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J Immunol Methods. 2014 Jun;408:123-31. doi: 10.1016/j.jim.2014.05.014. Epub 2014 Jun 5.

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

Author information

1
Division of Pediatric Rheumatology, University of Alabama at Birmingham, 1825 University Blvd,. Shelby Building, Rm. 371, Birmingham, AL 35233, United States. Electronic address: mzhang@peds.uab.edu.
2
Nemours/A. I. duPont Hospital for Children, 1600 Rockland Road, Wilmington, DE 19803, United States. Electronic address: zma@nemours.org.
3
Celgene Cellular Therapeutics, 7 Powder Horn Dr., Warren, NJ 07059, United States. Electronic address: nithiselliah@gmail.com.
4
Burnet Institute, 85 Commercial Road, Melbourne, Victoria 3004, Australia. Electronic address: weiss@burnet.edu.au.
5
Division of Pediatric Rheumatology, University of Alabama at Birmingham, 1825 University Blvd,. Shelby Building, Rm. 371, Birmingham, AL 35233, United States. Electronic address: agenin@peds.uab.edu.
6
Nemours Children's Hospital, 13535 Nemours Parkway, Orlando, FL 32827, United States. Electronic address: tfinkel@nemours.org.
7
Division of Pediatric Rheumatology, University of Alabama at Birmingham, 1825 University Blvd,. Shelby Building, Rm. 371, Birmingham, AL 35233, United States. Electronic address: rcron@peds.uab.edu.

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

KEYWORDS:

Calcium; Gene transfer; HIV-1; Nucleofection®; Plasmid; T cell activation

PMID:
24910411
PMCID:
PMC4120863
DOI:
10.1016/j.jim.2014.05.014
[Indexed for MEDLINE]
Free PMC Article
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