TCR-triggered extracellular superoxide production is not required for T-cell activation

Abstract

BackgroundIn the last decade, reactive oxygen species (ROS) production has been shown to occur upon T-cell receptor (TCR) stimulation and to affect TCR-mediated signalling. However, the exact reactive species that are produced, how ROS are generated and their requirement for T-cell activation, proliferation or cytokine production remain unclear, especially in the case of primary human T cells. Moreover, several groups have questioned that ROS are produced upon TCR stimulation.ResultsTo shed some light onto this issue, we specifically measured superoxide production upon TCR ligation in primary human and mouse T lymphocytes. We showed that superoxide is indeed produced and released into the extracellular space. Antioxidants, such as superoxide dismutase and ascorbate, abolished superoxide production, but surprisingly did not affect activation, proliferation and cytokine secretion in TCR-stimulated primary human T cells. It has been suggested that T cells produce ROS via the NADPH oxidase 2 (NOX2). Therefore, we investigated whether T-cell activation is affected in NOX2-deficient mice (gp91phox −/−). We found that T cells from these mice completely lack inducible superoxide production but display normal upregulation of activation markers and proliferation.ConclusionsCollectively, our data indicate that primary T cells produce extracellular superoxide upon TCR triggering, potentially via NOX2 at the plasma membrane. However, superoxide is not required for T-cell activation, proliferation and cytokine production.

Redox regulation of T-cell receptor signaling

“…Additionally, in non-single cell studies the generation of ROS from contaminating phagocytes present in T-cell preparations isolated from lymphoid tissues may lead to artifacts. In fact, we have found a significant release of O 2 ·-upon stimulation of T-cell suspensions (whose purity more than 96%) with isotype controls (Belikov et al, 2014). It is believed that this non-TCR-mediated O 2 ·-originates upon stimulation of Fc receptors on monocytes contaminating purified T-cell suspensions Belikov et al, 2014).…”

“…The importance of the intracellular GSH pool in T-cell activation has been also shown using inhibitors of GSH synthesis, which reduced CD3-mediated proliferation and IL-2 production (Gmunder et al, 1990;Suthanthiran et al, 1990;Smyth, 1991;Hehner et al, 2000). More recently, it has been shown that natural antioxidants such as SOD, catalase, and ascorbate do not have any effects on the proliferation of human primary T cells upon CD3 × CD28 stimulation (Belikov et al, 2014). Thus, after three decades of intense scrutiny, it is still controversial whether ROS support or suppress T-cell functions.…”

“…ROS production can be sustained and last up to 60 min upon stimulation (Belikov et al, 2014). Interestingly, kinetics of Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 7/4/15 5:30 AM ROS production appears to correlate with phosphorylation kinetics induced by TCR ligation (Arndt et al, 2013).…”

“…Studies from different groups have indeed shown that TCR triggering results in the generation of both O 2 ·-and H 2 O 2 in human and mouse T-cell blasts, primary T cells, and also in Jurkat T cells (Devadas et al, 2002;Kwon et al, 2003Kwon et al, , 2010Jackson et al, 2004;Remans et al, 2004;Jones et al, 2007;Gutscher et al, 2008;Kaminski et al, 2012;Sena et al, 2013;Belikov et al, 2014). The specificity of ROS production in T cells has long been a debated issue, for a review see Williams and Kwon (2004).…”

“…The data suggest that T cells produce O 2 ·-and H 2 O 2 rapidly (within few minutes) upon TCR stimulation (Devadas et al, 2002;Mishina et al, 2012;Belikov et al, 2014). ROS production can be sustained and last up to 60 min upon stimulation (Belikov et al, 2014).…”