Cancer Immunol Immunother. 2013 Mar;62(3):571-83.

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

Siegers GM, Ribot EJ, Keating A, Foster PJ.

Imaging Research Laboratories, Robarts Research Institute, Western University, London, ON, Canada. gsiegers@uwo.ca

 

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.

PMID: 23100099

 

Link to Cover Image, OncoImmunology:

https://www.landesbioscience.com/journals/oncoimmunology/toc/volume/2/issue/2/

The pdf of this issue also includes the description of the image and the link to the original article.

The link for the original manuscript in Cancer Immunology, Immunotherapy is:

http://link.springer.com/article/10.1007%2Fs00262-012-1353-y

 

Importance of the Research

Human gamma delta T cells (GDTc) are garnering great interest for their potential application as cellular therapy for a variety of different cancers; however, until our publication, only one group had been able to track infused GDTc in humans (using radioactive tracer). We sought to develop a protocol by which GDTc could be labeled with clinically approved iron-oxide nanoparticles for visualization via cellular MRI; however, GDTc were difficult to label and could not tolerate high doses of iron. While we demonstrate proof-of-principle that we can detect these iron-labeled cells in vitro, a different approach will be necessary to track GDTc in vivo.

Our paper also includes a novel GDTc expansion protocol, whereby clinically relevant numbers of cytotoxic GDTc can be obtained. We verified their cytotoxicity against prostate cancer, glioblastoma and leukemia cell lines. Of note, we can expand both Vdelta1 and Vdelta2 GDTc subsets obtained from fresh healthy donor blood. This is of importance, since most GDTc protocols only expand the more prevalent Vdelta2 cells. Another important achievement documented in this paper was the expansion of Vdelta2 GDTc from frozen peripheral blood mononuclear cells.

Our new expansion protocol for GDTc will pave the way for more intensive study of the lesser-understood peripheral Vdelta1 GDTc and perhaps lead to future clinical protocols for GDTc immunotherapy.

A schematic of the new protocol:

Gabrielle Siegers-1

 

Address correspondence regarding GDTc to:

Gabrielle M. Siegers, PhD

Robarts Research Institute

Western University

London, Ontario

Canada

gsiegers@uwo.ca

Tel: 519-661-2111 ext 87792

 

Address correspondence regarding cellular MRI to:

Paula J Foster, PhD

Robarts Research Institute

Imaging Research Laboratories

Western University

London, Ontario

Canada

pfoster@robarts.ca

Tel: 519-661-2111 ext 24040

Website: http://www.robarts.ca/paula-foster

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