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Keywords:

  • gamma delta T lymphocytes;
  • immunotherapy;
  • cancer;
  • monoclonal antibodies;
  • haematological oncology

Summary

  1. Top of page
  2. Summary
  3. Multifeatures of γδ T cells
  4. γδ T cells and lymphoid/immune cells: an atypical interaction
  5. Anti-tumour activity of γδ T cells
  6. Immunotherapy with γδ T cells
  7. How to improve the anti-tumour activity of γδ T cells
  8. Concluding remarks
  9. Acknowledgements
  10. Conflict of interest
  11. References

Gamma delta (γδ) Τ cells are non-conventional T lymphocyte effectors that can interact with and eradicate tumour cells. Several data demonstrate that these T cells, which are implicated in the first line of defence against pathogens, have anti-tumour activity against many cancers and suggest that γδ Τ cell-mediated immunotherapy is feasible and might induce objective tumour responses. Due to the importance of γδ Τ lymphocytes in the induction and control of immunity, a complete understanding of their biology is crucial for the development of a potent cancer immunotherapy. This review discusses recent advances in γδ Τ basic research and data from clinical trials on the use of γδ Τ cells in the treatment of different cancers. It analyses how this knowledge might be applied to develop new strategies for the clinical manipulation and the potentiation of γδ Τ lymphocyte activity in cancer immunotherapy.

Gamma delta (γδ) Τ cells are a small subset of human T lymphocytes (1–10% of all peripheral blood T cells) that express the γδ T cell receptors (γδ TCRs; Braza et al, 2010). These cells share effector functions with alpha beta (αβ) T cells as well as with natural killer (NK) cells, particularly the capacity to interact with dendritic cells (DCs; Devilder et al, 2006; D'Ombrain et al, 2007). Peripheral blood γδ T cells express mainly Vγ9Vδ2 TCRs and can be activated in vitro and in vivo in a non-Major Histocompatibility Complex (MHC)-dependent fashion by small non-peptidic phosphorylated metabolites that are referred to as natural phospho-antigens (PAgs) and are produced by many different microorganisms. Besides their role in the innate immune response against pathogens, Vγ9Vδ2 T cells may also have potent anti-tumour activity (Corvaisier et al, 2005; Liu et al, 2005; Viey et al, 2005; Burjanadze et al, 2007; Saitoh et al, 2008; Braza et al, 2011). However, the mechanisms whereby they recognize cancer cells are still poorly known. Vγ9Vδ2 T cells can recognize PAgs (such as isopentenyl pyrophosphate, IPP) that are accumulated in tumour cells (Gober et al, 2003). Moreover, Vγ9Vδ2 TCRs interact with F1-ATPase expressed at the tumour cell surface (Scotet et al, 2005), while γδ T cells recognize stress-induced molecules, such as MHC class I chain-related molecules A and B (MICA and MICB) as well as UL16-binding proteins (ULBP) 1–4 and Retinoic acid early transcript 1 (RAET1; Eagle & Trowsdale, 2007). MICA/B and ULBPs are expressed by different types of epithelial tumour cells (Groh et al, 1999; Pende et al, 2002) and are recognized by γδ T cells through NKG2D receptors in an MHC-unrestricted manner (Groh et al, 1998; Bauer et al, 1999) like NK cells, suggesting that they may exert anti-tumour effects even on target cells with reduced or absent expression of human leucocyte antigens and/or tumour antigens.

Based on recent data, we review here first the functions of γδ T cells and the immune regulatory networks through which their production/activity is finely modulated and then their possible use in cancer immunotherapy, particularly in combination with other compounds in order to improve their expansion and enhance their anti-cancer activity.

Multifeatures of γδ T cells

  1. Top of page
  2. Summary
  3. Multifeatures of γδ T cells
  4. γδ T cells and lymphoid/immune cells: an atypical interaction
  5. Anti-tumour activity of γδ T cells
  6. Immunotherapy with γδ T cells
  7. How to improve the anti-tumour activity of γδ T cells
  8. Concluding remarks
  9. Acknowledgements
  10. Conflict of interest
  11. References

A key role of γδ T cells consists of discriminating between the host and pathogens by reacting rapidly towards non-peptide antigens following infection and thereby activating the innate immune cells and thus facilitating the adaptive immune responses of αβ T cells (Fig 1). Circulating Vδ2+ T cells are involved in the elimination of some microbial pathogens, such as intracellular bacteria (Mycobacterium tuberculosis, Francisella tularensis, Borrelia burgdorferi), parasites (Plasmodium falciparum) and viruses (HIV and Influenza A; Casetti et al, 2008; Spencer et al, 2008; Ali et al, 2009; Henry et al, 2010; Qin et al, 2011; Shi et al, 2011). Many studies have shown that γδ T cells play a role in bridging the innate and adaptive immune responses. They have provided evidence that Vδ2+ cells, like αβ T cells, can contribute to the adaptive immune response and the maintenance of the immune homeostasis and have immunoregulatory functions as well (Born et al, 2006; Girardi, 2006; Konigshofer & Chien, 2006; Moser & Eberl, 2007). Moreover, a recent work has revealed that, surprisingly, human γδ T cells share some properties with professional antigen-presenting cells (APCs), like DCs (Brandes et al, 2009; Himoudi et al, 2012). Indeed, upon activation, γδ T cells efficiently process and display antigens and provide co-stimulatory signals that are sufficient to strongly induce proliferation, differentiation, target cell killing and cytokine production by antigen-experienced and naive CD8+ αβ T cells (Meuter et al, 2010). In addition, Vγ9Vδ2 T cells that had been stimulated with IPP seemed to be more efficient in antigen cross-presentation than DCs derived from monocytes (Brandes et al, 2009; Fig 1). Interestingly, recent works described a novel minor (<1%) subset of human interleukin 17 (IL-17)-producing (IL-17+) Vγ9Vδ2 T cells in the peripheral blood of healthy donors or in mice that play a critical role in the inflammatory responses following bacterial infections and in anti-tumour responses (Lockhart et al, 2006; Sutton et al, 2009; Ribot et al, 2010; Hayes & Laird, 2012). Moreover, Caccamo et al (2011) reported that, in children with bacterial meningitis, IL-17+ Vγ9Vδ2 T cells could account for 60–70% of all the γδ T cells in blood and cerebrospinal fluid and that their concentration decreased after successful antibacterial therapy. IL-17+ Vγ9Vδ2 T cells were generated in vitro using T helper 17 (Th17)-polarizing culture conditions (a cocktail of transforming growth factor-β, IL-1β, IL-6, IL-23) followed by an additional week of expansion in IL-2–rich medium (Caccamo et al, 2011). In such conditions, IL-6 plays a crucial role in the generation of IL-17+ γδ T cells, differently from that described for both human Vγ9Vδ2 and mouse γδ T cells (Ribot et al, 2009). Thus, the work by Caccamo et al (2011) provided new important in vivo data on the physiological importance of IL-17+ Vγ9Vδ2 T cells and an in vitro approach to facilitate their study. It is now important to identify the components of the inflammatory niches in which IL-17+ Vγ9Vδ2 T cells accumulate in vivo.

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Figure 1. Multiple functions of γδ T cells. (1) Following T cell receptor (TCR)-dependent activation, γδ T cells release perforin and granzymes, as an early step of their cytotoxic activity. (2) CD16+ effector γδ T cells bind to the Fc region of monoclonal antibodies that target tumour cells, thereby exercising their antibody-dependent cellular cytotoxicity (ADCC) function. (3) γ interferon (INFγ) and tumour necrosis factor α (TNFα) secretion by γδ T cells indicate their implication in immune defence/response networks. (4) Upon TCR engagement, Fas ligand (Fas-L) and TNF-related apoptosis-inducing ligand (TRAIL) expression is up-regulated in γδ T cells and as, a consequence, the Fas- or TRAIL-receptor (R) sensitive tumour cell killing by γδ T cells is enhanced. (5) Human γδ T cells can act as professional antigen-presenting cells because they can process and display antigens and can also provide co-stimulatory signals necessary for the induction of proliferation, differentiation and target cell killing. MHC, major histocompatibility complex.

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γδ T cells and lymphoid/immune cells: an atypical interaction

  1. Top of page
  2. Summary
  3. Multifeatures of γδ T cells
  4. γδ T cells and lymphoid/immune cells: an atypical interaction
  5. Anti-tumour activity of γδ T cells
  6. Immunotherapy with γδ T cells
  7. How to improve the anti-tumour activity of γδ T cells
  8. Concluding remarks
  9. Acknowledgements
  10. Conflict of interest
  11. References

Interaction of γδ T cells with CD8 αβ T cells and NK cells

CD8 T cells and NK cells are cytotoxic lymphocytes that recognize their targets in an MHC-restricted and -unrestricted manner, respectively, and thereby destroy infected and tumour cells. These cells are key effectors of the innate immune system (Jiang & Chess, 2004; Vivier et al, 2011). In mice and humans, the number of γδ T cells is tightly regulated. Indeed, their homeostasis is controlled not only by γδ T cells, but also by αβ T and NK cells. The most potent inhibitors of γδ T cell proliferation are CD8+ T cells and NK cells, at least in part through their ability to compete for IL-7 and IL-15, thus limiting their availability for γδ T cells (French et al, 2005; Do & Min, 2009; Fig 2). However, CD8+ T cells and NK cells may also positively regulate γδ T cells (Marcu-Malina et al, 2011). Indeed, NK cells that have been activated by bacterial antigens, such as Mycobacterium tuberculosis, can increase γδ T cell proliferation both by CD54-mediated cell–cell contact in the immune synapse and by production of cytokines, tumour necrosis factor α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-12 (Zhang et al, 2006; Fig 2).

image

Figure 2. Interactions between γδ T cells and lymphoid/immune cells. γδ T cells can interact with many cell types and the consequences of such interactions are not always beneficial for them. T regulatory (Treg) FoxP3+CD4+CD25+ and mesenchymal cells (MSC) have a negative impact on the proliferation of γδ T cells and their anti-tumour activity. The bidirectional activating interaction between dendritic cells (DC) and γδ T cells has a positive effect on DC maturation and γδ T cell proliferation. γδ T and CD8 αβ T/Natural Killer (NK) cells collaborate in the immune response against infections and tumours, whereas they compete in the regulation of their homeostasis and for interleukin (IL)-15. Double arrowheads show a reciprocal interaction, grey arrows indicate a positive impact on γδ T cells and pink arrows a negative impact.

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Reciprocal activating interaction between dendritic and γδ T cells

DCs are APCs that act as a bridge between innate and adaptive immunity (Banchereau & Steinman, 1998). Co-culture of immature DCs (iDCs) with peripheral blood Vδ2 T cells that were activated with IPP or with the aminobiphosphonate (ABP) pamidronate (PAM) leads to a significant increase in the expression of CD86 and MHC class I molecules and to the acquisition of functional features that are typical of activated DCs. DC activation induced by IPP- or PAM-stimulated γδ T cells is mostly mediated by TNF-α and interferon γ (IFN-γ) secreted by Vδ2 T cells (Fig 2). Reciprocally, the expression of lymphocyte activation markers (CD25 and CD69) and the production of TNF-α and IFN-γ by PAM-stimulated Vδ2 T cells are strongly up-regulated when they are co-cultured with iDCs. Similarly, co-culture with monocyte-derived DCs stimulates proliferation of isolated γδ T cells and IFN-γ secretion, but does not exert any effect on their cytotoxic activity (Brandes et al, 2005; von Lilienfeld-Toal et al, 2005). These findings indicate that iDCs, and to a lesser extent mature DCs, potentiate the production of Th1 and Th2 cytokines, but not the cytolytic responses of established Vγ9Vδ2 T cell clones and ex vivo memory Vγ9Vδ2 from peripheral blood stimulated by synthetic agonists. This suggests that, at optimal antigen doses, Vγ9Vδ2 T cells could promote efficient iDC licensing without killing. Moreover, these results show the existence of a bidirectional activating interaction between iDCs and γδ T lymphocytes and suggest that this early cross-talk might have a potential adjuvant role in the therapeutic activity of ABP drugs due to the ability of iDCs to selectively potentiate the cytokine response of memory Vγ9Vδ2 T cells (Conti et al, 2005; Devilder et al, 2006; Fig 2).

A negative impact of mesenchymal cells on γδ T cells

Mesenchymal stem cells (MSCs) are multipotent stromal cells that inhibit allorecognition, interfere with the function of DCs and T cells and generate a local immunosuppressive microenvironment by secreting cytokines (Ryan et al, 2007). MSC can suppress γδ T cell proliferation, cytokine production and cytolytic responses in vitro. This inhibition is mediated through the cyclooxygenase (COX-2)-dependent production of prostaglandin E2 (PGE2) by MSC and the PGE2 receptors EP2 and EP4 that are expressed by Vγ9Vδ2 T cells. COX-2 expression and PGE2 production by MSC are not constitutive, but are induced upon secretion of IFN-γ and TNF-α by activated Vγ9Vδ2 T cells (Fig 2). This regulatory cross-talk between MSC and Vγ9Vδ2 T cells through PGE2 could be important for the anti-tumour and anti-microbial activities of γδ T cells (Martinet et al, 2009). Indeed, cell-surface expression of EP2 and EP4 by γδ T cells enables soluble PGE2 to transduce a strong cyclic adenosine monophosphate (cAMP)-dependent activation of protein kinase A (PKA) type I to inhibit early signalling by these cytotoxic cells and block their physiological functions. Many tumours, particularly solid ones, contain MSC that secrete high amounts of PGE2 and might therefore escape Vγ9Vδ2 T cell cytotoxicity (Beyer & Schultze, 2006; Hall et al, 2007; Karnoub et al, 2007; Martinet et al, 2010; Iovino et al, 2011). However, the effect of MSC on the Vδ2+ T cell anti-tumour potential is variable with a tendency to abrogate their cytotoxic activity against several tumour cell lines (Martinet et al, 2009; Prigione et al, 2009).

Inhibition of phospho-antigen-mediated γδ T cell proliferation by CD4+ CD25+ FoxP3+ regulatory T cells

Regulatory T cells (Tregs) are a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens and control autoimmune diseases. This is an important ‘self-check’ built in the immune system to prevent excessive reactions (Hori et al, 2003). Tumour growth promotes the expansion of CD4+ CD25+ FoxP3+ Tregs, which can suppress various immune response pathways and might therefore contribute to the regulation of tumour immunity. Increasing evidence suggests that elevated percentages of Tregs are present in both peripheral blood and the tumour microenvironment of solid cancers and haematological malignancies (Gao et al, 2007). Recent work showed that in vitro co-culture of human Tregs and peripheral blood mononuclear cells from patients with cancer strongly inhibited PAg-induced proliferation of γδ T cells, whereas depletion of Tregs restored PAg-induced γδ T cell proliferation. Tregs do not suppress other effector functions of γδ T cells (such as cytokine production or cytotoxicity) and their inhibitory effect on γδ T cell proliferation is mediated via secretion of a soluble, non-protein factor (Kunzmann et al, 2009; Fig 2). These results might also explain the frequently observed γδ T cell proliferative anergy in patients with cancer.

Anti-tumour activity of γδ T cells

  1. Top of page
  2. Summary
  3. Multifeatures of γδ T cells
  4. γδ T cells and lymphoid/immune cells: an atypical interaction
  5. Anti-tumour activity of γδ T cells
  6. Immunotherapy with γδ T cells
  7. How to improve the anti-tumour activity of γδ T cells
  8. Concluding remarks
  9. Acknowledgements
  10. Conflict of interest
  11. References

γδ T cells may have a unique role in the immune surveillance against malignancies and also an advantage over αβ T cells because they can directly recognize molecules that are expressed on cancer cells without need of antigen processing and presentation (Chien & Konigshofer, 2007; Thedrez et al, 2007). Moreover, γδ T cells can migrate as infiltrating lymphocytes into solid tumours (Viey et al, 2008) and can recognize and eliminate cultured malignant cells (primary cells or cell lines) from myeloma (Burjanadze et al, 2007; Saitoh et al, 2008), non-Hodgkin lymphoma (NHL; Braza et al, 2011), prostate cancer (Liu et al, 2005, 2008), renal cell carcinoma (Viey et al, 2005; Kobayashi et al, 2007; Bennouna et al, 2008), colon carcinoma (Corvaisier et al, 2005) and squamous cell carcinoma (Alexander et al, 2008). A recent work reported that IL-17+ γδ T cells play a key role in the anti-tumour activity of Bacillus Calmette-Guérin (BCG) in bladder cancer by inducing the recruitment of neutrophils to the tumour (Takeuchi et al, 2011). In addition, IL-17+ γδ T cells might be involved in the chemotherapy-induced anti-cancer immune response. Indeed, chemotherapy in mice xenografted with tumour cells triggers a rapid and prominent invasion of IL-17+ γδ (Vγ4+ and Vγ6+) T lymphocytes that precedes the accumulation of cytotoxic T lymphocytes within the tumour. Adoptive transfer of γδ T cells restored the efficacy of chemotherapy in IL-17A−/− mice xenografted with the same tumour cells, whereas the anti-cancer effect of infused γδ T cells was lost when they were isolated from IL-17A−/− donors (Ma et al, 2011).

Moreover, activation of γδ TCRs promotes γδ T cell cytotoxicity through increased secretion of perforin/granzymes, IFN-γ and TNF-α (key effector molecules in the immune response against cancer) and up-regulates expression of Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) by γδ T cells that can thus eliminate Fas+ and TRAIL-R+ tumour cells (Kondo et al, 2008; Kang et al, 2009; D'Asaro et al, 2010; Fig 1). In addition, γδ T cells express CD16, which is a receptor for the Fc region of IgGs (Fcγ receptors) and promotes antibody-dependent cellular cytotoxicity (ADCC) in the presence of anti-tumour cell monoclonal antibodies (Tokuyama et al, 2008; Gertner-Dardenne et al, 2009; Braza et al, 2011; Fig 1). Finally, γδ T lymphocytes can induce also a robust NK cell-mediated anti-tumour cytotoxicity through engagement of the CD137 pathway (Maniar et al, 2010).

Immunotherapy with γδ T cells

  1. Top of page
  2. Summary
  3. Multifeatures of γδ T cells
  4. γδ T cells and lymphoid/immune cells: an atypical interaction
  5. Anti-tumour activity of γδ T cells
  6. Immunotherapy with γδ T cells
  7. How to improve the anti-tumour activity of γδ T cells
  8. Concluding remarks
  9. Acknowledgements
  10. Conflict of interest
  11. References

Several studies have reported a significant decrease in the absolute number of circulating Vγ9Vδ2 T cells and in their ability to produce TNFα or IFNγ in patients with cancer compared to healthy donors (Zheng et al, 2002; Argentati et al, 2003). These quantitative and qualitative defects in γδ T cells might contribute to reduce the immune surveillance and protection against tumours. However, γδ T cell proliferation and cytotoxicity can be restored in patients with solid tumours by treatment with zoledronate (Noguchi et al, 2011). All these data highlight that peripheral blood γδ T cells have an important role in the immune control of cancer and explain a growing interest for the clinical use of these cells (Table 1).

Table 1. Anti-tumour immunotherapeutic approaches using Vγ9Vδ2 T cells
Immunotherapeutic approachModelPathologyReferences
  1. IL-2, interleukin 2; DC, dendritic cell; TCRg, T cell receptor gamma.

  2. Summary of the immunotherapeutic trials carried in patients with different types of cancer and the methods used to expand Vγ9Vδ2 T cells.

In vivo administration of zoledronate and IL-2HumanMetastatic breast cancerMeraviglia et al (2010)
In vivo administration of zoledronate and IL-2HumanMetastatic prostate cancerDieli et al (2007)
Adoptive transfer of Vγ9Vδ2 T cells co-cultured with autologous DCs and amplified by using zoledronateronate and IL-2HumanHepatocellular carcinoma and colorectal carcinoma with hepatic metastasesCabillic et al (2010)
Adoptive transfer of Vγ9Vδ2 T cells amplified by using zoledronate and IL-2HumanVarious solid tumoursNoguchi et al (2011)
Adoptive transfer of Vγ9Vδ2 T cells amplified by using anti-TCRg antibodiesHumanMetastatic renal carcinomaSalot et al (2007)
Adoptive transfer of Vγ9Vδ2 T cells amplified by using BrHPP and IL-2HumanAdvanced renal carcinomaKobayashi et al (2007)
Adoptive transfer of Vγ9Vδ2 T cells amplified by using 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP) and zoledronate plus teceleukinHumanAdvanced renal carcinomaKobayashi et al (2011)

Two strategies could be developed to exploit the anti-tumour activity of γδ T cells: in vivo administration of compounds that activate γδ T cells or adoptive transfer of ex vivo activated and amplified γδ T cells.

As mentioned earlier, γδ T cell expansion can be efficiently stimulated by non-peptide PAgs and IL-2 in vitro. PAgs, such IPPs, are normally produced through the cholesterol pathway by microbial pathogens or by abnormal metabolic routes, such as the mevalonate and non-mevalonate 1-deoxy-d-xylulose-5-phosphate (DOXP) pathways. Synthetic stimulators of γδ T cells have also been described. They include phosphorylated bromohydrin (BrHPP, Phosphostim™, Innate pharma, Marseille, France) that mimics the biological properties of natural PAgs (Salot et al, 2007; Bennouna et al, 2008) and selectively stimulates γδ T lymphocytes. Other compounds, like ABPs (such as alendronate, pamidronate, or zoledronate) and alkylamines, indirectly activate γδ T lymphocytes as a consequence of the inhibition of farnesyl diphosphate synthase (a key enzyme of the mevalonate pathway) that leads to intracellular accumulation of endogenous PAgs (Thompson et al, 2006; Simoni et al, 2008; Li et al, 2009; Roelofs et al, 2009; Braza et al, 2010, 2011; Cabillic et al, 2010).

γδ T cell manipulation by administration of synthetic phospho-antigens

In vivo injection of BrHPP in Macaque fascicularis leads to a dose-dependent activation of Vγ9Vδ2 T cells that secrete an elevate quantity of Th1 cytokines. In addition, the combined administration of BrHPP and of weak doses of IL-2 in the same primate model induces amplification of memory effector Vγ9Vδ2 T cells in the peripheral blood. This proliferative response is transient, because the Vγ9Vδ2 T lymphocyte level return to baseline after 2 weeks. Moreover, repeated injections of BrHPP and IL-2 induce a progressively weaker Vγ9Vδ2 T cell expansion, suggesting an exhaustion of the response (Sicard et al, 2005).

γδ T cell manipulation by aminobisphosphonate administration

Injection of ABP (zoledronate, pamidronate) together with IL-2 in patients with metastatic cancers of different origins can increase the number of circulating γδ T cells, potentiate their anti-tumour activity and may lead to tumour reduction (Kunzmann et al, 2000; Wilhelm et al, 2003).

Specifically, zoledronate and low-dose IL-2 were administered to 10 patients with advanced metastatic breast cancer. The treatment was well-tolerated and promoted sustained maturation of effector γδ T cells in all patients (Meraviglia et al, 2010). Similarly, the number of circulating γδ T cells increased in patients with metastatic prostate cancer following administration of zoledronic acid alone or in combination with low doses of IL-2 (Dieli et al, 2007). Moreover, 25% of patients showed a significant shift of γδ T cells towards an activated effector memory phenotype with production of perforin and INF-γ and elevated TRAIL serum levels. However, in the majority of patients treated with zoledronate alone, the increased γδ T cell count and TRAIL level in serum were not maintained over time and they showed a progressive clinical deterioration (Galluzzo et al, 2007; Castella et al, 2011; Nussbaumer et al, 2011). Thus, γδ T cell number and TRAIL levels could be prognostic biomarkers of the efficacy of treatments against metastatic carcinoma.

Zoledronate can also exert immune modulatory activities that allow DCs from patients with multiple myeloma to effectively regulate the concurrent activation of γδ T cells and of MHC-restricted CD8+ αβ anti-tumour effector T cells (Castella et al, 2011). Moreover, by activating γδ T cells, zoledronate can prompt a DC-like cell-dependent activation of human NK cells (Nussbaumer et al, 2011). Altogether, these data suggest that ABPs and low-dose IL-2 can be used to stimulate the innate and specific immune response against cancer cells.

Adoptive transfer of Vγ9Vδ2 T cells

Adoptive transfer of γδ T cells following ex vivo expansion by using IL-2 and PAgs or ABPs could represent an alternative to their in vivo activation. Efficient Vγ9Vδ2 T cell expansion can be obtained by co-culturing peripheral blood mononucleated cells with autologous DCs that have been pre-treated with zoledronate (Cabillic et al, 2010; Noguchi et al, 2011). The infusion of expanded γδ T cells in patients with cancer might be preceded by injections of ABPs. Indeed, in mouse models of breast cancer, zoledronate can promote the chemotaxis of infused Vγ9Vδ2 T cells to the tumour. By blocking the mevalonate pathway, zoledronate leads to intracellular accumulation of IPP/triphosphoric acid I-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester (ApppI) mevalonate metabolites in tumour cells that are then secreted and possibly recognized by Vγ9Vδ2 T cells as PAgs. Without infusion of expanded Vγ9Vδ2 T cells, zoledronate does not inhibit tumour growth in these mouse models of breast cancer. These findings suggest that cancers producing elevated quantities of IPP/ApppI after zoledronate treatment are most likely to benefit from Vγ9Vδ2 T cell-mediated immunotherapy (Galluzzo et al, 2007; Benzaid et al, 2011). The large scale expansion of circulating γδ T cells from patients with cancer and their adoptive transfer have started to be assessed in clinical studies. For instance, Vγ9Vδ2 T cells from metastatic renal cell carcinoma were expanded in vitro using BrHPP and IL-2 and several billions of Vγ9Vδ2 T cells were injected back in to patients without adverse effects (Salot et al, 2007). Similarly, the results of another pilot study in patients with advanced renal cell carcinoma show that infusion of Vγ9Vδ2 T cells that were expanded with 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP, a synthetic pyrophosphomonoester antigen) and teceleukin (recombinant human IL-2) was well tolerated and induced anti-tumour effects in 50% of patients (Kobayashi et al, 2007). A phase I/II clinical trial in patients with advanced renal cell carcinoma assessed the outcomes of the administration of γδ T cells that were expanded with 2M3B1PP together with zoledronic acid and teceleukin. This study reported a good tolerance and objective clinical responses in some of the patients (Kobayashi et al, 2011).

Concomitant use of Vγ9Vδ2 T and dendritic cell-based therapies

Vaccination strategies using DCs (Cabillic et al, 2010) could be combined with Vγ9Vδ2 T lymphocyte-based therapy. One of the critical aspects for the establishment of an effective immune response is DC maturation. Vγ9Vδ2 T lymphocytes can induce DC maturation by producing cytokines and via the CD1 molecule (Devilder et al, 2006). In addition, a recent study has revealed unexpected properties (processing of exogenous proteins and presentation of peptide-MHC I complexes) of human γδ T cells as APCs in the induction of CD8 αβ T effector cells (Brandes et al, 2009).

Moreover, in macaques, a prime-boost with H-1 hybrid subunit candidate vaccines for tuberculosis combined with a synthetic PAg induces two waves of immune responses, first by γδ T cells and the second one by αβ T lymphocytes (Cendron et al, 2007). As human γδ T cells cross-react with mycobacteria and with cancer cells, the vaccine-PAg association may efficiently promote memory cells in both classes of lymphocytes, suggesting that this vaccine design could be used to obtain better vaccines not only against tuberculosis, but also against tumours. These data, which were obtained while investigating the possible adjuvant role of γδ T cells in an anti-tuberculosis vaccine, also bring support to the possible APC role of γδ T lymphocytes (Cendron et al, 2007; Brandes et al, 2009). Hence, Vγ9Vδ2 T lymphocytes could have an adjuvant effect on cellular immune responses, justifying their further exploration in immunotherapy.

How to improve the anti-tumour activity of γδ T cells

  1. Top of page
  2. Summary
  3. Multifeatures of γδ T cells
  4. γδ T cells and lymphoid/immune cells: an atypical interaction
  5. Anti-tumour activity of γδ T cells
  6. Immunotherapy with γδ T cells
  7. How to improve the anti-tumour activity of γδ T cells
  8. Concluding remarks
  9. Acknowledgements
  10. Conflict of interest
  11. References

It is crucial to understand how the innate anti-tumour properties of human γδ T cells, particularly as a complement to the more classical adaptive immune responses, could be better exploited for the treatment of a variety of malignancies. The amplification and activation of γδ T cells can be improved by using optimized activators and growth factors and by increasing NKG2D-mediated lysis. For instance, as IL-21 mediates the potentiation of the anti-tumour cytolytic and pro-inflammatory responses of human Vγ9Vδ2 T cells in adoptive immunotherapy (Thedrez et al, 2009), it could be combined with IL-2 to enhance γδ T cell-mediated anti-tumour responses. Activating anti-NKG2D antibodies might help co-stimulating γδ T cell expansion in vitro to obtain the large cell numbers required for adoptive cell transfer. Moreover, anti-CD2 antibodies and/or anti-apoptotic cytokines, like IL-15, could be used to prevent activating-induced cell death of γδ T cells during in vitro culture. In addition, as Tregs negatively regulate γδ T cell proliferation (Fig 2), Treg inhibition could be combined with subsequent γδ T cell expansion/activation to enhance γδ T cell-mediated immunotherapies. Indeed, recent data have shown that the ratio of Tregs to effector T cells predicts more accurately the inhibitory effects of Tregs on a target cell population than the number of Tregs in peripheral blood or tumour specimens (especially for rare effector populations, such as γδ T cells). The Tregs/γδ T cells ratio might also represent a promising independent prognostic factor for tumour recurrence and survival as this ratio, but not the absolute or relative number of circulating Tregs, was negatively correlated with the proliferation rate of PAg-induced γδ T cells in patients with cancer (Bui et al, 2006). Finally, the ADCC of γδ T cells could be enhanced by treatment strategies in which manipulation/infusion of γδ T cells is associated with therapeutic monoclonal antibodies (mAbs; Tokuyama et al, 2008; Gertner-Dardenne et al, 2009). For instance, anti-CD20 mAbs (such as rituximab, ofatumumab and GA101) increase the cytotoxic activity of expanded γδ T lymphocytes against allogenic or autologous follicular lymphoma cells. The strongest effect was observed with GA101, a type II anti-CD20 mAb with a glyco-engineered Fc portion that exhibits enhanced binding to the Fc receptor (Braza et al, 2011). Hence, by improving the expansion, cytotoxicity and survival of specific γδ T lymphocytes, their anti-tumour activity could be potentiated in order to develop an efficient tool for cancer immunotherapy.

Concluding remarks

  1. Top of page
  2. Summary
  3. Multifeatures of γδ T cells
  4. γδ T cells and lymphoid/immune cells: an atypical interaction
  5. Anti-tumour activity of γδ T cells
  6. Immunotherapy with γδ T cells
  7. How to improve the anti-tumour activity of γδ T cells
  8. Concluding remarks
  9. Acknowledgements
  10. Conflict of interest
  11. References

In conclusion, γδ T cells appear to be a promising therapeutic tool for the treatment of patients with cancer as they combine the properties of both adaptive and innate immunities and, differently from αβ T cells, they can recognize unusual and non-specific compounds. It might be also possible to combine in vivo activation of γδ T cells and adoptive transfer of ex vivo expanded γδ T cells, because several drugs that activate γδ T cells (PAgs or ABPs) are already in clinical trials. Moreover, a better knowledge of the different signalling molecules involved in γδ T cell expansion/activation and functions, such as the Toll-like receptors and NKG2D receptors, may help to improve and optimize the use of γδ T cells in anti-cancer therapies.

Recent advances concerning the knowledge of γδ T cell multiple functions in the innate immune response and also in the DC- and antigen-presentation system and their capacity to acquire antigen-presenting features have increased the interest of using γδ T cells for clinical treatment. However, it must be kept in mind that γδ T cells are part of the multicellular immune system, which is tightly regulated by numerous pathways and cell types, including regulatory cells. For instance, sustained stimulation of γδ T cells by non-peptide antigens might lead to their anergy and to depletion of an important compartment of the innate system. Also, questions remain about the poorly known nature of γδ T cell antigens, how they are recognized and their therapeutic relevance. Similarly, the mechanism underlying the cross talk between DC and γδ T cells is not clear. For instance, what are the receptors and ligands involved in this interaction and are there suitable in vivo models to study this cross talk? Moreover, the degree of developmental pre-programming versus functional plasticity of γδ T cells requires further investigation in mice and humans. Future studies should also address the possible advantage of combining γδ T cell-based therapies with other approaches and identify the right therapeutic window during which γδ T cell-based therapies can be more effective. Finally, beyond the basic immunology issues to be resolved, future research should aim at finding clinical-grade tools to manipulate the recently identified IL-17+ γδ T cell subtype. An attractive prospect is to interfere with their de novo differentiation.

Overcoming many of the mentioned obstacles and answering the unresolved questions should help to optimize the design of next-generation cancer therapies that take advantage of γδ T cells.

References

  1. Top of page
  2. Summary
  3. Multifeatures of γδ T cells
  4. γδ T cells and lymphoid/immune cells: an atypical interaction
  5. Anti-tumour activity of γδ T cells
  6. Immunotherapy with γδ T cells
  7. How to improve the anti-tumour activity of γδ T cells
  8. Concluding remarks
  9. Acknowledgements
  10. Conflict of interest
  11. References
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