title: Extensive expansion of primary human gamma delta T cells generates cytotoxic effector memory cells that can be labeled with Feraheme for cellular MRI.
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.
authors.affiliation: Imaging Research Laboratories, Robarts Research Institute, Western University, London, ON, Canada. gsiegers@uwo.ca
authors.forename: Gabrielle M
authors.initials: GM
authors.lastname: Siegers
authors.forename: Emeline J
authors.initials: EJ
authors.lastname: Ribot
authors.forename: Armand
authors.initials: A
authors.lastname: Keating
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Foster
mesh_terms: D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods; D005290:Ferric Compounds; D052203:Ferrosoferric Oxide; D006801:Humans; D007156:Immunologic Memory; D016130:Immunophenotyping; D016131:Lymphocyte Subsets / Q000276:immunology*; D008279:Magnetic Resonance Imaging / Q000379:methods*; D053758:Nanoparticles; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000378:metabolism*; D013194:Staining and Labeling; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000378:metabolism
chemical_list: D005290:Ferric Compounds; D016692:Receptors, Antigen, T-Cell, gamma-delta; C000499:ferric oxide; D052203:Ferrosoferric Oxide