Jurkat, an immortalized cell line derived from human leukemic T lymphocytes, has been employed as an excellent surrogate model of human primary T-cells for the advancement of T-cell biology and their applications in medicine. However, presumably due to its T-cell origin, Jurkat cells are very difficult to transfect. Thus, for the genetic modification of Jurkat cells, expensive and time-consuming viral vectors are normally required. Despite many previous efforts, non-viral vectors have not yet overcome the hurdles of low transfection efficiency and/or high toxicity in transfection of Jurkat cells. Here, we report that a simple addition of calcium ions (Ca 2+ ) into culture media at optimal concentrations can enhance the efficiency of the polyplex-mediated transfection using poly(ethylene imine) (PEI) by up to 12-fold when compared to the polyplex-only control. We show that calcium enhances the association between polyplex and Jurkat, which is at least partially responsible for the increase in transmembrane delivery of polyplex and consequential enhancement in expression of transgene. Other cations, Mg 2+ or Na + did not show similar enhancement. Interestingly, addition of Ca 2+ was rather detrimental for the transfection of lipoplex on Jurkat cells. Observation of significant enhancement in the transfection of non-viral vectors with a simple and physiologically relevant reagent like Ca 2+ in the engineering of hard-to-transfect cells such as Jurkat warrants further investigation on similar strategies.
Magnetic microbeads decorated with novel peptide ligands against human CD3ε can activate the Jurkat T cells via specific T cell receptor (TCR) signaling pathways linked to calcium flux, IL-2 secretion, and cell proliferation.
Despite the tremendous progress in immunotherapy regimens using T cells, efforts to modulate the functions of T cells are still significantly hampered by the lack of reliable methods to deliver various cargoes into the T cells. This ongoing challenge originates from the intrinsic resistance of T cells in taking up exogenous materials. Here, we strategically aimed to hijack the natural endocytosis of Interleukin-2 (IL2) by the activated T cells for the targeted association and intracellular delivery of cargoes in varying sizes. First, we carefully characterized the fluctuations in the expression levels of IL2 receptor (IL2R) subunits (CD25, CD122, and CD132) during the murine primary T cell cultures over 12 days. We identified the highest fraction of T cells that would express the high-affinity trimeric IL2R on Day 3. By examining the association and uptake efficiencies of IL2 molecules that are biotinylated via either random lysine-targeting chemical reaction (using NHS-PEG4-Biotin) or site-specific enzymatic modification (using Avitag sequence), we demonstrated that the most efficient delivery of cargo can be achieved by C-terminal conjugation. Upon confirmation of successful delivery of a small model cargo, streptavidin, we employed superparamagnetic iron oxide nanoparticles (SPIONs) as bigger model cargoes having core diameters of 50, 100, and 200 nm. We examined the association and intracellular delivery of the IL2-conjugated nanocargoes using flow cytometry, confocal laser scanning microscopy, and transmission electron microscopy. While cargoes of all tested sizes were successfully associated with the IL2Rexpressing T cells in comparable efficiencies, the uptake efficiencies were inversely proportional to the sizes of the cargoes. Nevertheless, our current definitive report confirms that nanocargoes with a practical maximum size limit around 100−200 nm can be intracellularly delivered into activated primary T cells using IL2R-mediated endocytosis, which opens a new horizon for engineering and manufacturing of various T cell immunotherapeutics.
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