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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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Abstract

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.

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Acknowledgments

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.

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The authors declare that they have no conflict of interest.

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  1. Imaging Research Laboratories, Robarts Research Institute, Western University, London, ON, Canada

    Gabrielle M. Siegers, Emeline J. Ribot & Paula J. Foster

  2. Cell Therapy Program, Princess Margaret Hospital, University of Toronto, Toronto, ON, Canada

    Armand Keating

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  1. Gabrielle M. Siegers
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Correspondence to Gabrielle M. Siegers.

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Siegers, G.M., Ribot, E.J., Keating, A. et al. Extensive expansion of primary human gamma delta T cells generates cytotoxic effector memory cells that can be labeled with Feraheme for cellular MRI. Cancer Immunol Immunother 62, 571–583 (2013). https://doi.org/10.1007/s00262-012-1353-y

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