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

• Nucleofection® of primary human CD4 T cells leads to sustained intracellular calcium levels up to 8 hours post transfection.
• Nucleofection® of primary human CD4 T cells leads to expression of T cell activation markers for 24 hours post transfection.
• Nucleofection® of primary human CD4 T cells results in T cell transcriptional activity at least 24 hours post transfection.

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 h later. The intracellular calcium level also increases after Nucleofection®, peaking after 1 h before recovering 8 h post transfection. Moreover, Nucleofection® leads to increased expression of T cell activation markers, CD154 and CD69, for more than 24 h, and enhances the activation effects of phytohemagglutinin (PHA) stimulation. In addition, transcriptional activity is increased in the first 24 h 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.