γδ T Cells as Immuno-Oncology Treatments in the Era of Precision Medicine

Hilal Arnouk1*and Bisini Panicker2

1College of Graduate Studies, Chicago College of Osteopathic Medicine, College of Dental Medicine-Illinois, Chicago College of Optometry, Midwestern University, Downers Grove, Illinois, United States

2Chicago College of Osteopathic Medicine, Midwestern University, Downers Grove, Illinois, United States

*Corresponding author: Hilal Arnouk, College of Graduate Studies, Chicago College of Osteopathic Medicine, College of Dental Medicine-Illinois, Chicago College of Optometry, Midwestern University, Downers Grove, Illinois, United States. E-mail: harnouk@midwestern.edu

Citation: Hilal Arnoukand Bisini Panicker (2020) γδ T Cells as Immuno-Oncology Treatments in the Era of Precision Medicine. J Can Res Adv Ther 1(2): 24-29.

Received Date: 9 August, 2020; Accepted Date: 13 August, 2020; Published Date: 14 August, 2020

γδ T Cell Biology:

Harnessing the potential of the immune system to treat cancers has been the goal of many scientific investigations in the last few decades. Recent advances in cancer biology and immunology have allowed for cancer immunotherapy to become a reality. The premise of cancer immunotherapy is to stimulate the patient’s own immune system to attack and reject the malignant cells, sparing normal surrounding tissues. Strategies that rely on the Dendritic Cells (DC) to Cytotoxic T Lymphocytes (CTL) axis to mount a specific immune response against different types of cancers have met variable success so far due largely to tumor evasion of recognition by the majority of T lymphocytes, known as αβ T lymphocytes, by several mechanisms including reduced expression levels of MHC Class I molecules [1] and secretion of immunosuppressive cytokines, such as Interleukin-10 (IL-10) and Transforming Growth Factor-β (TGF-β) [2, 3, 4] On the other hand, T lymphocytes that express the γδ TCR (γδ T cells) do not require antigen presentation by target cells. Instead, they recognize non-peptidic phospho-antigens (pyro-phospho-antigens), such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) and isopentenyl pyrophosphate(IPP), a metabolite of the mevalonate metabolic pathway, whichcan accumulate in tumor cells with increased metabolic activityof the mevalonate metabolic pathway (Figure 1). These phospho-antigens are usually recognized in complex with Butyrophilin 3A1(BTN3A1), also known as CD277 [5, 6].

Additionally, the C-type lectin Natural Killer Group 2D (NKG2D) on γδ T cells recognizes stress-induced self-antigens widely expressed on cancer cells, such as the MHC Class I-like stress-associated molecules MIC-A and MIC-B or the UL-16 binding proteins ULBP-1, 2, and 3 (Figure 1) [7, 8, 9] This recognition can mediate direct cytotoxicity and lysis of tumor cells without prior antigen exposure or priming [9] and secretion of anti-tumor cytokines, such as Interferon gamma (IFN-γ) and Tumor Necrosis Factor-α (TNF-α). Thus, giving γδ T cells the advantage of a rapid and uniform response similar to innate immune responses.

Human γδ T cells are divided into two major subsets, Vδ1 or Vδ2, according to their TCR-δ chain [10, 11]. Vδ1 is the dominant phenotype in mucosal areas [12, 13, 14], while Vδ2 is typically paired with Vγ9, making Vγ9Vδ2 the dominant phenotype of γδ T cells in the peripheral blood. Although the variable regions of the γ and δ chains exhibit diversity, circulating Vγ9Vδ2 repertoire has remarkably reduced diversity because of chronic positive selection due to exposure to phospho-antigens produced by host cells or resident microbes [15, 16].

Although γδ T cells normally constitute only 1-5% of peripheral blood T cells, they can be ex vivo-expanded and activated when cultured with IL-2 and Zoledronate (or Zoledronic acid) [17, 18, 19]. Zoledronate is an FDA-approved bisphosphonate mainly used to prevent bone fractures in patients with metastatic cancer or postmenopausal osteoporosis. Bisphosphonates interfere with the mevalonate metabolic pathway by inhibiting the farnesyl diphosphate synthase (FDPS), which leads to accumulation of the isopentenyl pyrophosphate (IPP) in cancer cells and subsequent TCR-dependent activation of γδ T cells, specifically the subpopulation carrying the Vδ2 receptor [20, 21, 22].

This method has been used to expand γδ T cells from cancer patients with hepatocellular [23], colorectal [23], prostate [24], lymphoid [25] and breast cancers [26]. However, γδ T cells from cancer patients are typically diminished in their numbers and have decreased proliferation in response to mitogens [27, 28]. Alternatively, we have demonstrated that γδ T cells can be expanded from healthy donors [29, 30] as these allogeneic donor effector cells are safe to transfer to cancer patients since γδ T cells do not recognize foreign MHC I antigens and are not likely to trigger graft versus host disease (GVHD). Collectively, these biological qualities of γδ T cells make them a promising option for cancer immunotherapy.

Cancer Immunotherapies Based on γδ T Cells:

Vγ9Vδ2 T cells were identified as tumor-infiltrating T lymphocytes (TILs) in a majority of colorectal cancer patients [31] where they seemed to correlate with favorable prognosis [32, 33]. Subsequent studies have shown that the ex vivo expansion of Vγ9Vδ2 T cells using Bromohydrin pyrophosphate (BrHPP) or Zoledronate yields effector memory cells with the phenotype CD45RA- CD45RO highCD27-. These expanded cells exhibited strong lytic activity toward colorectal carcinoma cell lines. The cytotoxicity was mainly dependent on the TCR receptor and on the NKG2D receptor as a costimulatory signal [23]. Similarly, BrHPP- or Zoledronate-stimulated γδ T cells were able to lyse tumor cells freshly isolated from hepatocellular carcinoma but not normal counterpart tissues [23]. Studies have shown that cytotoxicity was dependent on interactions between the DNAX Accessory Molecule-1 (DNAM-1) on γδ T cells and the Nectin-like molecule-5 (Necl-5) expressed on hepatocellular carcinoma cells, in addition to the recognition of MICA/B or ULBP 1-3 by the NKG2D receptor [34].

Ex vivo expansion of Vγ9Vδ2 T cells in the presence of Interleukin-12 resulted in large-scale expansion of human γδ T lymphocytes that are resistant to mitogen-induced apoptosis [35]. These apoptosis-resistant γδ T cells have proven effective against prostate cancer cells, such as DU145 and PC-3 cell lines [36]. Prostate cancer cell killing seemed to be dependent on γδ TCR and interactions between Integrin Beta Chain-2 (CD18) and Intercellular Adhesion Molecule-1 (ICAM-1) and is mediated by the perforin/granzyme pathway [36]. A modified protocol for ex vivoexpansion of Vγ9Vδ2 T cells used pulse Zoledronate stimulation to avoid the toxic effects of farnesyl diphosphate synthase (FDPS) inhibition by continuous exposure to Zoledronate [37]. This has enhanced γδ T cell purity and numbers compared with continuous Zoledronate stimulation. Moreove r, the expanded Vγ9Vδ2 T cells produced higher levels of perforin and degranulated in large numbers when exposed to PC-3 prostate cancer cells as evident by the increased expression of the CD107a, which is a lysosomal-associated membrane protein (LAMP-1) that is mobilized to the surface when cytotoxic T and NK cells degranulate for killing [38]. This resulted in a 2.5-fold increase in their anti-tumor cytolytic activity. Adoptive transfer of these effector cells halted the growth of PC-3 tumor cells xenotransplanted into the highly immunodeficient NSG mice, reducing tumor volume by 50% compared with those expanded using continuous Zoledronate stimulation [39].

Vδ2+ γδ T cells were identified in non-cancerous mammary ductal epithelial organoids, perhaps indicating their role in immunosurveillance and subsequent elimination of neoplastically-transformed breast ductal epithelial cells [40], given their ability to detect the changes associated with malignant transformation and stress-induced molecules, such as with MIC-A/B. Moreover, Vδ2+ γδ T cells derived from breast ductal organoids produced the anti-tumor cytokine, IFN-γ, while they efficiently killed the bisphosphonate-pulsed human breast carcinoma cell line, MDA-MB-468, which is triple negative for estrogen receptor, progesterone receptor, and HER2/neu [41]. Similarly, studies involving the peripherally-derived γδ T lymphocytes demonstrated cytotoxic activity against a variety of breast cancer cell lines both in vitro and in murine models. This anti-tumor activity seemed to be dependent on breast cancer subtype, TCR engagement, and MICA/B and ICAM1 expression levels [42, 43, 44]. However, it is worth mentioning that Vδ1+ T lymphocytes are the dominant subtype found in tumor-infiltrating T lymphocytes (TILs) of breast cancers, where they exert immune-suppressing effects, such as the suppression of naïve T cell proliferation and DC maturation and the secretion of immunosuppressant cytokines. This pro-tumorigenic activity seems to be mediated by interferon gamma-induced protein 10 (IP-10) secreted by breast tumor cells. IP-10 recruits Vδ1+ T cells to the tumor microenvironment where they promote tumor growth and spread [45].

Since Vγ9Vδ2 T cells can be ex vivo-expanded and are usually well-tolerated by recipients, highly enriched clinical-grade autologous Vγ9Vδ2 T cells were prepared using Zoledronate and re-infused into cancer patients in a number of adoptive transfer clinical studies to evaluate the safety and potential therapeutic effects. For instance, in two early phase clinical trials of Non-Small Cell Lung Cancer (NSCLC), stable disease was achieved in three out of ten patients in one study and six out of fifteen patients in the other NSCLC study [46, 47]. In advanced renal cell carcinoma (RCC), adoptive transfer of γδ T cells into eleven patients resulted in a prolonged tumor doubling time (DT) as measured by computed tomography (CT), which subsequently led to one complete remission and stable disease in five patients based on Evaluation Criteria in Solid Tumors (RECIST) [48]. Similar outcomes were achieved for metastatic RCC, where six out of ten patients showed stable disease [49]. Separately, intraperitoneal injection of ex vivo-expanded Vγ9Vδ2 T cells yielded a significant reduction in volume of malignant ascites, caused by peritoneal dissemination of gastric cancer, in two of the seven patients enrolled in this study [50]. In another pilot study, four patients with advanced refractory hematological malignancies (T-NHL, AML, secondary plasma cell leukemia, and multiple myeloma), who were not eligible for allogeneic transplantation, received γδ T cells from half-matched (haploidentical) family donors, resulting in three complete responses [51].

Alternatively, Vγ9Vδ2 T cells can be expanded in vivo by administration of FDA-approved amino-bisphosphonates and low dose IL-2 into the patients. Phase I/II of clinical trials demonstrated the safety and feasibility of this approach. In vivo stimulation of γδ T cells in breast cancer patients showed one case of partial remission and two cases of stable disease amongst three patients that sustained robust Vγ9Vδ2 T cell numbers over twelve months, which also correlated with declining levels of Cancer Antigen 15-3 (CA 15-3), a surrogate breast cancer biomarker [52]. γδ T cells in vivo stimulation with Zoledronate and IL-2 in nine hormone-refractory prostate cancer patients resulted in three instances of partial remission and five of stable disease [23]. Finally, in a study focused on lymphoid malignancies, which included a cohort of nine patients with relapsed/refractory Non-Hodgkin’s Lymphoma (NHL) or multiple myeloma (MM), partial remissions were achieved in three patients [53].

Although clinical studies have demonstrated the safety and some efficacy of γδ T cells immunotherapy, the overall response in patients has been less than ideal, which can possibly be explained by lymphocyte exhaustion or activation-induced cell death (AICD) upon repeated stimulation. As we usher in the age of precision medicine, efforts are being invested in developing Chimeric Antigen Receptor (CAR)-engineered γδ T Cells, as autologous or allogeneic, off-the-shelf cell therapy products (Figure 2) since these innate immune cells recognize their antigens in an MHC-independent manner. As a proof of concept, γδ T cells were successfully transduced with a second-generation CAR targeting GD2 and containing CD3-ζ and CD28 signaling domains and displayed GD2-specific anti-tumor cytotoxicity [54]. Combined with their capacity for migration to the tumor microenvironment and for uptake of tumor antigens and cross presentation, these CAR-transduced γδ T cells might offer significant advantages as personalized cancer treatments over the conventional αβ CAR-T cells, especially for patients with solid malignant tumors.

References

  1. Read SB, NV Kulprathipanja, GG Gomez, DB Paul, K. R. Winston, J. M. Robbins, and C. A. Kruse. Human Alloreactive CTL Interactions with Gliomas and with Those Having Upregulated Hla Expression from Exogenous Ifn-Gamma or Ifn-Gamma Gene Modification. J Interferon Cytokine Res. 2003;23(7):379-93.
  2. Ksendzovsky A, Feinstein D, Zengou R, Sharp A, Polak P, Lichtor T and Glick RP. Investigation of Immunosuppressive Mechanisms in a Mouse Glioma Model. J Neurooncol.  2009;93(1):107-14.
  3. Ashley DM, Kong FM, Bigner DD, Hale LP. Endogenous Expression of Transforming Growth Factor Beta1 Inhibits Growth and Tumorigenicity and Enhances Fas-Mediated Apoptosis in a Murine High-Grade Glioma Model. Cancer Res. 1998;58(2): 302-9.
  4. Jachimczak P, Bogdahn U, Schneider J, Beha C, Meixensberger J, Apfel R and Dorries R, et al. The Effect of Transforming Growth Factor-Beta 2-Specific Phosphorothioate-Anti-Sense Oligodeoxynucleotides in Reversing Cellular Immunosuppression in Malignant Glioma. J Neurosurg.  1993;78(6):944-51.
  5. Harly C, Guillaume Y, Nedellec S, Peigné CM, Mönkkönen H, Mönkkönen J and Li J, et al. Key Implication of Cd277/Butyrophilin-3 (Btn3a) in Cellular Stress Sensing by a Major Human γδ T-Cell Subset.Blood. 2012;120(11):2269-79.
  6. Palakodeti A, Sandstrom A, Sundaresan L, Harly C, Nedellec S, Olive D and Scotet E, et al. The Molecular Basis for Modulation of Human Vγ9vδ2 T Cell Responses by Cd277/Butyrophilin-3 (Btn3a)-Specific Antibodies. J Biol Chem. 2012;287(39): 32780-90.
  7. Groh V, Steinle A, Bauer S and Spies T. Recognition of Stress-Induced MHC Molecules by Intestinal Epithelial Gamma delta T Cells. Science. 1998;279(5357):1737-40.
  8. Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL and Spies T. Activation of Nk Cells and T Cells by Nkg2d, a Receptor for Stress-Inducible Mica. Science. 1999;285(5428):727-9.
  9. Groh V, Rhinehart R, Secrist H, Bauer S, Grabstein KH and T. Spies. Broad Tumor-Associated Expression and Recognition by Tumor-Derived Gamma Delta T Cells of Mica and Micb.  Proc Natl Acad Sci U S A. 1999;96(12) :6879-84.
  10. Hayday AC. Gamma Delta Cells: A Right Time and a Right Place for a Conserved Third Way of Protection.Anni Rev Immunol. 2000;18 :975-1026.
  11. Deniger DC, Moyes JS, Cooper LJ. Clinical Applications of Gamma Delta T Cells with Multivalent Immunity. Front Immunol. 2014: 5:636.
  12. LeFranc MP, Forster A, Baer R, Stinson MA and Rabbitts TH. Diversity and Rearrangement of the Human T Cell Rearranging Gamma Genes: Nine Germ-Line Variable Genes Belonging to Two Subgroups. Cell. 1986;45(2):237-46.
  13. Forster A, Huck S, Ghanem N, Lefranc MP and Rabbitts TH. New Subgroups in the Human T Cell Rearranging V Gamma Gene Locus. Embo j. 1987;6(7):1945-50.
  14. Strauss WM, Quertermous T and Seidman JG. Measuring the Human T Cell Receptor Gamma-Chain Locus. Science. 1987;237(4819):1217-9.
  15. Lafont V, Sanchez F, Laprevotte E, Michaud HA, Gros L, Eliaou JF, and Bonnefoy N. Plasticity of γδ T Cells: Impact on the Anti-Tumor Response. Front Immunol. 2014;5:622.
  16. Pauza CD and Cairo C. Evolution and Function of the Tcr Vgamma9 Chain Repertoire: It's Good to Be Public. Cell Immunol. 2015; 296(1):22-30.
  17. Kunzmann V, Bauer E, Wilhelm M. Gamma/Delta T-Cell Stimulation by Pamidronate. N Engl J Med. 1999;340(9):737-8.
  18. Kondo M, Sakuta K, Noguchi A, Ariyoshi N, Sato K, Sato S and Hosoi A, et al. Zoledronate Facilitates Large-Scale Ex Vivo Expansion of Functional Gammadelta T Cells from Cancer Patients for Use in Adoptive Immunotherapy. Cytotherapy. 2008;10(8):842-56.
  19. Sato K, Kimura S, Segawa H, Yokota A, Matsumoto S, Kuroda J, Nogawa M, et al. Cytotoxic Effects of Gamma delta T Cells Expanded Ex Vivo by a Third Generation Bisphosphonate for Cancer Immunotherapy. Int J Cancer. 2005;116(1):94-9.
  20. Bennouna Jaafar, Emmanuelle Bompas, Eve Marie Neidhardt, Frédéric Rolland, Irène Philip and Céline Galéa, et al. Phase-I Study of Innacell Γδ™, an Autologous Cell-Therapy Product Highly Enriched in Vγ9vδ2 T Lymphocytes, in Combination with Il-2, in Patients with Metastatic Renal Cell Carcinoma. Cancer Immunology Immunotherapy. 2008;57(11):1599-609.
  21. Gober HJ, Kistowska M, Angman L, Jeno P, Mori L and De LiberoG. Human T Cell Receptor Gammadelta Cells Recognize Endogenous Mevalonate Metabolites in Tumor Cells.  J Exp Med. 2003;197(2):163-8.
  22. Kunzmann V, Bauer E, Feurle J, Weissinger F, Tony HP and Wilhelm M. Stimulation of Gamma delta T Cells by Amino bisphosphonates and Induction of Anti plasma Cell Activity in Multiple Myeloma. Blood. 2000;96(2):384-92.
  23. Bouet-Toussaint F, Cabillic F, Toutirais O, Le Gallo M, Thomas de la Pintiere C and Daniel P, et al. Vgamma9vdelta2 T Cell-Mediated Recognition of Human Solid Tumors. Potential for Immunotherapy of Hepatocellular and Colorectal Carcinomas. Cancer Immunol Immunother. 2008;57(4):531-9.
  24. Dieli F, Vermijlen D, Fulfaro F, Caccamo N, Meraviglia S and Cicero G, et al. Targeting Human Gamma Delta T Cells with Zoledronate and Interleukin-2 for Immunotherapy of Hormone-Refractory Prostate Cancer. Cancer Res. 2007;67(15):7450-7.
  25. Wilhelm M, Kunzmann V, Eckstein S, Reimer P, Weissinger F, Ruediger T and Tony HP. Gamma delta T Cells for Immune Therapy of Patients with Lymphoid Malignancies. Blood. 2003;102(1):200-6.
  26. Santini D, Martini F, Fratto ME, Galluzzo S, Vincenzi B, Agrati C and Turchi F, et al. In Vivo Effects of Zoledronic Acid on Peripheral Gammadelta T Lymphocytes in Early Breast Cancer Patients.  Cancer Immunol Immunother. 2008;58(1):31-8.
  27. Gaafar A, Aljurf MD, Al-Sulaiman A, Iqniebi A, Manogaran PS, Mohamed GE and Al-Sayed A, et al. Defective Gammadelta T-Cell Function and Granzyme B Gene Polymorphism in a Cohort of Newly Diagnosed Breast Cancer Patients.  Exp Hematol. 2009;37(7):838-48.
  28. Bryant NL, Gillespie GY, Lopez RD, Markert JM, Cloud GA, Langford CP and Arnouk H, et al. Preclinical Evaluation of Ex Vivo Expanded/Activated γδ T Cells for Immunotherapy of Glioblastoma Multiforme. J Neurooncol.  2011;101(2):179-88.
  29. Bryant NL, Suarez-Cuervo C, Gillespie GY, Markert JM, Nabors LB and S Meleth, et al. Characterization and Immunotherapeutic Potential of Gammadelta T-Cells in Patients with Glioblastoma. Neuro Oncol. 2009;11(4):357-67.
  30. Knight A, Arnouk H, Britt W, Gillespie GY, Cloud GA, Harkins L and Su Y, et al. CMV-independent lysis of glioblastoma by ex vivo expanded/activated Vδ1+ γδ T cells. PLoS One. 2013;8(8):e68729.
  31. Corvaisier M, Moreau-Aubry A, Diez E, Bennouna J, Mosnier JF, Scotet E and Bonneville M. V Gamma 9v Delta 2 T Cell Response to Colon Carcinoma Cells. J Immunol. 2005;175(8):5481-8.
  32. Gentles AJ, Newman AM, Liu CL, Bratman SV, Feng W, Kim D and Nair, et al. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nat Med. 2015;1–12.
  33. Tosolini M, Pont F, Poupot M, Vergez F, Nicolau-Travers, Vermijlen ML and Sarry D, et al. Assessment of tumor-infiltrating TCRVγ9Vδ2 lymphocyte abundance by deconvolution of human cancers microarrays. Oncoimmunology. 2017;6:1–10.
  34. Toutirais O, Cabillic F, Le Friec G, et al. DNAX accessory molecule-1 (CD226) promotes human hepatocellular carcinoma cell lysis by γ9Vδ2 T cells. Eur J Immunol. 2009;39(5):1361-1368.
  35. Lopez RD, Xu S, Guo B, Negrin RS and Waller EK. Cd2-Mediated Il-12-Dependent Signals Render Human Gamma Delta-T Cells Resistant to Mitogen-Induced Apoptosis, Permitting the Large-Scale Ex Vivo Expansion of Functionally Distinct Lymphocytes: Implications for the Development of Adoptive Immunotherapy Strategies. Blood. 2000;96(12):3827-37.
  36. Liu Z, Guo BL, Gehrs BC, Nan L and Lopez RD. Ex Vivo Expanded Human Vgamma9vdelta2+ Gamma delta-T Cells Mediate Innate Antitumor Activity against Human Prostate Cancer Cells in Vitro. J Urol. 2005;173(5):1552-6.
  37. Wang H, Sarikonda G, Puan KJ, Tanaka Y, Feng J, Giner JL and Cao R, et al. Indirect Stimulation of Human Vγ2vδ2 T Cells through Alterations in Isoprenoid Metabolism.J Immunol.  2011;187(10):5099-113.
  38. Alter G, Malenfant JM, Altfeld M. Cd107a as a Functional Marker for the Identification of Natural Killer Cell Activity. J Immunol Methods 2004;294(1-2):15-22.
  39. Nada MH, Wang H, Workalemahu G, Tanaka Y and Morita CT. Enhancing Adoptive Cancer Immunotherapy with Vγ2vδ2 T Cells through Pulse Zoledronate Stimulation. J Immunother Cancer. 2017;5:9.
  40. Smyth MJ, Dunn GP and Schreiber RD. Cancer Immunosurveillance and Immunoediting: The Roles of Immunity in Suppressing Tumor Development and Shaping Tumor Immunogenicity. Adv Immunol. 2006:90;1-50.
  41. Zumwalde NA, Haag JD, Sharma D, Mirrielees JA, Wilke LG, Gould MN and Gumperz JE. Analysis of Immune Cells from Human Mammary Ductal Epithelial Organoids Reveals Vδ2+ T Cells That Efficiently Target Breast Carcinoma Cells in the Presence of Bisphosphonate. Cancer Prev Res (Phila). 2016;9(4):305-16.
  42. Bank I, Book M, Huszar M, Baram Y, Schnirer I and Brenner H. V Delta 2+ Gamma Delta T Lymphocytes Are Cytotoxic to the Mcf 7 Breast Carcinoma Cell Line and Can Be Detected among the T Cells That Infiltrate Breast Tumors. Clin Immunol Immunopathol. 1993;67(1):17-24.
  43. Guo BL, Liu Z, Aldrich WA, Lopez RD. Innate Anti-Breast Cancer Immunity of Apoptosis-Resistant Human Gamma delta-T Cells.  Breast Cancer Res Treat. 2005;93(2):169-75.
  44. Aggarwal R, Lu J, Kanji S, Das M, Joseph M, Lustberg B, Ray A, et al.  Human Vγ2vδ2 T Cells Limit Breast Cancer Growth by Modulating Cell Survival-, Apoptosis-Related Molecules and Microenvironment in Tumors. Int J Cancer.  2013;133(9):2133-44.
  45. Ye J, Ma C, Wang F, Hsueh EC, Toth K, Huang Y and Mo W, et al. Specific Recruitment of γδ Regulatory T Cells in Human Breast Cancer.Cancer Res. 2013;73(20):6137-48.
  46. Nakajima Jun, Tomohiro Murakawa, Takeshi Fukami, Shigenori Goto, Toru Kaneko, Yukihiro Yoshida, Shinichi Takamoto, and Kazuhiro Kakimi. A Phase I Study of Adoptive Immunotherapy for Recurrent Non-Small-Cell Lung Cancer Patients with Autologous γδ T Cells. European Journal of Cardio-Thoracic Surgery.  37(5):1191-97.
  47. Sakamoto Miki, Jun Nakajima, Tomohiro Murakawa, Takeshi Fukami, Yukihiro Yoshida, Tomonori Murayama, Shinichi Takamoto, Hirokazu Matsushita, and Kazuhiro Kakimi. Adoptive Immunotherapy for Advanced Non-Small Cell Lung Cancer Using Zoledronate-Expanded γδ T Cells: A Phase I Clinical Study. Journal of Immunotherapy. 2011;34(2):202-11.
  48. Kobayashi H, Tanaka Y, Yagi J, Minato N and Tanabe K. Phase I/II Study of Adoptive Transfer of γδ T Cells in Combination with Zoledronic Acid and Il-2 to Patients with Advanced Renal Cell Carcinoma. Cancer Immunol Immunother.  2011;60(8):1075-84.
  49. Bennouna J, Bompas E, Neidhardt EM, Rolland F, Philip I, Galea C, Salot S, et al. Phase-I Study of Innacell Gammadelta, an Autologous Cell-Therapy Product Highly Enriched in Gamma9delta2 T Lymphocytes, in Combination with Il-2, in Patients with Metastatic Renal Cell Carcinoma.Cancer Immunol Immunother. 2008;57(11):1599-609.
  50. Wada I, Matsushita H, Noji S, Mori K, Yamashita H, Nomura S and Shimizu N, et al. Intraperitoneal Injection of in Vitro Expanded Vγ9vδ2 T Cells Together with Zoledronate for the Treatment of Malignant Ascites Due to Gastric Cancer. Cancer Med. 2014;3(2):362-75.
  51. Wilhelm Martin, Manfred Smetak, Kerstin Schaefer-Eckart, Brigitte Kimmel, Josef Birkmann, Hermann Einsele and Volker Kunzmann. Successful Adoptive Transfer and in Vivo Expansion of Haploidentical γδ T Cells. Journal of Translational Medicine. 2014;12(1): 45.
  52. Meraviglia S, Eberl M, Vermijlen D, Todaro M, Buccheri S, Cicero G and La Mendola C, et al. In Vivo Manipulation of Vgamma9vdelta2 T Cells with Zoledronate and Low-Dose Interleukin-2 for Immunotherapy of Advanced Breast Cancer Patients.  Clin Exp Immunol. 2010;161(2):290-7.
  53. Wilhelm M, Kunzmann V, Eckstein S, Reimer P, Weissinger F, Ruediger T and Tony HP. Gamma delta T Cells for Immune Therapy of Patients with Lymphoid Malignancies. Blood. 2003;102(1):200-6.
  54. Capsomidis A, Benthall G, Van Acker HH, Fisher J, Kramer AM, Abeln Z and Majani Y, et al. Chimeric Antigen Receptor-Engineered Human Gamma Delta T Cells: Enhanced Cytotoxicity with Retention of Cross Presentation. Mol Ther. 2018;26:354–365.