Regulatory T Cells from IL-10-Deficient Mice Fail to Suppress Contact Hypersensitivity Reactions Due to Lack of Adenosine Production

ORIGINAL ARTICLE

Regulatory T Cells from IL-10-Deficient Mice Fail to Suppress Contact Hypersensitivity Reactions Due to Lack of Adenosine Production Sabine Ring1, Alexander H. Enk1 and Karsten Mahnke1 CD4 þ CD25 þ Foxp3 þ regulatory T cells (Tregs) produce immunosuppressive adenosine by degradation of adenosine triphosphate (ATP) by the ectonucleotidases CD39 and CD73. In this sequence of events, ATP is not only the substrate for generation of adenosine but it also activates the immunosuppressive functions of Tregs. To compare the effects of ATP on IL-10-deficient (IL-10/) Tregs with wild-type (wt) Tregs, we incubated both types of Tregs with ATP and assessed their phenotype and function. We show that IL-10/ Tregs failed to become activated by ATP and were impaired in adenosine production. As a consequence, IL-10/ Tregs were unable to block adherence of effector T cells to the endothelium in vitro. When testing the signaling of the ATP receptor P2X7 in IL-10/ Tregs, we recorded no elevation of intracellular calcium after engagement of P2X7 receptors, as compared with wt Tregs, thus indicating that IL-10/ Tregs fail to react normally to ATP and display impaired adenosine production, which explains their inability to suppress contact hypersensitivity responses. Therefore, when using IL-10/ Tregs in different disease models, one has to take into account that adenosine production is abrogated and reduced suppressive effects may not be exclusively attributable to the lack of IL-10 production. Journal of Investigative Dermatology (2011) 131, 1494–1502; doi:10.1038/jid.2011.50; published online 24 March 2011

INTRODUCTION Regulatory T cells (Tregs) are characterized by their suppressive capacity exerted by surface molecules and/or soluble factors (Vignali et al., 2008). Among the recently discovered suppressive means, we along with others have identified the production of adenosine as a mechanism, which to our knowledge is previously unreported, by which Tregs suppress T-cell proliferation in vitro and immune reactions in vivo (Borsellino et al., 2007; Deaglio et al., 2007; Ring et al., 2009). Tregs generate adenosine by degradation of adenosine triphosphate (ATP) through the ectonucleotidases CD39 and CD73. CD39 catalyzes the initial degrading step by converting ATP to adenosine diphosphate, and subsequently, adenosine is produced by CD73. Adenosine can then act as a suppressor of leukocyte activation, which has been demonstrated in humans and mice (Huang et al., 1997; Lappas et al., 2005; Odashima et al., 2005; Sitkovsky et al., 2008).

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Department of Dermatology, Ruprecht-Karls University of Heidelberg, Heidelberg, Germany Correspondence: Sabine Ring, Department of Dermatology, Ruprecht-Karls University of Heidelberg, Voßstrasse 11, Heidelberg 69115, Germany. E-mail: [email protected] Abbreviations: ATP, adenosine triphosphate; CHS, contact hypersensitivity; PMA, phorbol 12-myristate 13-acetate; Treg, regulatory T cell; wt, wild type Received 8 November 2010; revised 25 January 2011; accepted 29 January 2011; published online 24 March 2011

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In addition to serving as a substrate for adenosine generation by Tregs, ATP can also act as a proinflammatory signal, which was demonstrated by its capacity to activate the inflammasome (Di Virgilio, 2007). However, ATP can also have anti-inflammatory properties, as it directly activates Tregs. Exposure of Tregs to ATP results in drastically increased suppressive activity of Tregs in vitro, and we have shown that blockage of ATP receptors on Tregs abrogated their suppressive capacity in contact hypersensitivity (CHS) reactions in vivo (Ring et al., 2010). Thus, these data underline the importance of ATP in Treg activation and function. In addition to adenosine, another soluble factor, namely IL-10 is frequently attributed to the suppressive function of Tregs, because IL-10-deficient (IL-10/) Tregs fail to suppress colitis and experimental autoimmune encephalomyelitis (Asseman et al., 1999; Zhang et al., 2004). Similarly, in CHS reactions, IL-10/ Tregs were unable to suppress the inflammatory response, but interestingly, the injection of recombinant IL-10 did not re-establish the suppressive effects of IL-10/ Tregs (Ring et al., 2006). Thus, these data indicate that the lacking production of IL-10 may not be the only cause for the absent suppressive activity of IL-10/ Tregs. Therefore, we investigated the activation and generation of a second soluble suppressor adenosine in wild-type (wt) and IL10/ mice. We found that Tregs isolated from IL-10/ mice do not become activated by ATP and produce significantly reduced amounts of adenosine. As a consequence, IL-10/ Tregs failed to suppress the adherence of effector T cells on & 2011 The Society for Investigative Dermatology

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endothelial cells. Thus, when using IL-10/ Tregs in several disease models, one has to take into account that production of adenosine is severely reduced, and that therefore reduced suppressive effects of IL-10/ Tregs may not be attributable to the missing IL-10 production. Instead contribution of the altered adenosine production may have a substantial impact on suppression. RESULTS Tregs from IL-10/ mice are insensitive to ATP-mediated activation

In recent studies, we have demonstrated that exposure of Tregs to ATP in a micromolar range leads to activation and development of the full arsenal of inhibitory mechanisms of Tregs in vitro and in vivo, which is one prerequisite for conveying their suppressive function in CHS reactions (Ring et al., 2010). In addition, IL-10/ Tregs failed to suppress the elicitation phase of CHS reactions, and this lack of function was not exclusively due to the loss of IL-10 production, as injection of recombinant IL-10 did not reconstitute the suppressive effects of IL-10/ Tregs (Ring et al., 2006). Therefore, we asked whether ATP fails to activate IL-10/ Tregs, which would explain their inability to suppress CHS responses independent of the absent IL-10 production. We first analyzed whether naive IL-10/ Tregs become activated in vivo after hapten application when injected into sensitized mice, as previously shown by us for wt Tregs (Ring et al., 2010). Figure 1a and Supplementary Figure S1a online show that 24 hours after challenge, IL-10/ Tregs did not upregulate the characteristic activation markers CD69 and CD44, and also that CD62L, which decreases during activation, remained unchanged. In contrast, wt Tregs become strongly activated in the blood 24 hours after challenge (Figure 1a). Thus, IL-10/ Tregs remained in a naive status. As the activation of Tregs during CHS reactions depends on the amount of extracellular ATP (Ring et al., 2010), we next investigated the effect of ATP on IL-10/ Tregs in vitro. Here, IL-10/ Tregs were insensitive to externally added ATP and remained in a naive state (Figure 1b and Supplementary Figure S1b and c online). In contrast, wt Tregs became activated by ATP in a dose-dependent manner. Nevertheless, stimulation of the T-cell receptor using anti-CD3/anti-CD28 antibodies demonstrated that IL-10/ Tregs can indeed be activated by adequate stimuli, indicating that cells do not harbor a general failure in activation. Thus, we conclude that IL-10/ Tregs are specifically impaired in their ability to become activated by ATP. It is known that activation of Tregs is necessary to induce the full arsenal of inhibitory means, and we have shown that production of adenosine in particular is largely upregulated by ATP-activated Tregs. Therefore, we compared the ability of IL-10/ Tregs with that of wt Tregs to produce adenosine in response to ATP stimulation. We detected that production of adenosine was significantly reduced in IL-10/ Tregs than in wt Tregs (Figure 2a). Remarkably, activation of IL-10/ Tregs with anti-CD3/anti-CD28 restores their ability to degrade ATP into adenosine, indicating that IL-10/ Tregs do not harbor a generalized defect in adenosine production.

Moreover, the suppressive capacity of IL-10/ Tregs preincubated with ATP on the proliferation of effector T cells was strongly reduced (Figure 2b), and the inhibitory action of IL-10/ Tregs on adherence of effector T cells to endothelial cells, which is mediated by adenosine, was abrogated (Figure 2c). To further rule out that the absence of IL-10 solely accounts for these effects, we added rmIL-10 in the flow chamber experiments. Here, we show that substitution of IL10 alone was not sufficient to prevent the adherence of T cells to endothelial cells in the in vitro flow chamber (Figure 2c). Thus, these data show that it is not the lack of IL-10 production that causes the failure of IL-10/ Tregs to suppress the interaction between T cells and endothelial cells. Instead these data indicate that IL-10/ Tregs are insensitive to ATP-mediated activation, resulting in a reduced production of adenosine as compared with wt Tregs. As a functional consequence, the suppressive capacity of IL-10/ Tregs is severely impaired. The purinergic receptor P2X7 is required for ATP-mediated activation of Tregs

To further delineate ATP-induced effects in Tregs, we set out to analyze the respective ATP receptors involved in signaling. Lymphocytes recognize extracellular ATP mainly by the purinergic receptor P2X7. Thus, blocking of P2X7 on wt Tregs with the highly selective antagonist KN-62 prevented ATPmediated upregulation of the activation markers CD69 and CD44 in a concentration-dependent manner (Figure 3a). Similarly, shedding of CD62L, which was described previously (Scheuplein et al., 2009) was inhibited by KN-62 (Supplementary Figure S2a online). In contrast, Tregs incubated with ATP and the P2X1 antagonist NF-449 were not defective in becoming activated by ATP (Supplementary Figure S2b online), demonstrating the specific involvement of the P2X7 receptor in mediating the recorded effects in Tregs. As activation of the P2X7 receptor results in opening of ion channels permeable to Ca2 þ , Na þ , and K þ ions, we further analyzed the Ca2 þ influx into Tregs after addition of ATP with or without the P2X7 antagonist KN-62. Figure 3b and Supplementary Figure S2c online show that ATP-induced Ca2 þ influx into Tregs is P2X7 dependent. The relevance of this ATP signaling for Treg function could further be demonstrated in suppression assays, showing that the suppressive function of Tregs exposed to ATP was abrogated after application of the P2X7 receptor antagonist KN-62 (Figure 3c). Thus, the purinergic receptor P2X7 on Tregs is essential for ATP-induced activation and consequently for exciting their suppressive functions. IL-10/ Tregs are impaired in Ca2 þ -mediated intracellular signaling

As our data indicate that IL-10/ Tregs do not become activated by ATP, which normally triggers P2X7 receptors, we hypothesized that the insensitivity of IL-10/ Tregs to ATPmediated activation could be due to a depressed expression of P2X7 receptors. However, when analyzing the expression of P2X7 receptors by flow cytometry, we did not detect any substantial differences in wt Tregs and in IL-10/ Tregs www.jidonline.org 1495

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Figure 1. IL-10 Tregs are insensitive to ATP-mediated activation in vivo and in vitro. (a) Isolated IL-10 or wt Tregs were labeled with PKH-PE, analyzed by flow cytometry, and injected into sensitized mice. Twenty-four hours after challenge, Tregs were analyzed in different organs. Dot plots show the expression of CD69, CD62L, and CD44 on freshly isolated and re-isolated Tregs. One representative experiment out of three is shown. (b) IL-10/ and wt Tregs were stimulated with anti-CD3/anti-CD28, ATP, or left untreated. CD69 and CD44 were analyzed after 24 hours of culture. Dot plots show the expression of the respective markers, and numbers given in each dot plot indicate the percentages of CD69 þ Tregs or the MFI of CD44 on Tregs. One representative experiment out of four is shown. ATP, adenosine triphosphate; LN, lymph node; MFI, mean fluorescence intensity; PKH-PE, PKH-phycoerythrin; Treg, regulatory T cell; Unstim., unstimulated; wt, wild type.

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Figure 2. Reduced production of adenosine by IL-10/ Tregs. (a) Wt and IL-10/ Tregs were differently stimulated for 24 hours; supernatants were analyzed for adenosine using HPLC. The graph shows the mean adenosine production of Treg±SD of four independent experiments (*Po0.001). (b) CD4 þ CD25 T cells and Tregs were stimulated separately for 24 hours. Cells were washed, and CD4 þ CD25 T cells were co-cultured with differently pretreated wt Tregs in the stated ratios. Data show the mean proliferation of CD4 þ CD25 T cells±SD of four independent experiments (*Po0.001). (c) Endothelial cells were differently treated and superfused with effector T cells. The merged pictures of flowing CD4 þ CD25 T cells after 59 minutes (red) and after 60 minutes (green) of flow are shown. Non-moving, adherent cells appear yellow. Bar ¼ 50 mm. ATP, adenosine triphosphate; HPLC, high-pressure liquid chromatograph; TNFa, tumor necrosis factor-a; Treg, regulatory T cell; wt, wild type.

(Figure 4a), and these equal amounts of P2X7 receptors on the surface of Tregs persisted even after activation of cells by TCR stimulation (Supplementary Figure S3a online). Therefore, we concluded that the abrogated activation of IL-10/ Tregs is not due to reduced numbers of P2X7 receptors expressed on their surface. As the production of adenosine by IL-10/ Tregs was greatly reduced (Figure 2a), we next analyzed the expression of the ectonucleotidases CD39 and CD73, which are the main sources for generation of adenosine by Tregs. Figure 4b and Supplementary Figure S3b online show that the expression of CD39 and CD73 by IL-10/ Tregs was not changed when compared with wt Tregs. Therefore, similar to results obtained with the P2X7 receptor, the differential expression of CD39 or CD73 is not responsible for the reduced adenosine production recorded in IL-10/ Tregs. As the production of adenosine nevertheless was abrogated in IL-10/ Tregs, despite the normal expression of CD39 and CD73, we conclude that failure of IL-10/ Tregs to become activated by ATP also prevents stimulation of the catalytic activity of the ectonucleotidases. In light of these results, we further hypothesized that signaling of ATP receptors may be altered in IL-10/ Tregs. As we have demonstrated that engagement of P2X7 receptors by ATP results in a Ca2 þ influx into cells, we next analyzed the increase in intracellular Ca2 þ in IL-10/ Tregs in response to ATP. In Figure 4c and Supplementary Figure S4 online, we show significantly reduced Ca2 þ fluxes in IL-10/ Tregs stimulated with ATP as compared with wt Tregs.

However, Ca2 þ influx after treatment with PMA (phorbol 12-myristate 13-acetate)/ionomycin was comparable in both cell populations, indicating that IL-10/ Tregs do not suffer from a generalized defect in Ca2 þ -mediated signaling. Therefore, we conclude that in IL-10/ Tregs, ATP-induced elevation of intracellular calcium levels is impaired, which may affect the activation of Ca2 þ -mediated signaling further downstream. Extracellular IL-10 does not compensate for the lack of intracellular IL-10 in IL-10/ Tregs

We next tested whether addition of extracellular rmIL-10 to cultures of IL-10/ Tregs with ATP restores the ability of IL-10/ Tregs to respond to ATP signals. Analyzing the surface expression of activation markers and determining adenosine production in the supernatants of Treg cultures demonstrated no influence of rmIL-10 on IL-10/ Tregs (Figure 5a and c). These data are consistent with our results obtained in the flow chamber (Figure 2c), showing that addition of rmIL-10 to IL-10/ Tregs was not sufficient to restore their suppressive capacity. Analysis of the expression of IL-10 receptors on IL-10/ Tregs in comparison with wt Tregs revealed that both cell types express equal amounts of the receptor (data not shown), indicating that the unresponsiveness of IL-10/ Tregs to rmIL-10 is not due to differential expression of the IL-10 receptor in the respective Tregs. Vice versa, we could show that neutralization of extracellular IL-10 with anti-IL-10 in cultures of wt Tregs did not result in an unresponsiveness of wt Tregs to stimulation www.jidonline.org 1497

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by ATP (Figure 5b and d). Therefore, we conclude that the insensitivity of IL-10/ Tregs to activation by ATP (and consequently the reduced production of adenosine) is neither due to the amount of extracellular IL-10 nor caused by differential IL-10 receptor expression. Instead it is conceivable that differences in intracellular signaling transduced by P2X7 receptors may account for the reduced activation/suppression of IL-10/ Tregs. DISCUSSION Here, we have shown that IL-10/ Tregs are severely impaired in their activation by ATP and production of adenosine as compared with wt Tregs. These results are particularly important as IL-10/ Tregs are frequently used in experimental settings to determine the relative contribution of IL-10 production to suppression by Tregs. The pleiotropic suppressive effects of IL-10 in general are well known, and its production by Tregs seems to be crucial for suppression of colitis, experimental autoimmune encephalomyelitis, and also during tumor growth (Asseman et al., 1999; Zhang et al., 2004; Nishikawa and Sakaguchi, 2010). So far, IL-10 has been believed to be a major soluble suppressive factor produced by Tregs. However, more recently, we along with others have established that extracellularly generated adenosine also conveys suppressive effects (Borsellino et al., 2007; Deaglio et al., 2007; Sitkovsky 1498 Journal of Investigative Dermatology (2011), Volume 131

et al., 2008; Ring et al., 2009). For example, in CHS reactions, it suppresses the upregulation of E- and P-selectin and thus blocks inflammatory reactions (Ring et al., 2009). Moreover, in tumor environments, Treg-derived adenosine may be a key component that impedes anti-tumor immunity (Ohta et al., 2006; Pellegatti et al., 2008; Di Virgilio et al., 2009a). Thus, at least two different mechanisms of Treg-mediated suppression by soluble factors exist: IL-10 release and adenosine production. So far, Tregs from IL-10/ mice were frequently used to verify the relative contribution of Treg-derived IL-10 in suppression in various experimental settings (Asseman et al., 1999; Zhang et al., 2004). However, our data indicate that IL-10/ Tregs are not only defective in IL-10 production but also do not produce adenosine in response to ATP. Thus, IL-10/ Tregs may not be the right tool to specifically determine the effects of IL-10 during Treg-mediated immune suppression. Instead the ATP/adenosine turnover by Tregs has to be considered too. ATP acts as a ‘‘danger’’ signal that is also referred to as ‘‘damage-associated molecular pattern’’ (Di Virgilio, 2007). ATP is a molecule to signal cell death and destruction as it is present in high concentrations (1 mM) in almost all cells, but its extracellular concentration is kept at very low levels (below 20 nM). Therefore, a passive release of small amounts of ATP in response to cell injury is easy to detect by

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Figure 4. IL-10/ Tregs are impaired in calcium-mediated intracellular signaling. Expressions of the (a) purinergic receptor P2X7 and the (b) ectonucleotidases CD39 and CD73 were detected by flow cytometry on wt and IL-10/ Tregs. Data in panels a and b show the expression of the indicated molecule on Tregs each in one representative experiment out of five. (c) Intracellular calcium mobilization was determined in differently treated wt Tregs and IL-10/ Tregs. The fluorescence intensity corresponds to the amount of free intracellular calcium after 1 hour. One representative experiment out of three is shown. Bar ¼ 100 mm. ATP, adenosine triphosphate; PMA, phorbol 12-myristate 13-acetate; Treg, regulatory T cell; wt, wild type.

leukocytes and may serve as an activating trigger for proinflammatory cells (Di Virgilio et al., 2009b). ATP signals predominantly through the purinergic P2X7 receptor (Lister et al., 2007), which is expressed primarily by cells of hematopoietic origin. Our data showing that blockage of the P2X7 receptor by antibodies results in abrogated suppression, corroborate this notion and indicate that Tregs use the P2X7 receptor to sense ATP in the environment. The importance of this ATP-mediated activation for in vivo function of Tregs can be deducted from experiments investigating the role of Tregs in CHS responses. Here, ATP is produced at the site of antigen challenge, i.e., skin and blood vessels, which then causes activation and adenosine production of local Tregs. After blockage of P2X7 receptors on the surface of Tregs, the suppressive action is abrogated in vivo. This indicates that ATP-induced activation of Tregs is an important means by which inflammatory reactions, such

as the CHS response, trigger the suppressive function of Tregs (Ring et al., 2010). The further downstream signaling of P2X7 receptors in lymphocytes relies on pore formation and induction of calcium influx into cells (Corriden and Insel, 2010), but the detailed mechanisms as to how the signaling of P2X7 receptors is regulated in leukocytes are not completely understood. For example, pore size, which determines what ions can travel across the cell membrane, is variable and seems to be dependent on ATP concentration (Chused et al., 1996; Virginio et al., 1999). That is, low concentrations of ATP induce Na þ , Ca2 þ , and K þ fluxes, whereas high concentrations lead to sustained pore opening and increasing diameter, allowing molecules of up to 450 Da to travel. Moreover, even the downstream events following P2X7 receptor engagement seem to differ, depending on the strength of the signal. For example, prolonged opening mediates proinflammatory www.jidonline.org 1499

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cytokine release and cell death, whereas small amounts of ATP trigger cell proliferation and release of anti-inflammatory mediators (Adinolfi et al., 2005). Nevertheless, we tested ATP over a broad range of concentrations, but IL-10/ Tregs failed to display elevated calcium levels, produced only low amounts of adenosine, and did not upregulate CD69 in response to ATP. In this sequence of events, the increase in intracellular calcium is the first step after engagement of P2X7 receptors and it subsequently acts as a second messenger to induce cell-activating signals. Thus, as P2X7 receptors do not affect the intracellular calcium levels in IL-10/ Tregs in the first place, signaling events, which are normally induced by elevated intracellular calcium in wt Tregs, are not triggered in IL-10/ Tregs and cells remain inactive. However, as calcium and other ions, which may be able to cross ATP-induced pores, have pleiotropic effects in several signal transduction pathways in cells, the detailed mechanisms as to how the P2X7 receptor is impaired in signal transduction in IL-10/ Tregs have to be determined in future investigations. The cross-talk between P2X7 receptor signaling and cytokine release has been investigated inasmuch as ATP receptor engagement increased the production of IL-10, tumor necrosis factor-a, IL-1b, and IL-6 (Solini et al., 1999; Hide et al., 2000; Swennen et al., 2005; Ferrari et al., 2006; Seo et al., 2008). In particular for IL-10, an autocrine loop has been proposed in microglia cells. Here, stimulation of cells with lipopolysaccharide induces ATP release that act back on cells by inducing IL-10 release (Seo et al., 2008). However, we tested the opposite pathway by adding recombinant IL-10 to IL-10/ Tregs and 1500 Journal of Investigative Dermatology (2011), Volume 131

subsequently analyzing for activation, for adenosine production, and for P2X7 receptor expression of cells. Here, we did not record any changes in wt or 10/ Tregs, thus, indicating that ATP-induced effects on Tregs do not seem to be modulated by exogenous IL-10, and a feedback loop in which IL-10 and adenosine regulate each other is not responsible for the observed effects. However, one can only speculate on the underlying mechanisms. For example, IL-10 may be required intracellularly; therefore, exogenous IL-10 is ineffective. In addition, prolonged exposure of Tregs to autocrine produced IL-10, or additional so far unidentified soluble factors produced by bystander cells in vivo, may be necessary to maintain the responsiveness of Tregs to ATP. Despite the fact that the underlying mechanism remains to be elucidated, our data show that IL-10/ Tregs do not become activated by ATP and do not produce immunosuppressive adenosine in sizable amounts. Thus, the usage of IL-10/ Tregs to pinpoint the effects of IL-10 in Treg-mediated suppression needs to be revisited and effects mediated by adenosine have to be taken into account in the future. MATERIALS AND METHODS Mice Bl6 mice were purchased from Charles River (Sulzfeld, Germany) and housed at the central animal facility of the University of Heidelberg. IL-10/ mice on a C57BL/6 background were obtained from Jackson Laboratories (Bar Harbor, ME). All experiments were approved by the state of Baden-Wu¨rttemberg and performed in accordance with the governmental guidelines.

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Analysis of the activation status of injected naive Tregs during CHS reaction CD4 þ CD25 þ Tregs were isolated from lymph node and spleen cells using the CD4 þ CD25 þ Regulatory T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Tregs were labeled with the fluorescent dye PKH26, and 3  106 PKH26-labeled Tregs were injected intravenously into 2,4,6-trinitrochlorobenzene sensitized mice, followed by 2,4,6-trinitrochlorobenzene challenge on the ear. Mice were killed 24 hours after Treg injection, and activation statuses of Tregs were analyzed in the cell suspension of different organs by flow cytometry.

Determination of the activation status and detection of adenosine Overall, 2  105 Tregs were cultured in 200 ml per well in 96-well roundbottom plates and stimulated either with anti-CD3 and anti-CD28 (BD Biosciences, Heidelberg, Germany, 0.5 mg ml1 each) or with different amounts of ATP (Sigma-Aldrich, Taufkirchen, Germany) (75, 125, 200 mM). IL-10 (BD Biosciences; 10 or 25 ng ml1) or anti-IL10 (BD Biosciences, clone JES5-2A5; 1 or 10 mg ml1) was added in some experiments. Activation statuses of cells were analyzed after 24 hours by flow cytometry. The supernatants were analyzed for adenosine using a high-pressure liquid chromatograph. A volume of 20 ml aliquots of the samples was measured with a 0–7% acetonitrile/ H2O gradient mobile phase and absorption was measured at 260 nm.

Proliferation assay CD4 þ T cells and Tregs were stimulated separately with either antiCD3 and anti-CD28 (0.5 mg ml1 each) or ATP (125 mM) for 24 hours. Cells were washed thoroughly and co-cultured in different ratios without further stimulation. After 24 hours, 0.5 mCi ml1 [3H]thymidine (Amersham, Freiburg, Germany) was added for 18 hours, cells were harvested, and thymidine incorporation was determined using a Perkin-Elmer scintillation counter (Perkin-Elmer, Rodgau, Germany).

Flow chamber The murine EC line (bEND.3; ATCC, Wesel, Germany) was cultured on cover slips and stimulated with tumor necrosis factor-a (1 mg ml1) or tumor necrosis factor-a together with Tregs (5  105 per ml). After 24 hours, the cover slips were placed in a heated flow chamber (Chromaphor, Bottrop, Germany) and superfused with fluorescent dye-labeled CD4 þ T cells. The number of rolling and adherent cells per visible field was determined using video microscopy.

Detection of intracellular calcium mobilization Intracellular calcium mobilization was determined using the FluoForte Calcium Assay Kit (Enzo Life Sciences, Lo¨rrach, Germany). Tregs were resuspended in dye-loading solution, and 1.75  105 cells per 100 ml per well were cultured in a 96-well flat-bottom plate with or without KN-62. The plate was centrifuged with brake off and incubated for 1 hour at room temperature. PMA/ionomycin or ATP (200 mM) was added to the wells and the increase in fluorescence was monitored using a fluorescence microscope (excitation ¼ 490 nm/ emission ¼ 525 nm). Pictures were taken every 10 minutes for 1 hour. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS We thank Mrs Thome for excellent technical help. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 405 and Ma1924), the European Union (LSHC-2005-518178), and the Tumor Center HeidelbergMannheim (TZHDMA) to AH Enk and K Mahnke. S Ring and K Mahnke were supported by the Helmholtz Alliance against cancer. SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http:// www.nature.com/jid

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1502 Journal of Investigative Dermatology (2011), Volume 131

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