The impact of Nucleofection® on the activation state of primary human CD4 T cells

Abstract

Gene transfer into primary human CD4 T lymphocytes is a critical tool in studying the mechanism of T cell-dependent immune responses and human immunodeficiency virus-1 (HIV-1) infection. Nucleofection® is an electroporation technique that allows efficient gene transfer into primary human CD4 T cells that are notoriously resistant to traditional electroporation. Despite its popularity in immunological research, careful characterization of its impact on the physiology of CD4 T cells has not been documented. Herein, using freshly-isolated primary human CD4 T cells, we examine the effects of Nucleofection® on CD4 T cell morphology, intracellular calcium levels, cell surface activation markers, and transcriptional activity. We find that immediately after Nucleofection®, CD4 T cells undergo dramatic morphological changes characterized by wrinkled and dilated plasma membranes before recovering 1 hour later. The intracellular calcium level also increases after Nucleofection®, peaking after 1 hour before recovering 8 hours post transfection. Moreover, Nucleofection® leads to increased expression of T cell activation markers, CD154 and CD69, for more than 24 hours, and enhances the activation effects of phytohemagglutinin (PHA) stimulation. In addition, transcriptional activity is increased in the first 24 hours after Nucleofection®, even in the absence of exogenous stimuli. Therefore, Nucleofection® significantly alters the activation state of primary human CD4 T cells. The effect of transferred gene products on CD4 T cell function by Nucleofection® should be assessed after sufficient resting time post transfection or analyzed in light of the activation caveats mentioned above.

Optimization of methods for the genetic modification of human T cells

“…Although this electroporation protocol generates high efficacy of gene input, the viability of electroporated T cells after 24 h was only in the range of 15–40% (data not shown). Our cell death observations are very similar to other T‐cell electroporation studies 6 , 23 . Additionally, cell death continued to progress 2–3 days post electroporation and correlated with increased amount of plasmid DNA used.…”

“…Electroporation is one of the most effective methods for the introduction of DNA into human T cells. The main drawback of this method is the reduced cell viability and phenotypic changes 6 , 8 , 23 . Additionally, electroporated cells, particularly primary T cells, only transiently express delivered sequences; therefore, this method is not as convenient compared with the development of stable cell lines via viral transductions.…”

“…However, concentrated lentiviruses preps give the best transduction efficiency (percent integration) and number of gene copies (determined via YFP MFI) relative to unconcentrated preparations. Electroporation may also be used, however, this method is highly toxic to cells, and should be used as an alternative method 6 , 8 , 23 . Additionally, stable cell lines produced via viral transductions are more convenient to use, and give reproducible results when compared with the need to repeatedly electroporate T‐cell lines with variable efficiencies between experiments.…”

“…Performing these types of experiments utilizing electroporated cells is not cost‐effective and will require multiple electroporations for one replicate. Moreover, electroporation is not the optimal method for T‐cell analysis owing to the increased activation phenotype of T cells induced by electroporation 23 . A recent study demonstrated that electroporated primary human CD4 + T cells had enhanced intracellular calcium levels, increased surface activation markers, augmented transcriptional activity and amplified sensitivity to phytohemagglutinin 23 .…”

“…Moreover, electroporation is not the optimal method for T‐cell analysis owing to the increased activation phenotype of T cells induced by electroporation 23 . A recent study demonstrated that electroporated primary human CD4 + T cells had enhanced intracellular calcium levels, increased surface activation markers, augmented transcriptional activity and amplified sensitivity to phytohemagglutinin 23 . These observations indicate that assays examining T‐cell function (that is, cytokine production, calcium mobilization, surface protein expression) may give misleading results when electroporation is used and are best performed in virally transduced T cells.…”