γδ T cells, named after their distinctive γδ T cell receptor (TCR) usage, comprise approximately 5% of all T lymphocytes (1). Similar to conventional αβ T cells, γδ T cells recognize targets and exert direct cytotoxic effector functions by secreting granzymes or perforin (2, 3) and inducing immune responses of other cells by secreting cytokines (4), thereby participating in host protection against various pathogens or tumors. Unlike αβ T cells, which recognize peptides on the major histocompatibility complex (MHC) (5), γδ T cells recognize other surface molecules (6). In humans, Vδ2⁺ T cells recognize the butyrophilin family 2A1 and 3A1 complex (BTN2A1–BTN3A1 complex) linked by phosphoantigens (7), and Vδ1⁺ T cells recognize MHC class I chain-related molecule A (8). Because these surface molecules are upregulated in the presence of infection or cellular damage (9, 10), γδTCR-mediated target recognition of γδ T cells resembles that of pattern recognition receptors. Therefore, γδ T cells function as linkers between innate and adaptive immune responses (11) and act as the first-line defense system of the body during early infection.

In addition to infection, γδ T cells have demonstrated their importance in immune responses related to tumors (12, 13). γδ T cells not only localize in peripheral organs (14) but also circulate through blood and lymphatics (15). Therefore, they play critical roles in tumor immune responses in solid cancers, such as lung (16) or colorectal cancer (17), as well as in hematopoietic malignancies (18). Particularly for solid cancers, high infiltration of γδ T cells represents a good prognosis marker (19). Therefore, many research groups have investigated γδ T cell–based immunotherapeutic procedures for cancer treatment (20, 21). Based on their diverse target recognition mechanism, a strong tendency toward activation via various types of stimulation, subsequent cytotoxic effector functions (22, 23), and MHC-independent target recognition mechanism (6), the possibility exists that γδ T cells can be effective immunotherapeutic agents that can target tumors that do not respond to current therapeutic procedures (24–26). Therefore, several research groups are investigating γδ T cell–based cancer therapy targeting various tumor models.

Glioblastoma (GBM) is a malignant tumor that occurs in the brain and is the most common yet lethal malignancy among central nervous system (CNS) tumors (27). A lack of distinctive risk factors (28) combined with nonspecific symptoms (29) make GBM difficult to diagnose in the early phase, thereby decreasing the survival rate. Many research groups have performed extensive investigations to identify an effective treatment for GBM. As a result, various mechanical (30, 31), chemical (32), and immunological (33) treatment approaches have been developed for GBM. Although some treatments have shown meaningful increases in patient survival rates (34, 35), many of those procedures did not show substantial results (26, 36, 37). Therefore, identification of novel therapeutic procedures is critical for effective treatment of GBM.

In this review, we will summarize the immunologic signatures of γδ T cells, focusing on their roles in anti-tumoral immune responses. Then, we will discuss current immunotherapeutic approaches in GBM treatment and challenges arising from the tumor microenvironment (TME) of GBM. Additionally, we will discuss current approaches to target GBM using γδ T cells and the limitations of γδ T cell–based treatments. Finally, we will suggest possible solutions to overcome those challenges in γδ T cell–based GBM immunotherapy.

γδ T cells are a small subset of T cells that express the γδTCR instead of the conventional αβTCR. Even though they comprise a small population of circulating lymphocytes (38), γδ T cells localize in peripheral organs and barrier sites such as the skin, mucosal tract of the intestine or reproductive organs, and pulmonary tract (39) and comprise 15–30% of intraepithelial lymphocytes in the human gut (40). γδ T cells are further subdivided into various subsets according to their Vγ (mouse) or Vδ (human) usage, and Vγ or Vδ utilization determines their localization. In mice, γδ T cells expressing Vγ1 or Vγ4 (Tonegawa nomenclature) circulate through the bloodstream, Vγ5 is localized in the skin, Vγ6 is localized in the dermis and meninges, and Vγ7 is localized in the gut (39). In humans, Vδ2⁺ γδ T cells circulate in the blood, whereas Vδ1⁺ and Vδ3⁺ γδ T cells have resident features (13). Even though they make up a small portion of the T cell population (1), their various effector functions and distinct tissue localization make γδ T cells a first-line immune system defense mechanism by directly suppressing pathogenic infection and working as both innate and adaptive immune cells.

γδ T cells recognize various types of surface molecules, unlike conventional αβ T cells that recognize peptides loaded on the MHC. For example, human Vδ1⁺ γδ T cells recognize the CD1d molecule (41), Vγ8Vδ3⁺ T cells recognize stress-induced annexin A2 (42), and Vγ9Vδ1⁺ T cells recognize ephrin type-A receptor 2 induction by AMP-activated protein kinase (43). In addition to these tissue-localizing human γδ T cells, Vγ9Vδ2⁺ T cells circulating in the peripheral blood recognize the BTN2A1-BTN3A1 complex in the presence of phosphoantigens (7, 44). Because γδTCRs recognize stress-induced molecules expressed on the target cell surface, recognition of γδTCRs resembles that of pattern recognition receptors (45). Therefore, γδ T cells possess invariant or semi-variant signatures, unlike αβ T cells, which have to recognize various peptides; therefore, TCR diversity is critical (46). In addition to the γδTCR, γδ T cells recognize a broad spectrum of surface molecules via NK receptors (NKRs) and exert effector functions synergistically with γδTCR ligation (47). In addition to γδTCR and NKR-mediated target recognition and effector function, γδ T cells may exert a cytolytic function via death ligands (Fas-ligand or TRAIL) (48, 49). With these multi-faceted target recognition mechanisms, γδ T cells play important roles in the first-line protection of various tissues (50, 51).

γδ T cells exert multiple effector functions and share those effector functions with conventional αβ T cells. For example, γδ T cells lyse target cells by granzyme and perforin production (52), similar to cytotoxic CD8⁺ T cells. Additionally, γδ T cells secrete various cytokines, including IFNγ and TNFα, demonstrating that γδ T cells can modulate the immune system through cytokine production (53). Furthermore, similar to effector CD4⁺ T cells, γδ T cells polarize into distinct subtypes and concomitantly produce cytokines that affect the surrounding immune microenvironment. Among murine γδ T cells, IL-17-producing γδ T cells and IFN-γ-producing γδ T cells differentially develop in the thymus (54) and perform distinct roles (55, 56). In contrast, human Vγ9Vδ2⁺ T cells show functional plasticity (57, 58) according to their exposure to cytokines during TCR stimulation. This functional plasticity of γδ T cells makes them multi-faceted effectors that exert both protective and damaging effects in disease conditions, including cancers (1).

GBM is a malignant brain tumor that is classified as WHO grade IV. Annually, approximately 10 out of every 100,000 people are diagnosed with GBM (73). Though the overall incidence is relatively low compared with other types of cancers, GBM is the most common malignant tumor occurring in the CNS (74) and has one of the worst prognoses of all cancer types. GBM patients survive less than 1 year without treatment, and the 5-year survival rate is less than 10% even with intensive care (34). GBM typically occurs in old adults, but it can also occur in children (75). GBM more commonly occurs in male patients than in female patients (76), and female GBM patients have better responses to standard treatment (radiotherapy + temozolomide) (77). Several studies of the risk factors of GBM have revealed that high-dose ionizing radiation (78–80) and rare genetic disorders, such as neurofibromatosis (81), increase GBM incidence. However, other risk factors, including smoking, alcohol uptake, and exposure to pesticides or steroidal hormones were not correlated with GBM onset (28). Common symptoms of GBM are headache, seizures, and cognitive and behavioral impairment (29). Because these symptoms are nonspecific, patients usually miss the opportunity for early therapeutic intervention.

Recent research revealed that GBM starts in the subventricular zone of the brain and spreads to the cortex (82). GBM originates from three cell types: neural stem cells (NSCs), NSC-derived astrocytes, and oligodendrocyte precursor cells. Among these, NSC and NSC-derived astrocytes are the more frequent cells of origin that induce GBM (83). Moreover, GBM consists of glioma stem cells (GSCs), which develop into a heterogenous cell population responsible for increasing GBM tumor burden (84). GSCs contribute to GBM’s resistance to chemoradiotherapy and high recurrence rate (85).

Current studies on molecular and genetic signatures have enabled researchers to classify GBM into various subtypes. According to the WHO classification, IDH-wildtype GBM is characterized by TERT promoter mutation, epidermal growth factor receptor (EGFR) amplification, and a combination of chromosome 7 duplication and chromosome 10 loss (86). Using gene expression patterns, researchers further classified GBM into four different subtypes: proneural, neural, mesenchymal, and classical (87, 88). Not only do these subtypes express different morphological signatures and distinct genes (89), but they also show different susceptibility toward therapeutics. Classical subtypes, which possess a TP53 mutation, show susceptibility to radiotherapy and concurrent chemotherapy with temozolomide (90). By contrast, the mesenchymal GBM subtype shows resistance to radiotherapy and chemotherapy (91, 92). Although GBM cells are classified into various subtypes, the subtypes are not stable because transitions between subtypes frequently occur, most commonly to the mesenchymal subtype from other subtypes. Ionizing radiation (91, 93) from radiotherapy and hypoxic stress (94) that arises during tumor progression instigate this transition to the mesenchymal subtype. In addition to the four subtype-based GBM classifications, epigenetic signatures can differentiate GBM types. The methylation status of the O (6)-methylguanine-DNA methyltransferase (MGMT) promoter can be used to categorize GBM tumor cells as MGMT promoter methylated or unmethylated. The classification by MGMT promoter methylation is important for GBM patient prognosis because MGMT-expressing GBM cells are more resistant to DNA alkylating agents, such as temozolomide. Therefore, those patients with MGMT promoter methylation in GBM tumor cells respond better to temozolomide treatment and live longer (95).

Currently, the Stupp protocol is the standard care for GBM. The protocol reduces tumor burden by resecting GBM to the extent feasible followed by radiotherapy and concomitant chemotherapy using temozolomide, a DNA alkylating agent administered orally or intravenously (96). Although this therapeutic approach improved overall survival, GBM still has a poor prognosis due to the recurrence of tumors after treatment, which leads to a lower survival rate. This high recurrence rate is a result of the intrinsic characteristics of GBM, the unique anatomical and immunological features of the brain, and the limitations of the current treatment procedures. First, GBM cancer cells undergo a mesenchymal transition during tumor progression or due to radiation therapy. This mesenchymal transition is driven by hypoxia-inducible factors (97), and the high hypoxic signature of GBM can promote mesenchymal transition. Cancer cells exhibiting a mesenchymal signature can invade through the surrounding normal brain tissue (98), which makes it difficult to determine the boundary of the GBM and renders complete resection of the tumor impossible. Furthermore, GSCs in brain tumors undergo self-renewal and differentiation (99), thereby contributing to tumor recurrence if not completely removed (100). In addition, the brain is protected by the blood–brain barrier (BBB), which hinders active involvement of the external immune system (101). As a result, brain tumors are classified as immunologically cold cancers with limited infiltration of lymphoid cells, particularly T cells (102). These characteristics lead to the ineffectiveness of various therapeutic procedures in the context of GBM treatment (26), even though those procedures have proven effective in other types of cancers (103). Moreover, brain-residing microglia (104) and neurons (105) maintain an anti-inflammatory immune environment, which hinders a robust tumor-suppressive immune response even when immune cells infiltrate the GBM. Lastly, the standard of care for GBM patients does not use target-specific therapeutic agents and may lead to off-target toxicity in the surrounding normal cells. GBM surgical resection leads to the loss of normal tissues surrounding the tumor, and radiation therapy can deplete brain immune cells or trigger mutations in normal brain tissue, potentially leading to the initiation of new tumor foci. It can also promote the mesenchymal transition of existing cancer cells, increasing resistance to drugs and radiation therapy (106). Temozolomide can affect normal cells as well, including immune cells. Most importantly, brain tumors with an unmethylated MGMT promoter exhibit resistance to temozolomide (107). In 2014, it was discovered that the addition of anti–vascular endothelial growth factor therapy, which inhibits angiogenesis, had a synergistic effect with conventional treatment methods in recurrent gliomas. However, the improvement in patient survival resulting from this combination therapy was found to be modest (108). Similarly, although the utilization of a novel treatment method, called tumor-treating fields (35), has led to a meaningful improvement in overall survival in brain tumor patients, overall patient survival rates remain low (109). To overcome the current limitations of brain tumor therapy, it is crucial to devise novel therapeutic approaches that not only effectively remove tumors but also facilitate the involvement of the immune system to prevent tumor recurrence. Consequently, research has emphasized the necessity of immunotherapy, a treatment modality that focuses on enhancing the immune response against brain tumors.

For successful GBM therapy using γδ T cells, the current limitations of γδ T cells must be addressed and novel therapeutic procedures that fully utilize the benefits of γδ T cells must be devised ( Figure 4 ). Rather than expanding patient γδ T cells by zoledronic acid, allogeneic γδ T cell transfer from a healthy donor to the patient is gaining interest (139) ( Figure 4A ). γδ T cells have already proven their safety in allograft transfers, with low risk of graft-versus-host diseases and rejection (131) ( Figure 4A ). With an allograft transfer, global suppression of γδ T cells induced by GBM and chemotherapy will be reduced. In addition to allograft transfers, further engineering of allogeneic γδ T cells can lead to synergistic effects ( Figures 4B-D ). CAR-T cell-based GBM treatment currently shows a modest effect (140), possibly due to the low persistence of CAR-T cells in peripheral blood. It is known that a weak-not high-level of tonic signaling is required for better in-vivo persistence and superior antitumor function (141). Anti-EGFRviii CAR-T cells were used for GBM treatment, although this target is not expressed in peripheral blood and cannot provide tonic signaling to T cells ( Figure 4B ). However, the issues caused by the lack of tonic signaling can be resolved by expressing the CAR in human Vγ9Vδ2⁺ γδ T cells, which can receive tonic signaling by γδTCR and have endogenous butyrophilin expression ( Figure 4B ). Introduction of the CAR to γδ T cells provides an additional route by which γδ T cells can target tumor cells, which prevents tumor cells from escaping immune surveillance by antigen loss. In conventional CAR-T cells, which introduce CAR molecules to conventional T cells, tumor cells may escape CAR-T cell surveillance by downregulating the target of the CAR. However, if the CAR is introduced to human Vγ9Vδ2⁺ γδ T cells, tumor cells cannot evade surveillance even after antigen downregulation because γδ T cells can target tumor cells via TCR and other NK receptors. By reducing the chance of tumor cell immune escape, CAR–γδ T cells may represent an improvement over conventional CAR-T cell ( Figure 4B ).

The introduction of engineering expands the opportunities of γδ T cell-based therapy beyond the CAR ( Figures 4C, D ). For example, γδ T cells can be engineered to overcome the immune-suppressive GBM environment. Liu et al. suggested engineering a novel switch receptor that switches the immune-suppressive PD-1 signaling into immune-activating CD28 signaling (142) ( Figure 4C ). Introducing the receptor augmented the anti-tumor immune response of CAR-T cells. In GBM that express PD-L1 (72), engineering γδ T cells by introducing the switch receptor can overcome immunosuppression and may even exploit the suppressive microenvironment. A similar approach to the switch receptor mediation can also be applied to TGF-β to overcome immunosuppression ( Figure 4C ). It is well known that TGF-β is highly expressed in GBM (143), and TGF-β signaling reduces the γδ T cell anti-tumoral immune response by making these cells anti-inflammatory (58). The introduction of a switch receptor that changes the TGF-β signal into other pro-inflammatory signals may help γδ T cells overcome TGF-β-induced immunosuppression. Noh et al. recently introduced a TGF-β-targeting switch receptor that can change TGF-β signaling into IL-7 signaling, and expression of the receptor improved tumor control in the CAR-T-based B-cell lymphoma suppression model (144). Therefore, similar concepts can be applied when designing γδ T cell-based GBM treatment.

Engineered γδ T cells can have synergistic effects when combined with other treatments. Recently, novel genetically engineered human Vγ9Vδ2⁺ γδ T cells were used in a GBM clinical trial ( Figure 4D ). The current standard care therapy for GBM includes temozolomide; however, this treatment affects immune cells, which may lose functionality, because temozolomide does not specifically target tumor cells. In this situation, γδ T cells engineered to express MGMT retain their functionality under temozolomide treatment (145). A clinical trial for GBM treatment using adoptive transfer of human γδ T cells expressing MGMT (ClinicalTrials.gov Identifier: NCT04165941) in combination with temozolomide is currently in progress. In summary, although γδ T cell therapy alone cannot control GBM, it still has therapeutic potential. γδ T cells can overcome current limitations with engineering and combination therapy and may become an effective therapeutic agent for GBM treatment.

Developing novel expansion methods to block the skewing of γδ T cells toward the pro-tumoral population can be an effective and plausible solution for γδ T cell adoptive transfer ( Figure 4E ). Several studies have previously demonstrated procedures to skew γδ T cells toward anti-tumoral populations. For example, the addition of IL-21 helps human Vγ9Vδ2⁺ γδ T cells to produce pro-inflammatory cytokines and exert increased cytotoxicity by irreversibly polarizing Vγ9Vδ2⁺ γδ T cells to express Th1-like signatures (146). This Th1-polarizing condition may show strong synergy with another expansion protocol devised by Choi et al., which uses artificial antigen-presenting cells to expand human Vγ9Vδ2⁺ T cells (147). The expansion strategy proposed by Harmon et al. also showed that addition of IL-15, IL-18, anti-CD2 antibody, and anti-CD3 antibody effectively expanded human Vδ1⁺ T cells and polarized them toward an anti-tumoral population (148). Because the plasticity of γδ T cells in the TME is a major issue that hinders γδ T cell therapy, development of an improved expansion protocol to block this plasticity is crucial for effective γδ T cell therapy.

Engineering γδ T cells to cross the BBB is another effective strategy to increase the infiltration of γδ T cells into GBM ( Figure 4F ). Recent findings from Kendirli et al. show that various factors, ranging from transcription factors to chemokine receptors, regulate T cell migration to the CNS (104). Using genome-wide CRISPR screening, the authors found that knockout of integrin α4, CXCR3, and GRK2 significantly reduced T cell trafficking to the CNS, while ETS1 knockout significantly upregulated T cell trafficking to the CNS. Therefore, modulation of molecules related to T cell trafficking to the CNS in γδ T cells can facilitate infiltration of these cells into GBM.

γδ T cells, with their versatile effector functions, have the potential to be a promising therapeutic agent to target tumors. Their ability to target tumor cells via various mechanisms, including γδTCRs, NK receptors, Fc receptors, and death receptors, decreases the possibility of tumor cells evading surveillance. Their ability to produce pro-inflammatory cytokines and spread antigens via direct antigen presentation to the adaptive immune system helps γδ T cells overcome the immunosuppression of the TME and induce optimal anti-tumoral immune responses. Additionally, because they do not recognize MHC molecules and do not risk inducing graft-versus-host disease when transferred from donors to MHC-mismatched patients, γδ T cells can possibly be used in allogeneic adoptive transfer therapy. Therefore, γδ T cells have the potential to be a novel therapeutic agent for GBM, a malignant brain tumor with the highest WHO grade and therefore the worst prognosis. Understanding the immunological signatures of the GBM TME is critical for optimal function of γδ T cells in the GBM TME and subsequent tumor suppression. The immunosuppressive microenvironment, BBB, and MHC-deficient GSCs are the major factors that suppress effective immunotherapy. Although γδ T cells have the potential to overcome some of these limitations, several obstacles still exist, hindering effective therapy and the achievement of successful treatment for GBM. Therefore, for successful γδ T cell–based immunotherapy, it is critical to devise strategies to overcome those limitations. With further studies to determine the signatures of the GBM TME and γδ T cells themselves, in combination with the augmentation of their abilities and improvement of current limitations, γδ T cells can become an innovative therapeutic agent for GBM.

IK: Conceptualization, Writing – original draft, Writing – review & editing. YK: Conceptualization, Writing – original draft. HL: Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing.