Cancer Immunology, Immunotherapy

, Volume 62, Issue 3, pp 571–583 | Cite as

Extensive expansion of primary human gamma delta T cells generates cytotoxic effector memory cells that can be labeled with Feraheme for cellular MRI

  • Gabrielle M. SiegersEmail author
  • Emeline J. Ribot
  • Armand Keating
  • Paula J. Foster
Original article


Gamma delta T cells (GDTc) comprise a small subset of cytolytic T cells shown to kill malignant cells in vitro and in vivo. We have developed a novel protocol to expand GDTc from human blood whereby GDTc were initially expanded in the presence of alpha beta T cells (ABTc) that were then depleted prior to use. We achieved clinically relevant expansions of up to 18,485-fold total GDTc, with 18,849-fold expansion of the Vδ1 GDTc subset over 21 days. ABTc depletion yielded 88.1 ± 4.2 % GDTc purity, and GDTc continued to expand after separation. Immunophenotyping revealed that expanded GDTc were mostly CD27-CD45RA- and CD27-CD45RA+ effector memory cells. GDTc cytotoxicity against PC-3M prostate cancer, U87 glioblastoma and EM-2 leukemia cells was confirmed. Both expanded Vδ1 and Vδ2 GDTc were cytotoxic to PC-3M in a T cell antigen receptor- and CD18-dependent manner. We are the first to label GDTc with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles for cellular MRI. Using protamine sulfate and magnetofection, we achieved up to 40 % labeling with clinically approved Feraheme (Ferumoxytol), as determined by enumeration of Perls’ Prussian blue-stained cytospins. Electron microscopy at 2,800× magnification verified the presence of internalized clusters of iron oxide; however, high iron uptake correlated negatively with cell viability. We found improved USPIO uptake later in culture. MRI of GDTc in agarose phantoms was performed at 3 Tesla. The signal-to-noise ratios for unlabeled and labeled cells were 56 and 21, respectively. Thus, Feraheme-labeled GDTc could be readily detected in vitro via MRI.


Gamma delta T cell expansion Gamma delta T cell cytotoxicity Iron labeling Preclinical cellular immunotherapy 



We would like to extend a heartfelt thank you to our healthy donors, without whom this work would not have been possible. Thanks to Catherine McFadden for advice on cell labeling as well as Gelareh Zadeh and Kelly Burrell for the U87 glioblastoma cells. Additionally, we thank Martin Rutter at Miltenyi Biotec for timely assistance and the Haeryfar laboratory at Western University for lending us the MACS Midi magnet for our depletions. We thank Judith Sholdice for EM imaging. A.K. holds the Gloria and Seymour Epstein Chair in Cell Therapy and Transplantation at University Health Network and the University of Toronto. P.F. was funded by the Ontario Institute for Cancer Research, One Millimeter Cancer Challenge Program.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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Supplementary material 6 (DOC 32.5 kb)


  1. 1.
    Hayday AC (2000) [gamma][delta] cells: a right time and a right place for a conserved third way of protection. Annu Rev Immunol 18:975–1026PubMedCrossRefGoogle Scholar
  2. 2.
    Kabelitz D, Wesch D, He W (2007) Perspectives of gammadelta T cells in tumor immunology. Cancer Res 67:5–8PubMedCrossRefGoogle Scholar
  3. 3.
    Lamb LS Jr, Lopez RD (2005) gammadelta T cells: a new frontier for immunotherapy? Biol Blood Marrow Transplant 11:161–168PubMedCrossRefGoogle Scholar
  4. 4.
    Ensslin AS, Formby B (1991) Comparison of cytolytic and proliferative activities of human gamma delta and alpha beta T cells from peripheral blood against various human tumor cell lines. J Natl Cancer Inst 83:1564–1569PubMedCrossRefGoogle Scholar
  5. 5.
    Zheng BJ, Chan KW, Im S, Chua D, Sham JS et al (2001) Anti-tumor effects of human peripheral gammadelta T cells in a mouse tumor model. Int J Cancer 92:421–425PubMedCrossRefGoogle Scholar
  6. 6.
    Viey E, Lucas C, Romagne F, Escudier B, Chouaib S et al (2008) Chemokine receptors expression and migration potential of tumor-infiltrating and peripheral-expanded Vgamma9Vdelta2 T cells from renal cell carcinoma patients. J Immunother 31:313–323PubMedCrossRefGoogle Scholar
  7. 7.
    Knight A, Mackinnon S, Lowdell MW (2012) Human Vdelta1 gamma-delta T cells exert potent specific cytotoxicity against primary multiple myeloma cells. Cytotherapy 14:1110–1118Google Scholar
  8. 8.
    Wright A, Lee JE, Link MP, Smith SD, Carroll W et al (1989) Cytotoxic T lymphocytes specific for self tumor immunoglobulin express T cell receptor delta chain. J Exp Med 169:1557–1564PubMedCrossRefGoogle Scholar
  9. 9.
    Freedman MS, D’Souza S, Antel JP (1997) gamma delta T-cell-human glial cell interactions. I. In vitro induction of gammadelta T-cell expansion by human glial cells. J Neuroimmunol 74:135–142PubMedCrossRefGoogle Scholar
  10. 10.
    Vollenweider I, Vrbka E, Fierz W, Groscurth P (1993) Heterogeneous binding and killing behaviour of human gamma/delta-TCR+ lymphokine-activated killer cells against K562 and Daudi cells. Cancer Immunol Immunother 36:331–336PubMedCrossRefGoogle Scholar
  11. 11.
    Kunzmann V, Bauer E, Feurle J, Weissinger F, Tony HP et al (2000) Stimulation of gammadelta T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma. Blood 96:384–392PubMedGoogle Scholar
  12. 12.
    Siegers GM, Dhamko H, Wang XH, Mathieson AM, Kosaka Y et al (2011) Human Vdelta1 gammadelta T cells expanded from peripheral blood exhibit specific cytotoxicity against B-cell chronic lymphocytic leukemia-derived cells. Cytotherapy 13:753–764Google Scholar
  13. 13.
    Siegers GM, Felizardo TC, Mathieson AM, Kosaka Y, Wang XH et al (2011) Anti-leukemia activity of in vitro-expanded human gamma delta T cells in a xenogeneic Ph+ leukemia model. PLoS ONE 6:e16700PubMedCrossRefGoogle Scholar
  14. 14.
    Gioia C, Agrati C, Casetti R, Cairo C, Borsellino G et al (2002) Lack of CD27-CD45RA-V gamma 9V delta 2+ T cell effectors in immunocompromised hosts and during active pulmonary tuberculosis. J Immunol 168:1484–1489Google Scholar
  15. 15.
    Dieli F, Poccia F, Lipp M, Sireci G, Caccamo N et al (2003) Differentiation of effector/memory Vdelta2 T cells and migratory routes in lymph nodes or inflammatory sites. J Exp Med 198:391–397PubMedCrossRefGoogle Scholar
  16. 16.
    Liu Z, Guo BL, Gehrs BC, Nan L, Lopez RD (2005) Ex vivo expanded human Vgamma9Vdelta2+ gammadelta-T cells mediate innate antitumor activity against human prostate cancer cells in vitro. J Urol 173:1552–1556PubMedCrossRefGoogle Scholar
  17. 17.
    Dieli F, Vermijlen D, Fulfaro F, Caccamo N, Meraviglia S et al (2007) Targeting human {gamma}delta} T cells with zoledronate and interleukin-2 for immunotherapy of hormone-refractory prostate cancer. Cancer Res 67:7450–7457PubMedCrossRefGoogle Scholar
  18. 18.
    Yamaguchi T, Fujimiya Y, Suzuki Y, Katakura R, Ebina T (1997) A simple method for the propagation and purification of gamma delta T cells from the peripheral blood of glioblastoma patients using solid-phase anti-CD3 antibody and soluble IL-2. J Immunol Methods 205:19–28PubMedCrossRefGoogle Scholar
  19. 19.
    Yamaguchi T, Suzuki Y, Katakura R, Ebina T, Yokoyama J et al (1998) Interleukin-15 effectively potentiates the in vitro tumor-specific activity and proliferation of peripheral blood gammadeltaT cells isolated from glioblastoma patients. Cancer Immunol Immunother 47:97–103PubMedCrossRefGoogle Scholar
  20. 20.
    Fujimiya Y, Suzuki Y, Katakura R, Miyagi T, Yamaguchi T et al (1997) In vitro interleukin 12 activation of peripheral blood CD3(+)CD56(+) and CD3(+)CD56(-) gammadelta T cells from glioblastoma patients. Clin Cancer Res 3:633–643PubMedGoogle Scholar
  21. 21.
    Lamb LS Jr (2009) Gammadelta T cells as immune effectors against high-grade gliomas. Immunol Res 45:85–95PubMedCrossRefGoogle Scholar
  22. 22.
    Kobayashi H, Tanaka Y, Yagi J, Osaka Y, Nakazawa H et al (2007) Safety profile and anti-tumor effects of adoptive immunotherapy using gamma-delta T cells against advanced renal cell carcinoma: a pilot study. Cancer Immunol Immunother 56:469–476PubMedCrossRefGoogle Scholar
  23. 23.
    Kobayashi H, Tanaka Y, Shimmura H, Minato N, Tanabe K (2010) Complete remission of lung metastasis following adoptive immunotherapy using activated autologous gammadelta T-cells in a patient with renal cell carcinoma. Anticancer Res 30:575–579PubMedGoogle Scholar
  24. 24.
    Nakajima J, Murakawa T, Fukami T, Goto S, Kaneko T et al (2010) A phase I study of adoptive immunotherapy for recurrent non-small-cell lung cancer patients with autologous gammadelta T cells. Eur J Cardiothorac Surg 37:1191–1197PubMedCrossRefGoogle Scholar
  25. 25.
    Bennouna J, Bompas E, Neidhardt EM, Rolland F, Philip I et al (2008) 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 57:1599–1609PubMedCrossRefGoogle Scholar
  26. 26.
    Nicol AJ, Tokuyama H, Mattarollo SR, Hagi T, Suzuki K et al (2011) Clinical evaluation of autologous gamma delta T cell-based immunotherapy for metastatic solid tumours. Br J Cancer 105:778–786PubMedCrossRefGoogle Scholar
  27. 27.
    Mallett CL, McFadden C, Chen Y, Foster PJ (2012) Migration of iron-labeled KHYG-1 natural killer cells to subcutaneous tumors in nude mice, as detected by magnetic resonance imaging. Cytotherapy 14:743–751Google Scholar
  28. 28.
    de Chickera S, Willert C, Mallet C, Foley R, Foster P et al (2012) Cellular MRI as a suitable, sensitive non-invasive modality for correlating in vivo migratory efficiencies of different dendritic cell populations with subsequent immunological outcomes. Int Immunol 24:29–41PubMedCrossRefGoogle Scholar
  29. 29.
    Zhang X, de Chickera SN, Willert C, Economopoulos V, Noad J et al (2011) Cellular magnetic resonance imaging of monocyte-derived dendritic cell migration from healthy donors and cancer patients as assessed in a scid mouse model. Cytotherapy 13:1234–1248PubMedCrossRefGoogle Scholar
  30. 30.
    Gonzalez-Lara LE, Xu X, Hofstetrova K, Pniak A, Chen Y et al (2010) The use of cellular magnetic resonance imaging to track the fate of iron-labeled multipotent stromal cells after direct transplantation in a mouse model of spinal cord injury. Mol Imaging BiolGoogle Scholar
  31. 31.
    Jirak D, Kriz J, Strzelecki M, Yang J, Hasilo C et al (2009) Monitoring the survival of islet transplants by MRI using a novel technique for their automated detection and quantification. MAGMA 22:257–265PubMedCrossRefGoogle Scholar
  32. 32.
    Heyn C, Ronald JA, Mackenzie LT, MacDonald IC, Chambers AF et al (2006) In vivo magnetic resonance imaging of single cells in mouse brain with optical validation. Magn Reson Med 55:23–29PubMedCrossRefGoogle Scholar
  33. 33.
    Oweida AJ, Dunn EA, Karlik SJ, Dekaban GA, Foster PJ (2007) Iron-oxide labeling of hematogenous macrophages in a model of experimental autoimmune encephalomyelitis and the contribution to signal loss in fast imaging employing steady state acquisition (FIESTA) images. J Magn Reson Imaging 26:144–151PubMedCrossRefGoogle Scholar
  34. 34.
    Bernas LM, Foster PJ, Rutt BK (2010) Imaging iron-loaded mouse glioma tumors with bSSFP at 3 T. Magn Reson Med 64:23–31PubMedCrossRefGoogle Scholar
  35. 35.
    Foster PJ, Dunn EA, Karl KE, Snir JA, Nycz CM et al (2008) Cellular magnetic resonance imaging: in vivo imaging of melanoma cells in lymph nodes of mice. Neoplasia 10:207–216PubMedGoogle Scholar
  36. 36.
    Perera M, Ribot EJ, Percy DB, McFadden C, Simedrea C et al (2012) In vivo magnetic resonance imaging for investigating the development and distribution of experimental brain metastases due to breast cancer. Trans Oncol 5:217–225Google Scholar
  37. 37.
    Heyn C, Ronald JA, Ramadan SS, Snir JA, Barry AM et al (2006) In vivo MRI of cancer cell fate at the single-cell level in a mouse model of breast cancer metastasis to the brain. Magn Reson Med 56:1001–1010PubMedCrossRefGoogle Scholar
  38. 38.
    Ribot EJ, Foster PJ (2012) In vivo MRI discrimination between live and lysed iron-labeled cells using balanced steady state free precession. Eur Radiol (in press)Google Scholar
  39. 39.
    Garden OA, Reynolds PR, Yates J, Larkman DJ, Marelli-Berg FM et al (2006) A rapid method for labelling CD4+ T cells with ultrasmall paramagnetic iron oxide nanoparticles for magnetic resonance imaging that preserves proliferative, regulatory and migratory behaviour in vitro. J Immunol Methods 314:123–133PubMedCrossRefGoogle Scholar
  40. 40.
    Beer AJ, Holzapfel K, Neudorfer J, Piontek G, Settles M et al (2008) Visualization of antigen-specific human cytotoxic T lymphocytes labeled with superparamagnetic iron-oxide particles. Eur Radiol 18:1087–1095PubMedCrossRefGoogle Scholar
  41. 41.
    Iida H, Takayanagi K, Nakanishi T, Kume A, Muramatsu K et al (2008) Preparation of human immune effector T cells containing iron-oxide nanoparticles. Biotechnol Bioeng 101:1123–1128PubMedCrossRefGoogle Scholar
  42. 42.
    Janic B, Rad AM, Jordan EK, Iskander AS, Ali MM et al (2009) Optimization and validation of FePro cell labeling method. PLoS ONE 4:e5873PubMedCrossRefGoogle Scholar
  43. 43.
    Arbab AS, Janic B, Jafari-Khouzani K, Iskander AS, Kumar S et al (2010) Differentiation of glioma and radiation injury in rats using in vitro produce magnetically labeled cytotoxic T-cells and MRI. PLoS ONE 5:e9365PubMedCrossRefGoogle Scholar
  44. 44.
    Lewin M, Carlesso N, Tung CH, Tang XW, Cory D et al (2000) Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat Biotechnol 18:410–414PubMedCrossRefGoogle Scholar
  45. 45.
    Liu L, Ye Q, Wu Y, Hsieh WY, Chen CL et al (2012) Tracking T-cells in vivo with a new nano-sized MRI contrast agent. Nanomedicine [Epub ahead of print]Google Scholar
  46. 46.
    Neri S, Mariani E, Meneghetti A, Cattini L, Facchini A (2001) Calcein-acetyoxymethyl cytotoxicity assay: standardization of a method allowing additional analyses on recovered effector cells and supernatants. Clin Diagn Lab Immunol 8:1131–1135PubMedGoogle Scholar
  47. 47.
    Mallett CL, Foster PJ (2011) Optimization of the balanced steady state free precession (bSSFP) pulse sequence for magnetic resonance imaging of the mouse prostate at 3T. PLoS ONE 6:e18361PubMedCrossRefGoogle Scholar
  48. 48.
    Lamb LS Jr, Musk P, Ye Z, van Rhee F, Geier SS et al (2001) Human gammadelta(+) T lymphocytes have in vitro graft vs leukemia activity in the absence of an allogeneic response. Bone Marrow Transplant 27:601–606PubMedCrossRefGoogle Scholar
  49. 49.
    Halary F, Pitard V, Dlubek D, Krzysiek R, de la Salle H et al (2005) Shared reactivity of V{delta}2(neg) {gamma}{delta} T cells against cytomegalovirus-infected cells and tumor intestinal epithelial cells. J Exp Med 201:1567–1578PubMedCrossRefGoogle Scholar
  50. 50.
    Schilbach K, Frommer K, Meier S, Handgretinger R, Eyrich M (2008) Immune response of human propagated gammadelta-T-cells to neuroblastoma recommend the Vdelta1+ subset for gammadelta-T-cell-based immunotherapy. J Immunother 31:896–905PubMedCrossRefGoogle Scholar
  51. 51.
    Couzi L, Pitard V, Sicard X, Garrigue I, Hawchar O et al (2012) Antibody-dependent anti-cytomegalovirus activity of human gammadelta T cells expressing CD16 (FcgammaRIIIa). Blood 119:1418–1427PubMedCrossRefGoogle Scholar
  52. 52.
    Knight A, Madrigal AJ, Grace S, Sivakumaran J, Kottaridis P et al (2010) The role of Vdelta2-negative gammadelta T cells during cytomegalovirus reactivation in recipients of allogeneic stem cell transplantation. Blood 116:2164–2172PubMedCrossRefGoogle Scholar
  53. 53.
    Dokouhaki P, Han M, Joe B, Li M, Johnston MR et al (2010) Adoptive immunotherapy of cancer using ex vivo expanded human gammadelta T cells: a new approach. Cancer Lett 297:126–136PubMedCrossRefGoogle Scholar
  54. 54.
    Correia DV, Fogli M, Hudspeth K, da Silva MG, Mavilio D et al (2011) Differentiation of human peripheral blood Vdelta1+ T cells expressing the natural cytotoxicity receptor NKp30 for recognition of lymphoid leukemia cells. Blood 118:992–1001PubMedCrossRefGoogle Scholar
  55. 55.
    Poggi A, Zocchi MR, Carosio R, Ferrero E, Angelini DF et al (2002) Transendothelial migratory pathways of V delta 1+ TCR gamma delta+ and V delta 2+ TCR gamma delta+ T lymphocytes from healthy donors and multiple sclerosis patients: involvement of phosphatidylinositol 3 kinase and calcium calmodulin-dependent kinase II. J Immunol 168:6071–6077PubMedGoogle Scholar
  56. 56.
    Janssen O, Wesselborg S, Heckl-Ostreicher B, Pechhold K, Bender A et al (1991) T cell receptor/CD3-signaling induces death by apoptosis in human T cell receptor gamma delta+ T cells. J Immunol 146:35–39PubMedGoogle Scholar
  57. 57.
    Lopez RD, Xu S, Guo B, Negrin RS, Waller EK (2000) 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 96:3827–3837PubMedGoogle Scholar
  58. 58.
    Viey E, Laplace C, Escudier B (2005) Peripheral gammadelta T-lymphocytes as an innovative tool in immunotherapy for metastatic renal cell carcinoma. Expert Rev Anticancer Ther 5:973–986PubMedCrossRefGoogle Scholar
  59. 59.
    Kondo M, Sakuta K, Noguchi A, Ariyoshi N, Sato K et al (2008) Zoledronate facilitates large-scale ex vivo expansion of functional gammadelta T cells from cancer patients for use in adoptive immunotherapy. Cytotherapy 10:842–856PubMedCrossRefGoogle Scholar
  60. 60.
    Wilhelm M, Kunzmann V, Eckstein S, Reimer P, Weissinger F et al (2003) Gammadelta T cells for immune therapy of patients with lymphoid malignancies. Blood 102:200–206PubMedCrossRefGoogle Scholar
  61. 61.
    Pennington DJ, Silva-Santos B, Shires J, Theodoridis E, Pollitt C et al (2003) The inter-relatedness and interdependence of mouse T cell receptor gammadelta+ and alphabeta+ cells. Nat Immunol 4:991–998PubMedCrossRefGoogle Scholar
  62. 62.
    Caccamo N, Meraviglia S, Ferlazzo V, Angelini D, Borsellino G et al (2005) Differential requirements for antigen or homeostatic cytokines for proliferation and differentiation of human Vgamma9Vdelta2 naive, memory and effector T cell subsets. Eur J Immunol 35:1764–1772PubMedCrossRefGoogle Scholar
  63. 63.
    Groh V, Rhinehart R, Secrist H, Bauer S, Grabstein KH et al (1999) Broad tumor-associated expression and recognition by tumor-derived gamma delta T cells of MICA and MICB. Proc Natl Acad Sci USA 96:6879–6884PubMedCrossRefGoogle Scholar
  64. 64.
    Novotna B, Jendelova P, Kapcalova M, Rossner P Jr, Turnovcova K et al (2012) Oxidative damage to biological macromolecules in human bone marrow mesenchymal stromal cells labeled with various types of iron oxide nanoparticles. Toxicol Lett 210:53–63PubMedCrossRefGoogle Scholar
  65. 65.
    Brekelmans P, van Soest P, Voerman J, Platenburg PP, Leenen PJ et al (1994) Transferrin receptor expression as a marker of immature cycling thymocytes in the mouse. Cell Immunol 159:331–339PubMedCrossRefGoogle Scholar
  66. 66.
    Keating A, Bernstein ID, Papayannopoulou T, Raskind W, Singer JW (1983) EM-2 and EM-3: two new Ph’+ myeloid cell lines. In: PA GDM (ed) Symposia on molecular and cellular biology, new series; UCLA. Alan R. Liss, New York, pp 513–520Google Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Gabrielle M. Siegers
    • 1
    Email author
  • Emeline J. Ribot
    • 1
  • Armand Keating
    • 2
  • Paula J. Foster
    • 1
  1. 1.Imaging Research Laboratories, Robarts Research InstituteWestern UniversityLondonCanada
  2. 2.Cell Therapy Program, Princess Margaret HospitalUniversity of TorontoTorontoCanada

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