Redox regulation of T-cell receptor signaling.

Biol. Chem. 2015; 396(5): 555–569

Review Luca Simeoni* and Ivan Bogeski

Redox regulation of T-cell receptor signaling Abstract: T-cell receptor (TCR) triggering by antigens activates a sophisticated intracellular signaling network leading to transcriptional activation, proliferation and differentiation of T cells. These events ultimately culminate in adaptive immune responses. Over recent years it has become evident that reactive oxygen species (ROS) play an important role in T-cell activation. It is now clear that ROS are involved in the regulation of T-cell mediated physiological and pathological processes. Upon TCR triggering, T cells produce oxidants, which originate from different cellular sources. In addition, within inflamed tissues, T cells are exposed to exocrine ROS produced by activated phagocytes or other ROS-producing cells. Oxidative modifications can have different effects on T-cell function. Indeed, they can stimulate T-cell activation but they can be also detrimental. These opposite effects of oxidation likely depend on different factors such as ROS concentration and source and also on the differentiation status of the T cells. Despite the well-stablished fact that ROS represent important modulators of T-cell activation, the precise molecular mechanisms of their action are far from clear. Here, we summarize the present knowledge on redox regulation of T-cell function with a particular emphasis on the redox regulation of TCR signaling. Keywords: calcium; mitochondria; NADPH oxidase; redox; ROS; T-cell; T-cell activation; TCR signaling; thiol switch. DOI 10.1515/hsz-2014-0312 Received December 12, 2014; accepted February 27, 2015; previously published online March 10, 2015

*Corresponding author: Luca Simeoni, Institute of Molecular and Clinical Immunology, Otto von Guericke University, Leipziger Straße 44, D-39120 Magdeburg, Germany, e-mail: [email protected] Ivan Bogeski: Department of Biophysics, School of Medicine, Saarland University, D-66421 Homburg, Germany

Introduction The immune system is equipped with a variety of antimicrobial factors including complement proteins, antibodies and small chemically active molecules (e.g., reactive oxygen species, ROS) to fight against invading pathogens. During the last years, it has become clear that, in addition to their microbicidal activity, these substances possess important immunoregulatory functions that are required to guarantee a correct spatial and temporal proceeding of the immune response. In particular, ROS have recently emerged as important modulators of activation, proliferation, and apoptosis in many cell types (Valko et al., 2007). Among all ROS, superoxide anion radical (O2·-) and hydrogen peroxide (H2O2) are probably the biologically most important ones (Droge, 2002). Within this review, we use the abbreviation ROS for simplicity. However, in most cases ROS in T cells are mainly represented by O2·- and H2O2. They can be generated by both enzymatic and nonenzymatic systems, including mitochondria and NOX complexes, in the intracellular as well as in the extracellular space (Bedard and Krause, 2007; Bogeski et al., 2011; Sies, 2014; Zorov et al., 2014). O2·- is generated upon transfer of electrons to the free molecular oxygen. O2·- is highly unstable and is rapidly transformed into H2O2 either spontaneously or by superoxide dismutase (SOD). H2O2 can react with the thiol of cysteine residues resulting in the formation of sulfenic acid (Kettenhofen and Wood, 2010). This posttranslational modification – known as sulfenylation – is reversible (e.g., by GSH) and can modulate the function of the targeted protein. Additional reversible cysteine modifications include glutathionylation, S-nitrosylation, S-acylation, sulfenylamide formation, and the generation of disulfide bridges. Therefore, cysteine residues have recently become the focus of intensive investigation, as they function as ‘switches’ upon oxidation/reduction of thiol groups. The emerging idea is that reversible oxidation of cysteines is akin to phosphorylation of tyrosine, threonine, and serine residues and represents a novel global regulatory mechanism during signal transduction. In this review, we discuss the role of ROS as modulators of T-cell activation with a particular emphasis on

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556 L. Simeoni and I. Bogeski: ROS in TCR signaling oxidative modification on cysteine of molecules involved in TCR signaling. A number of studies have shown that imbalance in redox homeostasis characterizes many immune-related diseases (Staal et al., 1992; Chrobot et al., 2000; Kovacic and Jacintho, 2001; Reyes et al., 2005; Hultqvist et al., 2009; Kesarwani et al., 2013; Padgett et al., 2013). Nevertheless, despite intensive investigations during the last decade, mechanistic insights of how oxidation regulates T-cell signaling have not yet been completely revealed.

T-cell activation T cells are organized in highly specialized subsets (e.g., CD4+ helper, CD8+ cytotoxic, and CD4+CD25+ regulatory T cells) coordinating both humoral and cellular immune responses. They express a specific receptor for antigens on their cell surface called T-cell receptor (TCR). Antigens are presented in complex with MHC molecules on antigen presenting cells (APCs) such as dendritic cells, macrophages, and B cells. Upon recognition of the antigens by the TCR, a signal is triggered within T cells, thus leading to transcriptional activation, proliferation, differentiation, and immune responses. Alterations in TCR-mediated signaling are at the basis of many human diseases such as

autoimmunity, immunodeficiency, and cancer. Therefore, in recent years efforts were made to investigate how the TCR-mediated signaling network is regulated. In this section we summarize how TCR signaling is regulated. For more details on T-cell activation we recommend the following excellent reviews: Acuto et al. (2008); Chakraborty and Weiss (2014); Samelson (2011); SmithGarvin et al. (2009).

Initiation of TCR signaling: the critical role of Lck and Zap-70 The TCR is a multi-protein complex including an αβTCR heterodimer (or γδTCR in a minor subset of T cells), which recognizes antigens, and the signal transducing units CD3γε, CD3δε, and TCRζζ. In their cytoplasmic domains, CD3 and TCRζ chains possess a particular amino acid sequence, called immunoreceptor tyrosine-based activation motifs (ITAMs), containing two core tyrosines that are required for signaling (Figure 1). Upon recognition of the antigen, tyrosines within ITAMs are phosphorylated by members of the Src-family tyrosine kinases such as Lck (lymphocyte-specific protein tyrosine kinase). Activation of Lck is regulated upon the phosphorylation of two conserved tyrosine residues, Y394

Figure 1: Schematic representation of TCR-mediated signaling. TCR ligation by antigens induces Lck activation, ITAM phosphorylation, and the recruitment and activation of Zap-70. Zap-70 in turn phosphorylates LAT, thus leading to the formation of the LAT signalosome. This complex triggers additional signaling cascades leading to NFAT, AP1, and NFkB activation, thus resulting in IL-2 production and T-cell activation.


L. Simeoni and I. Bogeski: ROS in TCR signaling 557

and Y505. Auto-trans-phosphorylation of Y394, located in the kinase domain, activates Lck, whereas the phosphorylation of the inhibitory Y505, located in the C-terminus, by the non-receptor tyrosine kinase Csk inhibits Lck’s enzymatic activity. A number of tyrosine phosphatases have been reported to dephosphorylate the regulatory sites within Lck. There is evidence that CD45 play a major role as positive regulator of Lck activity upon dephosphorylation of the inhibitory Y505, which will in turn result in Lck activation (for a review on the role of phosphatases in the regulation of TCR signaling see Mustelin et al., 2005). Other phosphatases such as Shp1 and LYP/PEP have been shown to be important inhibitors of Lck activity by dephosphorylating Y394. The reason why much attention has been given to the analysis of the regulation of Lck activity is that, among the Src-family kinases, Lck appears to be the most important in T cells. In fact, Lck-/- mice show a severe block in T-cell development (Molina et al., 1992) and Lck-deficient Jurkat T cells display impaired proximal TCR signaling (Goldsmith and Weiss, 1987). Active Lck in turn phosphorylates the ITAMs (Figure 1). Phosphorylation of the ITAMs allows the recruitment of ZAP-70, a protein tyrosine kinase with two tandem SH2 domains, to the activated receptor (Wang et al., 2010). Binding to the ITAMs results in the perturbation of the auto-inhibited conformation of Zap-70. Phosphorylation of the activatory Y493 by Lck or Zap-70 itself results in full activation. The additional phosphorylation of Y315 and Y319 within Zap-70 further stabilizes the active enzyme and additionally serves as docking sites for other effector molecules including Vav and Lck. Similarly to Lck, Zap-70 is also dephosphorylated and inactivated by phosphatases such as Shp1 and LYP/PEP (Mustelin et al., 2005). Zap-70 is required during thymic development, which is completely blocked in Zap-70-deficient mice (Negishi et al., 1995). Additionally, Zap-70 is required for signaling downstream of the TCR in mature T cells. Indeed, a Zap70-deficient Jurkat T-cell variant (P116) displays impaired TCR-mediated signaling (Williams et al., 1998).

Diversification of the signal: the LAT signalosome Activated ZAP-70 in turn phosphorylates the two adaptor proteins LAT and SLP-76, which form the backbone of a signaling complex required for the diversification of the signal, thus leading to transcriptional activation, cytoskeleton reorganization, proliferation and differentiation of T cells (Figure 1) (Wange, 2000). A crucial event triggered by the LAT signalosome is the activation of PLCγ-1, which

cleaves PIP2 thus generating the second messengers DAG and IP3. DAG activates PKCs, which in turn orchestrate the activation of the CBM complex including Bcl10, MALT1 and CARMA1 leading to NF-κB activation (Lin and Wang, 2004). Conversely, DAG is also crucial for the initiation of Ras-Erk signaling, which is indispensable for the activation of the transcription factor AP1 (Roose and Weiss, 2000). Ras activation is triggered upon recruitment of RasGRP1 to the plasma membrane via DAG. RasGRP1 will in turn enhance the generation of active Ras leading to the activation of the Raf-Mek-Erk cascade. However, for sustained Ras signaling and T-cell activation the activity of Sos1 is also required (Roose et al., 2007; Das et al., 2009; Warnecke et al., 2012; Poltorak et al., 2014). IP3, however, activates the IP3 receptors (IP3R) at the endoplasmic reticulum (ER) membrane and causes depletion of the intracellular ER Ca2+ stores, thereby triggering accumulation of the ER Ca2+ sensor proteins STIM1 and STIM2 (Figure 1) (Liou et al., 2005; Zhang et al., 2005). Clustered STIM proteins directly couple and activate Ca2+-influx through Ca2+ release-activated Ca2+ (CRAC) channels in a process known as store-operated Ca2+ entry (SOCE) (Hoth and Penner, 1992; Parekh and Putney, 2005). In T cells CRAC channels are mainly formed by Orai1 (Feske et al., 2006) although heteromeric channels can assemble with both Orai2 and Orai3 (Lis et al., 2007; Schindl et al., 2009). Humans with mutations in Orai1 or STIM1 have severely impaired SOCE and consequently defective T-cell proliferation and cytokine production (Feske, 2009). Ca2+ signaling will lead to the activation of the transcription factor NFAT. In summary, the signal triggered at the plasma membrane will culminate in the activation of NFkB, AP-1, and NFAT, which will in turn regulate IL-2 transcription in the nucleus. IL-2 production will further support T-cell proliferation and immune responses.

T-cell activation is sensitive to oxidation In the 1980s, different groups demonstrated that oxidation is crucial for T-cell proliferation. Initial data, generated using compounds known to scavenge free radicals, demonstrated that PMA-induced proliferation of human peripheral lymphocytes was inhibited upon scavenging of hydroxyl radicals (Novogrodsky et al., 1982). Similarly, Ceredig and colleagues showed that several antioxidant compounds inhibit the proliferation of T cells stimulated with both alloantigen and PMA and ionomycin (Chaudhri et al., 1986, 1988). Additional data suggested that antioxidants affect T-cell proliferation by inhibiting IL-2 expression (Gerber et al., 1985; Chaudhri et al., 1988; Fidelus,


558 L. Simeoni and I. Bogeski: ROS in TCR signaling 1988; Sekkat et al., 1988; Dornand and Gerber, 1989; Roth and Droge, 1991; Tatla et al., 1999). Collectively, these initial observations indicated that oxygen radicals are involved in the regulation of T-cell activation. Nevertheless, additional studies have complicated this simple view. Indeed, it has been shown that H2O2 may inhibit and, under particular conditions, also stimulate lymphocyte proliferation (Roth and Droge, 1987; Patterson et al., 1988; Los et al., 1995a). Also, studies from Tsan and colleagues showed that increasing amount of intracellular antioxidants such as glutathione (GSH) increased the proliferation of murine splenic lymphocytes in response to mitogens (Fidelus and Tsan, 1986, 1987; Fidelus et al., 1987). 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. The observed discrepancies may be the result of different T-cell subsets used in these studies such as T-cell lines, T-cell blasts, human peripheral blood T cells, murine splenocytes and thymocytes, CD4+, CD8+, memory or naïve T cells, which may have a different intracellular redox equilibrium and hence have different sensitivities to oxidation. Indeed, it has been observed that T cells isolated from the intestinal lamina propria express significantly higher levels of GSH compared to peripheral blood T cells and hence display a different sensitivity to H2O2 (Reyes et al., 2005). Additionally, the different stimulation conditions used in these studies, including PMA and ionophores, lectins such as ConA and PHA, agonistic antibodies such as CD3 alone or in combination with the costimulatory molecule CD28, IL-2, alloreactions, may also activate different signaling pathways that may be more or less sensitive to oxidation. Finally, also the different concentrations (in some cases ranging above physiological levels) and chemical properties of antioxidants or inhibitors used in the studies, which may have off-targets effects, could help to explain the conflicting results.

immune cells such as phagocytes, T cells also produce ROS. Studies from different groups have indeed shown that TCR triggering results in the generation of both O2·and H2O2 in human and mouse T-cell blasts, primary T cells, and also in Jurkat T cells (Devadas et al., 2002; Kwon et al., 2003, 2010; Jackson 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). One of the main problems in this regard is that, in comparison with professional phagocytes, T cells generate much lower amounts of ROS upon TCR ligation. This makes the measurement of specific TCR-mediated ROS a difficult task. For example, extracellular O2·- production by antiCD3/CD28 bead activated T cells could not be measured using electron paramagnetic resonance (EPR) but could be detected by the H2O2-sensitive genetically encoded protein sensor HyPer [Bogeski et al. unpublished data (Mishina et al., 2012)]. Additional problems may be encountered when measuring specific TCR-mediated ROS production. Indeed, under culture conditions cell stress may induce alterations of the redox status and lead to unspecific detection of ROS in the absence of TCR stimulation. 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 O2·- 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 O2·- originates upon stimulation of Fc receptors on monocytes contaminating purified T-cell suspensions (Williams and Kwon, 2004; Belikov et al., 2014). The recent development of new tools such as genetically encoded biosensors, which detect intracellular H2O2 production or GSH/GSSG ratio, certainly represents an important advance in the analysis of specific ROS production in T cells (Gutscher et al., 2008; Mishina et al., 2012). Using these methods, it has been demonstrated that TCR engagement leads to a discrete generation of intracellular ROS and to a shift in the GSH/GSSG ratio.

The Ws of ROS production in T cells: when, where, what, and why

The data suggest that T cells produce O2·- and H2O2 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). Interestingly, kinetics of

The observation that antioxidants and H2O2 affected T-cell activation raised the question whether, similarly to other

When and where are ROS generated?



ROS production appears to correlate with phosphorylation kinetics induced by TCR ligation (Arndt et al., 2013). These observations reinforce the idea that O2·- and H2O2 may play a role in the regulation of signaling events during T-cell activation. The rapid induction of ROS upon TCR stimulation appears to depend on NADPH oxidases (NOX) (Jackson et al., 2004; Belikov et al., 2014). The NOX family of enzymes consists of seven members (NOX 1–5 and two dual oxidases, DUOX 1 and 2). Many cells express some or more subtypes of NOX. In T cells NOX2 and DUOX1 are, most likely, the predominant NOX isoforms (Jackson et al., 2004; Kwon et al., 2010). Once activated these enzymes assemble at the plasma membrane and generate O2·- by transferring electrons from the cytosolic NADPH to the free molecular oxygen. Subsequently, the highly unstable O2·- is rapidly transformed into H2O2 (Bedard and Krause, 2007). NOX2 is an important source of ROS in T cells as NOX2-deficient T cells display strongly reduced O2·- and H2O2 production (Jackson et al., 2004; Belikov et al., 2014). However, whether NOX2-derived ROS are required for T-cell activation is still debated. In fact, NOX2-deficient T cells display normal T-cell activation and proliferation (Belikov et al., 2014). Also DUOX1 has been implicated in H2O2 production in T cells. Suppression of DUOX1 expression by siRNAs strongly decreased anti-CD3-mediated oxidation of H2DCFDA (Kwon et al., 2010). It is believed that DUOX1 mediates a positive feed-back loop that promotes proximal TCR signaling by inactivating Shp-2 in Jurkat T cells and CD4+ T-cell blasts. Also mitochondria produce O2·- and H2O2, which appear to play an important function in T cells [for the interested reader we recommend a recently published review on the role of mitochondrial oxidative signaling in T-cell activation, Kaminski et al. (2013)]. The functional role of these organelles and their oxidant production in T cells was reported some decades ago. These initial reports suggested that mitochondria, via their O2·- and H2O2, induce at least two apoptotic pathways thereby controlling apoptosis of activated T cells. Several excellent reviews, which we highly recommend to the interested reader, describe the role of redox signaling in T-cell apoptosis (Hildeman et al., 1999, 2003; Akhand et al., 2002; Ueda et al., 2002; Tripathi and Hildeman, 2004; Sena and Chandel, 2012). How do mitochondria produce ROS? TCR ligation is followed by a rapid increase in cytosolic Ca2+. A minor fraction of this Ca2+ is released by the ER, while the bigger portion comes from Ca2+ entry across the plasma membrane through the Orai Ca2+ channels (Feske, 2009;

Kummerow et al., 2009; Hogan et al., 2010). We and others have shown that following SOCE, mitochondria neighboring the plasma membrane and the ER are able to uptake significant amounts of Ca2+ (Hoth et al., 1997, 2000; Gilabert and Parekh, 2000; Glitsch et al., 2002; Parekh, 2003; Quintana et al., 2007, 2011; Santo-Domingo and Demaurex, 2010; Quintana and Hoth, 2012). High Ca2+ levels within the mitochondrial matrix drive mitochondrial dehydrogenases and result in increased electron supply for the electron transfer chain (ETC), which in turn, results in increased ATP production (Santo-Domingo and Demaurex, 2010). However, the increased electron supply of the ETC simultaneously increases the probability for some of these electrons to leak into the inner mitochondrial membrane and reduce molecular oxygen into O2·- (Bogeski et al., 2006, 2011; Murphy, 2009). It has been proposed that at least eight possible locations exist within the ETC where this phenomenon can occur (Sena and Chandel, 2012). In most cases, electrons will leak into the mitochondrial matrix, but it is very likely that some electrons will also end up into the mitochondrial intermembrane space. O2·- can be converted by mitochondrial SODs to H2O2. Mitochondrial ROS generation appears to be temporally delayed compared to NOX-mediated ROS production and peaks 1–2 h after activation (Kaminski et al., 2012). In the last few years, several studies redefined the role of mitochondria and mitochondrial redox signals in T-cell physiology. According to these works, mitochondrial ROS are not only needed for cell death but, similar to Ca2+, are also required for proper T-cell activation and expansion. In this context, Sena et al. showed that ETC complex III-originating ROS, produced upon T-cell activation, are essential for NFAT activation as well as IL-2 production (Murphy and Siegel, 2013; Sena et al., 2013). The findings of Sena et al. are in line with a study by Kaminski and colleagues who, a few years earlier, reported that complex-I-oxidants control IL-2 and IL-4 production and activation of NF-κB and AP-1 (Kaminski et al., 2010, 2013). The authors of the latter study further supported their conclusions by examining T cells isolated from atopic dermatitis patients. Blockade of mitochondrial complex-I in these cells reduced disease-associated hyper-expression of IL-4 (Kaminski et al., 2010, 2013). In addition, a number of recent studies identified novel molecular players, which all affect mitochondrial ROS production and thereby T-cell activation as well as apoptosis (Kaminski et al., 2007, 2013; Silic-Benussi et al., 2010). In summary, it now clear that mitochondria have a complex and very important role in controlling T-cell function. In this regard, mitochondrial redox and Ca2+ homeostasis and their interplay are of vital importance. It is very likely that, in the near future,


560 L. Simeoni and I. Bogeski: ROS in TCR signaling additional mitochondrial molecules will be identified as essential regulators of T-cell function and of adaptive immune responses.

What are the targets of ROS? Studies performed in the Jurkat T-cell line suggested that the addition of H2O2 induce mitogen- activated protein kinases (MAPK) activation (Griffith et al., 1998; Lee and Esselman, 2002). Nevertheless, it became also clear that H2O2 does not regulate MAPK activation directly. In fact, inhibition of the Src kinases, PLCγ, or PKCs abrogated H2O2-mediated activation of Erk, p38, and JNK (Lee and Esselman, 2002). Moreover, Griffith et al. showed that H2O2mediated Erk activation requires Zap-70. Indeed, treatment of the Zap-70-deficient Jurkat T-cell variant with hydrogen peroxide failed to induce Erk phosphorylation (Griffith et al., 1998). Collectively, these data suggest that H2O2 activate MAPK through molecules involved in TCR signaling. It is has been well-known for many years that protein tyrosine phosphatases (PTPs) are targeted by H2O2, which reacts with a cysteine residue located in the catalytic center and hence inhibit PTPs (Finkel, 1998). Therefore, accordingly to a previously proposed model, H2O2 may support signaling initiation in T cells by inactivating PTPs and thus, by enhancing the activation of protein tyrosine kinases (PTKs) (Reth, 2002). T cells express at least 45 different PTPs and most of them are negative regulators of lymphocyte activation (Mustelin et al., 2005). CD45 and Shp1, among others, have been shown to be crucial in the regulation of TCR proximal signaling (Mustelin et al., 2005). CD45 may function as both positive and negative regulator of TCR signaling. In fact, previous and more recent data have demonstrated that CD45 can dephosphorylate both the activatory Y394 and the inhibitory Y505 of Lck (D’Oro et al., 1996; Hui and Vale, 2014). Conversely, it is clear that Shp1 is a negative regulator of T-cell activation that dephosphorylates the activatory tyrosines of Lck and Zap-70 (Unkeless and Jin, 1997). Shp1-deficiency results in T-cell hyperactivation and autoimmunity (Tsui et al., 1993; Pani et al., 1996). Both CD45 and Shp1 have been reported to be inactivated by H2O2 (Secrist et al., 1993; Cunnick et al., 1998) and more recently, it has also been shown that Shp1 and the related PTP Shp2 are sulfenylated upon antigen stimulation in murine CD8+ T cells (Michalek et al., 2007). In agreement with these data, a study from the group of M.S. Williams has also shown that Shp2 is oxidized upon CD3-mediated stimulation in Jurkat and human T-cell blasts (Kwon et al., 2005). It has been proposed that Shp2

oxidation is required to enhance Vav1 and ADAP phosphorylation, thus promoting integrin activation and T-cell adhesion. Nevertheless, Shp1 was found not to be oxidized in this study. The reason for this discrepancy is not clear. It is possible that both Shp1 and Shp2 are oxidized upon stimulation of primary resting T cells as suggested by Michalek et al., whereas Shp2, but not Shp1, is selectively oxidized in activated T cells as suggested by Kwon et al. Therefore, PTPs may show different sensitivity to oxidation depending on the activation status of the cells. These data also suggest that Shp1 and Shp2 may be located in different cell compartments with a different redox state in naïve vs. activated T cells. Similar observations for B cells show that Shp1 and Shp2 are sulfenylated. Stimulation of the BCR results in O2·- and H2O2 production and also in Shp1/Shp2 sulfenylation in primary B cells (Capasso et al., 2010; Crump et al., 2012). Reduction in ROS production affected Shp1 sulfenylation (inactivation) and impaired B-cell signaling, activation and function (Capasso et al., 2010). These results further support the model proposed by M. Reth suggesting that H2O2 takes part in the amplification of BCR signaling (Reth, 2002; Rolli et al., 2002). Thus, sulfenylation/ inactivation of PTPs like Shp1 and Shp2 appears to be a general mechanism for signal initiation and amplification downstream of the antigen receptor in both T and B lymphocytes (Figure 2). Does H2O2 regulate signaling only by inactivating PTPs? A growing body of evidence suggests that H2O2 may directly influence the activity of PTKs, in particular by oxidizing cysteine residues that are not located in the catalytic site (Lee et al., 2011; Yoo et al., 2011; Paulsen et al., 2012). For example, the regulation of the activity of Src, the prototype of Src-family kinases, via oxidationmediated cysteine modification has become the focus of intense research (Chiarugi, 2008; Giannoni et al., 2010; Corcoran and Cotter, 2013; Giannoni and Chiarugi, 2014). More recently, it has been shown that Lyn, another member of the Src-family kinases that is expressed in different immune cells, functions as a redox sensor that regulates the recruitment of neutrophils to wounds (Yoo et al., 2011). Collectively, these data suggest that, in addition to tyrosine-based regulation, Src-family kinases have another regulatory mechanism based on oxidation of cysteine residues. T cells express two members of the Src-family, namely Lck and Fyn. Little is known about the redox regulation of Src-family kinases expressed in T cells. It appears that Fyn autophosphorylation is enhanced upon GSH treatment in a cell-free system (Hehner et al., 2000). Also Lck seems to be sensitive to oxidation both in vitro (Kanner et al., 1992;



Nakamura et al., 1993; Trevillyan et al., 1999) and in vivo during HIV infection (Stefanova et al., 1996). Two decades ago it was shown that, similarly to the corresponding cysteine residues of Src, C378, C465, and C476 of Lck are crucial for its enzymatic activity, as C to A mutants of these residues displayed impaired autophosphorylation and in vitro kinase activity (Veillette et al., 1993). However, the functional significance of Lck and Fyn oxidation has yet to be elucidated (Figure 2). Little is known also about the redox-mediated regulation of Zap-70, the other tyrosine kinase crucial for the initiation of TCR signaling. About two decades ago, it was shown that, similarly to Lck and Fyn, Zap-70 structure is

altered in T cells from HIV patients (Stefanova et al., 1996). This appears to be an oxidation-mediated event as reducing agents were able to restore protein structure. Another study has shown that treatment with peroxide impaired CD3-mediated phosphorylation of Zap-70 (Chakravarti and Abraham, 2002). A more recent study has identified C39, which is located in the phosphotyrosine-binding pocket of the N-terminal SH2 domain of Zap-70, as a possible target of oxidation (Visperas et al., 2015). The proposed model postulates that H2O2 oxidizes this cysteine, thus preventing the recruitment of the Zap-70 SH2 domain to the phosphorylated ITAMs and ultimately attenuating TCR signaling (Figure 2).

Figure 2: Mechansims of action of ROS in TCR signaling. T-cell receptor (TCR) engagement induces a signaling cascade which leads to ROS production (red stars) from NADPH oxidase 2 (NOX2) at the plasma membrane, dual NADPH oxidase 1 (DUOX1) at the ER membrane, and the mitochondria. Possible targets of ROS including LAT, ZAP70, SHP-1, Lck, cofillin, CD45, and Orai1 Ca2+ channels are depicted (marked with a red star).


562 L. Simeoni and I. Bogeski: ROS in TCR signaling A variety of other signaling molecules have been shown to possess cysteine residues that are sensitive to oxidation. Among these is the transmembrane adaptor protein LAT, which, as mentioned above, is a crucial organizer of TCR signaling. Initial evidence that LAT is sensitive to oxidation came from studies investigating altered responses in synovial fluid T cells isolated from patients suffering from rheumatoid arthritis (RA) (Gringhuis et al., 2000). T cells from RA patients are hyporesponsive to TCR stimulation and display a defective TCR-mediated LAT phosphorylation. This defect appears to be due to the displacement of LAT from the plasma membrane, where LAT coordinates TCR signaling (Figures 1 and 2). It has additionally been shown that RA T cells display reduced intracellular GSH levels, which consequently indicates higher oxidation (oxidative stress) in these cells. A key finding of this study was that treatment of T cells with N-acetyl-L-cysteine (NAC), which elevates the intracellular GSH levels, restores the membrane localization and phosphorylation of LAT and ultimately T-cell activation. An additional study from the same group has shown that three cysteines, C26 and C29, which are present at the end of the transmembrane α-helix region or just proximal of the α-helix, respectively, and C117 located approximately in the middle of the cytoplasmic domain, are sensitive to oxidation (Gringhuis et al., 2002). Indeed, mutations at cysteine residues C117 and C26/C29 confer redox insensitivity to LAT, which remains localized in the plasma membrane upon GSH depletion in Jurkat T cells. The proposed model suggests that, under oxidative conditions, C117 forms a disulfide bond with either C26 or C29, thus affecting the conformation of LAT and hence interfering with the integration of the α-helical structure of LAT into the plasma membrane. Another study has shown that ROS participate in the formation of lipid rafts (Lu et al., 2007). Reduction of ROS levels by Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) and NAC affects the TCR-mediated formation of lipid platform containing signaling molecules such as LAT, PLCγ1, and PKCθ, whereas the ROS-inducer TBHP (tert-butyl hydroperoxide) enhances raft formation. However, the mechanism of how ROS induce the formation of signaling platform is still unknown. Recently, it has been shown that H2O2 have complex effects on T-cell Ca2+ homeostasis and consequently on cytokine production and viability (Bogeski et al., 2010). In a concentration-dependent manner, H2O2 caused inhibition of Orai1 and Orai2 but not of Orai3 and also induced activation of non-selective cation channels, possibly from the TRP (transient receptor potential) family. The cysteine content of Orai3 is slightly different when compared with Orai1.

By creating loss-of-function and gain-of-function cysteine mutants of Orai1 and 3, it has been identified that Orai1’s cysteine 195 is the major redox sensor of the channel. For more details regarding redox regulation of ion channels in different immune and non-immune cell types we recommend the following literature: Bogeski et al. (2011); Takahashi et al. (2011); Bogeski and Niemeyer (2014); O-Uchi et al. (2014a, b); Kozai et al. (2014); Nunes and Demaurex (2014); Paula-Lima et al. (2014); Sahoo et al. (2014); Stojilkovic et al. (2014); Todorovic and Jevtovic-Todorovic (2014). In addition, components of the actin cytoskeleton have been found to be sensitive to oxidation (Fratelli et al., 2002; Klemke et al., 2008). Actin reorganization upon TCR engagement is essential for T-cell activation and also for the migration of T cells through the body (Burkhardt et al., 2008). Cofilin is one of the key orchestrators of actin remodeling that regulates both the disassembly of existing filaments and the formation of new filaments (Samstag et al., 2013). Cofilin function is controlled by the posttranslational modification of S3. Phosphorylation of this residue reduces its actin-binding capacity, whereas dephosphorylation induces actin remodeling. TCR × CD28 stimulation induces cofilin dephosphorylation via Ras and PI3K signaling. Cofilin also contains four cysteine residues that can be potentially oxidized (Samstag et al., 2013). Indeed recent data have shown that C139 is sulfonylated, whereas C39 and C80 are possibly involved in the formation of an intramolecular disulfide bridge under oxidative stress (Klemke et al., 2008; Samstag et al., 2013). Cofilin oxidation parallels with the loss of S3 phosphorylation, thus impairing its ability to remodel actin and ultimately leading to T-cell hyporesponsiveness.

What are the downstream targets of ROS? As mentioned above, triggering of the TCR at the plasma membrane results in IL-2 production, thus further driving proliferation and expansion of T-cell clones. At the transcriptional level, IL-2 mRNA expression is induced by the activity of three transcription factors NFAT, AP-1, and NF-κB (Rao, 1994). A number of studies suggest that H2O2 play an important role in the activation of the transcription factor NF-κB (Los et al., 1995a, b; Lahdenpohja and Hurme, 1998; Hehner et al., 2000). Two mechanisms for redox regulation of NF-κB activation have been proposed, which have been reviewed elsewhere (Kabe et al., 2005; Gloire et al., 2006). First, H2O2 can regulate NF-κB activation indirectly by stimulating the activity of tyrosine kinases (such as Lck and Zap-70), which are involved in the regulation of IκB. Subsequently, phosphorylated



IkB is released from p50/p65, which in turn translocates into the nucleus, thus activating gene transcription. It has additionally been shown that the lipid phosphatase SHIP-1 plays also a role in H2O2-mediated NF-κB activation, although the exact mechanism of this regulation has not yet been elucidated. Alternatively, H2O2 can directly oxidize the IKKβ kinase likely on cysteine 179, thus inactivating NF-κB. However, this mechanism does not seem to occur in T cells. In addition to H2O2 other oxidants such as hypochlorous acid, singlet oxygen and reactive nitrogen species have also been shown to regulate NF-κB activation (Gloire et al., 2006). Also in this case, whether these mechanisms are active in T cells is still unknown. Recent data have shown that T cells from mice with reduced mitochondrial O2·- display normal NF-κB activation (Sena et al., 2013). Similarly, enhanced expression of a mitochondrial SOD (MnSOD) did not affect NF-κB activation (Gill and Levine, 2013). These data suggest that NF-κB activation does not depend on mitochondrial ROS. Members of the MAPKs, which are required for the activation of AP-1, also seem to be regulated in a redoxdependent manner. It has been shown that the ROS scavenger L-NAC and the inhibitor of NADPH oxidases DPI suppress ConA-mediated proliferation and JNK kinase activity in murine thymocytes (Pani et al., 2000). Devadas et al. showed that CD3-mediated Erk phosphorylation depends on hydrogen peroxide but not on superoxide anion in both T-cell hybridoma and T-cell blasts (Devadas et al., 2002). Furthermore, H2O2 stimulation of Jurkat T cells induces not only Erk phosphorylation (Griffith et al., 1998; Lee and Esselman, 2002) but also activates JNK, and p38 (Lee and Esselman, 2002). In contrast to these data, it has been shown that T cells from mice with reduced mitochondrial O2·- display normal CD3xCD28-mediated Erk activation (Sena et al., 2013). Similarly, enhanced expression of MnSOD did not affect the phosphorylation of Erk, whereas JNK activation was enhanced in Jurkat T cells (Gill and Levine, 2013). Interestingly, the expression of a cytosolic SOD (Cu, Zn-SOD) had no effect (Gill and Levine, 2013). Similarly to the last study, it has been shown that oxidation appears to promote JNK and p38, but not Erk activation upon CD3 × CD28 stimulation in both Jurkat and primary human T cells (Hehner et al., 2000). Finally, TCR-mediated NFAT activation appears to be dependent on mitochondrial O2·- in mouse primary T cells (Sena et al., 2013) but not on O2·- and H2O2 in both T-cell hybridoma and T-cell blasts (Devadas et al., 2002). Collectively, these data suggest that somehow ROS play a role in the transcriptional activation of the IL-2 promoter. Whether the modulatory effects of ROS on IL-2 production are exerted directly on transcription factors or

indirectly on signaling molecules upstream of transcription are yet not completely understood. Some conflicting data have also been generated in the case of ROS-mediated IL-2 transcription. It is important to keep in mind that oxidants act within the intricate cellular network and hence ROS applied extracellularly, originated from different subcellular sources, or produced at different stages during T-cell activation may be differentially involved in the transcriptional regulation of the IL-2 gene.

Why are ROS important for T cells? The data presented above clearly highlight a crucial function for TCR-mediated ROS production in the regulation of T-cell activation. In addition to endogenously produced ROS, T cells are also exposed to H2O2 produced by activated macrophages and neutrophils at the site of inflammation. These exogenous oxidants represent important messengers that may regulate cross-talks between immune cells under both physiological and pathological conditions. Recent data support this hypothesis. In fact, NOX2-mediated ROS production by macrophages suppresses the activation of autoreactive T cells in a collageninduced arthritis mouse model (Gelderman et al., 2007). Neutrophils also suppress human T-cell proliferation during endotoxin-induced acute systemic inflammation by releasing H2O2 (Pillay et al., 2012). A large body of evidence suggests that alterations of the T-cell redox homeostasis, might be involved in the pathogenesis of immune-related diseases such as AIDS (Staal et al., 1992; Herzenberg et al., 1997; Gil et al., 2003), viral infections (Chrobot et al., 2000; Kesarwani et al., 2013), cancer (Kovacic and Jacintho, 2001; Valko et al., 2007), intestinal inflammation (Reyes et al., 2005), systemic lupus erythematosus (Caza et al., 2012; Perl, 2013; Doherty et al., 2014; Kato and Perl, 2014), and other autoimmune diseases (Hultqvist et al., 2009; Kesarwani et al., 2013; Padgett et al., 2013; Ortona et al., 2014).

Conclusions and future perspectives During recent years it has become evident that TCR triggering increases the level of intracellular ROS (such as O2·- and H2O2) via NOX enzymes and mitochondria. It is also clear that alteration in the redox equilibrium is crucial for the regulation of T-cell activation, differentiation, cytokine secretion, and apoptosis. However,


564 L. Simeoni and I. Bogeski: ROS in TCR signaling despite intense scrutiny, the molecular details of how ROS regulate signaling in T cells remain still largely elusive. Recent data have demonstrated that antigen stimulation increases H2O2 level and induces sulfenylation of central signaling molecules in naive CD8+ T cells (Michalek et al., 2007). Accordingly, inhibition of sulfenylation reduced TCR signaling, T-cell activation, and proliferation. Therefore, one of the major goals for the future is the identification of the molecular targets of ROS in T cells. Recently, new tools to analyze protein thiol oxidation have become available (Poole, 2008; Lindemann and Leichert, 2012; Ckless, 2014). These methods have been shown to be useful for profiling thiol oxidation and for thiol redox proteome analyses in cell lines (Leonard et al., 2009; Seo and Carroll, 2009; Kettenhofen and Wood, 2010). By applying the available tools for the analysis of the redox proteome, it will be possible to identify new oxidation targets in T cells. This could lead to the development of new immunomodulatory compounds for the treatment of immune-related diseases such as autoimmunity and chronic inflammation. The second major development that might bring significant advances in our understanding of how redox signals modulate immunity is the development of genetically encoded redox sensors and their use in animal models (Belousov et al., 2006; Gutscher et al., 2008; Meyer and Dick, 2010; Mishina et al., 2012; Albrecht et al., 2014; Breckwoldt et al., 2014; Ermakova et al., 2014; Sies, 2014; Peralta et al., 2015). With such tools in hand, we could finally observe and record the spatial and temporal parameters of T-cell redox homeostasis in vivo under physiological and pathological conditions. Acknowledgments: This work was supported by the German Research Foundation (DFG) grants SI861/3-1, SFB854 (project B19), BO3643/3-1, SFB1027 (project C4) and the HOMFOR excellent research grant by the Medical School, University of Saarland.

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Dynamic Redox Regulation of IL-4 Signaling.

The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling.

Redox Signaling through DNA.

Redox signaling in neurodegeneration.

Redox regulation of ion channels.

Redox regulation of cardiac hypertrophy.

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MiR393 regulation of auxin signaling and redox-related components during acclimation to salinity in Arabidopsis.

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Regulation of the NMDA receptor by redox phenomena: inhibitory role of ascorbate.

The growth hormone secretagogue receptor: its intracellular signaling and regulation.

B lymphocyte antigen receptor signaling: initiation, amplification, and regulation.

Endothelins and sarafotoxins: physiological regulation, receptor subtypes and transmembrane signaling.

Wnt signaling-mediated redox regulation maintains the germ line stem cell differentiation niche.

Nrf2--A regulator of keratinocyte redox signaling.

Redox modulatory site of the NMDA receptor-channel complex: regulation by oxidized glutathione.

Redox regulation of differentiation and de-differentiation.