Gamma Delta T Cell: A Unique Member of the T Cell Family with a Significant Role in Cancer Immunotherapy

1. Introduction

Cancer has posed a formidable challenge to researchers for decades. Since the earliest days of cancer research, scientists have been striving to develop treatments that specifically target cancer cells while sparing the surrounding healthy tissues. Recent advancements in cancer biology and emerging technologies suggest that this goal is on the cusp of becoming a reality. At the forefront of these advancements is immunotherapy, which harnesses the body’s immune system to seek out and destroy cancer cells, leaving healthy tissues unharmed.

The underlying principle of immunotherapy is to leverage the body’s natural defense mechanisms to treat cancer more effectively. However, like many other treatments, immunotherapy has been met with variable success across different cancer types. When exposed to treatment pressures, many cancers evolve mechanisms to evade immune detection. Traditional immunotherapies often rely on Antigen-Presenting Cells (APCs) to prime αβ T lymphocytes to attack cancer cells specifically. This process involves recognizing Major Histocompatibility Complex (MHC) Class I molecules on the surface of cancerous cells. However, many cancers can downregulate the expression of MHC Class I molecules, evading αβ T lymphocytes and preventing these immune cells from exerting their cytotoxic effects [1]. Additionally, cancer cells can escape immune surveillance through various strategies, such as secreting the immunosuppressive cytokines Interleukin-10 (IL-10) and Transforming Growth Factor β (TGF-β) into the tumor microenvironment, dampening the immune response and leading to unchecked cancer growth [2, 3, 4]. This complex interplay between cancer cells and the immune system poses significant challenges to conventional immunotherapy strategies.

2. The biology of Gamma Delta T cell

To overcome challenges faced by the traditional cancer immunotherapy approaches, researchers have been exploring other creative solutions, including a subset of immune cells known as γδ T lymphocytes. These cells offer a promising approach to cancer immunotherapy as they do not rely on the traditional MHC Class I pathway for antigen recognition. γδ T lymphocytes are a unique subset of T cells characterized by their distinct γδ T-cell receptors (TCRs), which distinguish them from the more common αβ T cells. Instead of requiring antigen presentation from MHC Class I molecules, they are activated by conserved stress-induced self-antigens on cancer cells. The C-type lectin Natural Killer Group 2D (NKG2D) receptor on γδ T-cell recognizes these stress-induced ligands, such as MHC Class I-chain (MIC) molecules, MIC-A and MIC-B, along with UL-16 binding proteins ULBP-1, ULBP-2, and ULBP-3 on cancer cells directly and without the need for antigen presentation or priming [5]. Thus, granting γδ T lymphocytes the ability to elicit an immunological response akin to that of the innate immune system. Consequently, γδ T cells can identify and react to a wider variety of cancer cells, including those that have evolved defenses against other immune cell types. Moreover, as a subset of T cells, γδ T cells can also establish memory responses, enabling them to attack cancer cells upon subsequent exposures.

Human γδ T cells are divided into two subsets: Vδ1 and Vδ2 [6, 7]. The Vδ1 phenotype is primarily found in epithelial tissues, such as the skin, gut, and respiratory tract, where they play a critical role in immune surveillance, serving as first responders to cellular stress and transformation. Vδ1 T cells possess the C-type Natural Killer Group 2D (NKG2D) receptor, which recognizes stress-induced self-antigens, including MIC-A and MIC-B. These antigens are typically overexpressed on cancer cells, marking them for attack [8, 9, 10]. Stimulation of the NKG2D receptor, on γδ T cells, upon binding to these stress ligands, on cancer cells, can mediate direct cytotoxicity and lysis of tumor cells. Additionally, this interaction activates the release of anti-tumor cytokines, such as Interferon gamma (IFN-γ) and Tumor Necrosis Factor-α (TNF-α) [11, 12, 13].

Conversely, the second phenotype, Vδ2, is predominantly found in the peripheral blood. This subset typically binds with a second receptor, Vγ9, forming the Vγ9Vδ2 phenotype that circulates throughout the body. These cells can recognize non-peptidic phospho-antigens, such as isopentenyl pyrophosphate (IPP), which is a metabolite of the mevalonate metabolic pathway, crucial for cellular metabolism and proliferation. In normal cells, IPP concentrations are low, and these cells remain undetected by Vγ9Vδ2 cells. However, in cancer cells, the mevalonate metabolic pathway is often upregulated to meet increased energy demands, leading to IPP accumulation and its recognition as a tumor antigen [14]. This metabolic adaptation presents a distinct opportunity for Vγ9Vδ2 T cells, which can identify and eradicate tumor cells via both TCR-dependent and TCR-independent mechanisms. This unique ability makes Vγ9Vδ2 T cells an attractive option for immunotherapy, especially when tumors become resistant to other therapeutic modalities.

Despite the promising capabilities of γδ T cells, one of the current challenges in using them for immunotherapy is their relatively low abundance in the human body. Normally, Vγ9Vδ2 cells constitute about 1–5% of peripheral blood T cells, which can be even lower in cancer patients. To address this pitfall, protocols were developed to expand these cells ex vivo. One successful strategy involves using agonist molecules to stimulate their proliferation. Zoledronate, which is already an FDA-approved bisphosphonate used to prevent bone fractures [15, 16, 17] can be used to expand Vγ9Vδ2 T cells from cancer patients with hepatocellular [18], colorectal [18], prostate [19], lymphoid [20], and breast cancers [21, 22]. Zoledronate’s mechanism of action is to inhibit the enzyme, farnesyl diphosphate synthase (FDPS), which is part of the mevalonate metabolic pathway leading to the accumulation of IPP in cancer cells and subsequent TCR-dependent activation of Vγ9Vδ2 T cells. Similarly, Bromohydrin pyrophosphate (BrHPP) also interferes with the mevalonate metabolic pathway (Figure 1) [23, 24, 25].

In cancer patients, levels of γδ T cells are often low due to exposure to rigorous radiation and chemotherapy regimens, making ex vivo expansion a necessary solution. Studies have also shown that Vγ9Vδ2 T cells can be expanded from healthy donors and transferred to cancer patients with minimal adverse effects [22, 26, 27, 28]. This is largely possible because these cells do not rely on MHC recognition for antigen detection. Consequently, donor-derived allogeneic γδ T cells are unlikely to trigger graft-versus-host disease (GVHD), a major concern in other forms of cell-based therapy.

3. Cancer immunotherapies based on γδ T cells

Research on agonist-expanded Vγ9Vδ2 T cells has been conducted across various cancer types. In colorectal cancer (CRC), Vγ9Vδ2 T cells have been identified as tumor-infiltrating lymphocytes (TILs), and their presence seems to correlate with favorable outcomes. Ex vivo expanded Vγ9Vδ2 T cells exhibit strong lytic activity against colorectal carcinoma cell lines, relying on both the TCR and the NKG2D receptor for costimulatory signaling [18, 29, 30, 31]. Interestingly, expanding Vγ9Vδ2 T cells with BrHPP or Zoledronate generates effector memory cells capable of continuously eliminating CRC cell lines upon re-exposure. Similarly, BrHPP- or Zoledronate-stimulated γδ T cells effectively lyse hepatocellular carcinoma cells while sparing healthy surrounding tissues [18]. Further research indicates that this cytotoxicity occurs through both TCR-dependent and independent mechanisms, involving interactions between the NKG2D receptor and MIC A/B, as well as between the DNAX Accessory Molecule-1 (DNAM-1) receptor and the Nectin-like molecule-5 (Necl-5) on hepatocellular carcinoma cells [32].

While expanded γδ T cells show great promise, they tend to undergo mitogen-induced apoptosis more readily than traditional αβ T lymphocytes. To address this challenge, researchers attempted expanding Vγ9Vδ2 T cells in the presence of Interleukin-12 (IL-12), which selectively protects a subset of cells from apoptosis, leading to the proliferation of these apoptosis-resistant cells. These resistant Vγ9Vδ2 T cells demonstrated effectiveness against prostate cancer cell lines, such as the DU145 and PC-3 cell lines [33, 34]. These cells appear to kill cancer cell lines via TCR-dependent interactions, as well as through interactions between Integrin Beta Chain-2 (DC18) and Intercellular Adhesion Molecule-1 (ICAM-1), facilitated by the perforin/granzyme pathway [34]. Additionally, a modified protocol for ex vivo expansion using pulsed Zoledronate stimulation was developed to mitigate the toxicity produced by inhibiting farnesyl diphosphate synthase using a continuous exposure protocol [35]. Compared to continuous exposure, the pulsed γδ T cells showed increased purity and quantities. Moreover, pulse-expanded Vγ9Vδ2 T cells produced higher levels of perforin and degranulated their granzymes in greater numbers when co-cultured with PC-3 prostate cancer cells, resulting in a 2.5-fold increase in their anti-tumor cytolytic activity [36]. In immunocompromised mice with PC-3 tumor xenotransplants, the pulsed Vγ9Vδ2 T cells reduced tumor size by 50% compared to those receiving continuously expanded Vγ9Vδ2 T cells [37].

Furthermore, the Vδ2 subset of γδ T cells has been found in mammary ductal epithelial organoids, indicating their role in immunosurveillance within these tissues. These cells produce the anti-tumor cytokine IFN-γ while efficiently killing human breast cancer cells that are triple-negative for estrogen and progesterone receptors, as well as HER2/neu [38, 39]. Additional studies have also shown that Vγ9Vδ2 T cells exhibit cytotoxic activity against various breast cancer cell lines both in vitro and in murine models. This anti-tumor activity appears to depend on breast cancer subtype, TCR engagement, and the expression of MIC A/B and ICAM-1 [40, 41, 42]. While these results are promising, it is important to note that the predominant γδ T cell subset, Vδ1, found in TILs of breast cancer, exerts immunosuppressive effects, including the suppression of naïve T cell proliferation, dendritic cell maturation, and the secretion of immunosuppressive cytokines. The pro-tumor effects seem to be mediated by interferon gamma-induced protein 10 (IP-10), secreted by tumor cells. IP-10 recruits Vδ1 T cells to the tumor microenvironment, where they suppress anti-tumor immune responses and promote tumor growth [43].

Since ex vivo expanded Vγ9Vδ2 T cells are well-tolerated, several clinical studies have used highly enriched autologous Vγ9Vδ2 T cells prepared with Zoledronate, which were then re-infused into cancer patients to evaluate safety and potential therapeutic effects. In two early-phase clinical trials for Non-Small Cell Lung Cancer (NSCLC), stable disease was observed in three out of ten patients in one study and six out of fifteen patients in another [44, 45]. In advanced renal cell carcinoma (RCC), the transfer of γδ T cells into eleven patients resulted in prolonged tumor doubling time (DT), leading to one complete remission and stable disease in five patients [46]. Similar outcomes were achieved in metastatic RCC, where six out of ten patients showed stable disease [23]. In a clinical trial involving gastric cancer, intraperitoneal injection of ex vivo expanded Vγ9Vδ2 T cells led to a significant reduction in the volume of malignant ascites due to peritoneal dissemination in two of the seven patients enrolled [47]. In another trial, four patients with advanced refractory hematological malignancies received γδ T cells from haploidentical family donors, resulting in three complete responses [48].

Alternatively, Vγ9Vδ2 T cells can also be expanded in vivo rather than strictly ex vivo. This is typically done through the administration of the same FDA-approved amino-bisphosphonates used in ex vivo expansion, along with a low dose of IL-2. Phase I/II clinical trials demonstrated that this approach is both safe and feasible. Indeed, in vivo stimulation of Vγ9Vδ2 T cells in three breast cancer patients resulted in one case of partial remission and two cases of stable disease. This treatment was administered for over twelve months and correlated with declining levels of Cancer Antigen 15–3 (CA 15–3), a surrogate breast cancer biomarker [49]. Additionally, in vivo expansion with Zoledronate and IL-2 in nine hormone-refractory prostate cancer patients resulted in three instances of partial remission and five instances of stable disease [18]. Finally, in a trial focusing on nine patients with relapsed/refractory Non-Hodgkin’s Lymphoma or multiple myeloma, partial remissions were achieved in three patients [20].

4. Conclusion

Although several clinical trials have demonstrated the safety and feasibility of γδ T cell immunotherapies, the overall response in patients has been variable and inconsistent. There might be several potential contributing factors. For instance, the agonists used in in vivo expansion may have off-target effects, such as systemic toxicity in a subset of patients. Additionally, in several clinical trials, γδ T cell infiltration and activation have been limited, possibly due to the immunosuppressive effects of the tumor microenvironment on γδ T cells [50]. There is also limited knowledge about γδ T cell checkpoint inhibition and their exact mechanisms for suppressing tumor growth. Currently, efforts are invested in developing Chimeric Antigen Receptor (CAR)-engineered γδ T cells. These genetically engineered immune cells, whether autologous or allogeneic, have the potential to be off-the-shelf cell therapy products due to their ability to recognize antigens in an MHC-independent manner. One study successfully transduced γδ T cells with a second-generation CAR targeting GD2, demonstrating precise antitumor cytotoxicity against cancer cells expressing this specific antigen [51]. Despite the challenges and limitations observed in early clinical trials, these CAR-transduced γδ T cells could offer significant advantages as personalized cancer treatments. As the understanding of cancer biology expands and new technologies continue to develop, cancer treatments are becoming increasingly personalized, and γδ T cell therapy may be on the brink of a significant breakthrough.