GFP Affects Human T Cell Activation and Cytokine Production following In Vitro Stimulation

There are many Green Fluorescent Proteins (GFPs) originating from diverse species that are invaluable to cell biologists today because of their ability to provide experimental visualization of protein expression. Since their initial discovery, they have been modified and improved to provide more stable variants with emission ranges spanning a wide array of colors. Due to their ease of expression both in-vitro and in-vivo, they are an attractive choice for use as markers in molecular biology. GFPs are generally assumed to have negligible effects on the cells to which they have been introduced. However, a growing number of reports indicate that this is not always the case. Consequently, because of GFP's ubiquitous use, it is important to document the nature and extent of unintended effects. In this report, we find that GFP affects T cell activation, leading to defects in clustering, upregulation of the activation marker CD25 and IL-2 cytokine production following stimulation in human primary T cells that also express TurboGFP. We utilized a reporter assay which has been routinely used to assay the NF-κB pathway and found reduced NF-κB activitation in stimulated HEK293 and HeLa cells that were co-transfected with TurboGFP, suggesting that GFP interferes with signaling through the NF-κB pathway. These findings indicate that the utilization of GFP-tagged vectors may negatively impact in vitro experiments in T cells, emphasizing the critical importance of controls to identify any GFP-induced effects.