Loading of Primary Human T Lymphocytes with Citrate-Coated Superparamagnetic Iron Oxide Nanoparticles Does Not Impair Their Activation after Polyclonal Stimulation

Abstract

For the conversion of immunologically cold tumors, characterized by a low T cell infiltration, into hot tumors, it is necessary to enrich T cells in the tumor area. One possibility is the use of magnetic fields to direct T cells into the tumor. For this purpose, primary T cells that were freshly isolated from human whole blood were loaded with citrate-coated superparamagnetic iron oxide nanoparticles (SPIONCitrate). Cell toxicity and particle uptake were investigated by flow cytometry and atomic emission spectroscopy. The optimum loading of the T cells without any major effect on their viability was achieved with a particle concentration of 75 µg Fe/mL and a loading period of 24 h. The cellular content of SPIONCitrate was sufficient to attract these T cells with a magnet which was monitored by live-cell imaging. The functionality of the T cells was only slightly influenced by SPIONCitrate, as demonstrated by in vitro stimulation assays. The proliferation rate as well as the expression of co-stimulatory and inhibitory surface molecules (programmed cell death 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain containing 3 (Tim-3), C-C motif chemokine receptor 7 (CCR7), CD25, CD45RO, CD69) was investigated and found to be unchanged. Our results presented here demonstrate the feasibility of loading primary human T lymphocytes with superparamagnetic iron oxide nanoparticles without influencing their viability and functionality while achieving sufficient magnetizability for magnetically controlled targeting. Thus, the results provide a strong fundament for the transfer to tumor models and ultimately for new immunotherapeutic approaches for cancer treatment.

Improving Tumor Retention of Effector Cells in Adoptive Cell Transfer Therapies by Magnetic Targeting

“…Although effective for lymphoid cell loading, MNP functionalization complicates synthesis and cell handling which would introduce increased variability in MNP-lymphoid cell preparation. Indeed, we and others have described that lymphoid cell loading with minimally functionalized MNPs could be sufficient to achieve magnetic retention [78,79,81]. Our microscopy analysis and iron uptake measurements showed that positively charged MNPs (3-aminopropyl-triethoxysilane (APS)-MNPs) were most associated with all types of lymphoid cells [78].…”

“…Since these cells are barely metabolically active and present a low number of mitochondria in the early stages, these data indicate that MNPs could promote some metabolic activity in resting lymphoid cells. In line with this observation, Mühlberger et al observed a slight decrease in the percentage of naïve T cells after MNP treatment, both in resting cells and in polyclonally-stimulated cells, hinting that MNP loading may facilitate T cell activation [81]. It has been recently reported that activation of pattern recognition receptors (PRR), which include Toll-like receptor (TLRs) and play a role in the initiation of innate immune responses, can regulate TCR signals and T cell functions [107].…”

“…Magnetic retention increased with the amount of MNPs associated with the cells as well as with the force of the magnetic gradient applied [78]. Others also found that similar amounts of MNP were necessary to load lymphoid cells to magnetically retain them [80,81]. Indeed, additional studies confirm that three parameters are critical for magnetic targeting: (1) amount of MNP associated to cells; (2) force of the magnetic gradient; (3) length in time of EMF application [73,153].…”

“…We and others have recently demonstrated the feasibility of this approach and analyzed the effects of the combined use of magnetic nanoparticles and the application of an external magnetic field, to promote the accumulation and retention of lymphoid cells [77][78][79][80][81]. The following sections of this review will highlight some of the recent findings on the effects of MNPs on lymphoid cells, their specific targeting through the application of external magnetic fields and the possibility to use this platform to solve some of the limitations such as the low efficiency in lymphocyte trafficking, that some current antitumor immunotherapies present.…”

“…It is well described that positively charged NPs interact to a greater extent with cell membranes, possibly due to electrostatic interactions [114,[120][121][122][123][124][125][126]. Muhlberger et al used negatively charged MNPs and found sufficient association with T cells to elicit retention by a magnetic field, indicating that MNP interactions with lymphoid cells are not solely dependent on charge [80,81]. The activation status of the lymphoid cells is also likely to affect its capacity to interact with MNPs.…”