. by rearranging the TCR g chain (Vg9) and TCR d chain (Vd2) gene regions (gd T-iPSCs).Notably, these gd T-iPSCs can differentiate into hematopoietic progenitor cells, which could theoretically provide a more potent collection of cells for new cancer research and a nearly infinite source of regenerating cells (138).Similarly, Zeng et al. successfully reprogrammed the gd T-iPSC line of Vg9Vd2 T cells and these cells were modified into NK-like gd T cells, termed "gd natural killer T" (gd NKT) cells (139).

TABLE 1
Mechanisms of gd T cell effects on tumors.Within the TMEs, gd T cells exert intermediate anti-tumor effects by interacting with B cells, dendritic cells, ab T cells, and NK cells.gd T cells can be used as antigen- (91)enting cell to activate ab T cells(68).They can increase the amount of IFN-g secreted by ab T to regulate the TME by inducing recruitment of CTL, NK cells, and Th1, inducing M1-type polarization of macrophages (12), activating dendritic cells to induce their maturation(88), upregulating the expression of MHC class I in tumor cells to improve anti-tumor immune response(89)and preventing pro-tumor T helper cells from functioning (Treg, Th17 and/or Th2).Additionally, epithelial Vg5 T cells induce B cell transformation and secretion of large amounts of Ig E, CCR5-expressing Vg9Vd2 T cell subsets promote antibody production and class switching(90), leading to the development of an immediate adaptive immune response in skin malignancies brought on by chemicals(91).

TABLE 2
Clinical attempts at tumor immunotherapy using gd T cells.