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