1330

Laurie Prigent et al.

DOI: 10.1002/eji.201343920

Eur. J. Immunol. 2014. 44: 1330–1340

The aryl hydrocarbon receptor is functionally upregulated early in the course of human T-cell activation Laurie Prigent ∗1 , Marc Robineau ∗1 , St´ephane Jouneau1,2 , Claudie Morzadec1 , Laetitia Louarn1 , Laurent Vernhet1 , Olivier Fardel1,3 and Lydie Sparfel1 1

UMR INSERM U1085, Institut de Recherche sur la Sant´e, l’Environnement et le Travail (IRSET), Universit´e de Rennes 1, Rennes, France 2 Service de Pneumologie, Centre Hospitalier Universitaire, Rennes, France 3 Pˆ ole Biologie, Centre Hospitalier Universitaire, Rennes, France The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates immunosuppression caused by a variety of environmental contaminants, such as polycyclic aromatic hydrocarbons or dioxins. Recent evidence suggests that AhR plays an important role in T-cell-mediated immune responses by affecting the polarization and differentiation of activated T cells. However, the regulation of AhR expression in activated T cells remains poorly characterized. In the present study, we used purified human T cells stimulated with anti-CD3 and anti-CD28 Abs to investigate the effect of T-cell activation on AhR mRNA and protein expression. The expression of AhR mRNA increased significantly and rapidly after T-cell activation, identifying AhR as an immediate-early activation gene. AhR upregulation occurred in all of the T-cell subtypes, and is associated with its nuclear translocation and induction of the cytochromes P-450 1A1 and 1B1 mRNA expression in the absence of exogenous signals. In addition, the use of an AhR antagonist or siRNA-mediated AhR knockdown significantly inhibited IL-22 expression, suggesting that expression and functional activation of AhR is necessary for the secretion of IL-22 by activated T cells. In conclusion, our data support the idea that AhR is a major player in T-cell physiology.

Keywords: Aryl hydrocarbon receptor r Human T lymphocytes r T-cell activation



Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction The aryl hydrocarbon receptor (AhR) is a widely expressed cytosolic protein belonging to the basic helix-loop-helix superfamily of transcriptional factors. It plays important physiological roles, and its absence is associated with a large spectrum of hepatic and skin defects as well as abnormalities in vascular and hemato-

Correspondence: Dr. Lydie Sparfel e-mail: [email protected]  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

poietic development in transgenic AhR-deficient mice [1]. Discovered almost 30 years ago as a specific binding receptor for aromatic hydrocarbons [2], the AhR has been studied primarily for its responsiveness to environmental contaminants present in tobacco smoke, polluted city air, and certain foods [3]. Among these contaminants, halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) and nonhalogenated



These authors contributed equally to this work.

www.eji-journal.eu

Eur. J. Immunol. 2014. 44: 1330–1340

polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) are the most commonly associated with a variety of toxic effects, including carcinogenic and immunosuppressive effects, and contribute to smoking-related diseases [4]. Most of these effects have been linked to the activation of AhR and subsequent transcription of AhR-regulated genes. Indeed, after ligand binding, AhR translocates into the cell nucleus where it dimerizes with the AhR nuclear translocator protein and interacts with xenobiotic responsive elements located upstream in the promoters of target genes, including the xenobiotic metabolizing enzymes of the cytochrome P-450 superfamily, (cytochromes P-450 (CYPs)) 1A1 and 1B1 [5]. Increasing experimental evidence supports an important role for AhR in the regulation of immune responses. Early studies on AhRdeficient mice reported altered lymphocyte numbers in the spleen, increased incidence of infection [6], and resistance to dioxininduced immunosuppression [7], suggesting that T-cell-mediated immune responses are among the most sensitive targets of dioxin toxicity. Other studies have suggested that AhR ligation in Th cells does not lead to immunosuppression but, instead, enhances the secretion of various mediators implicated in immune pathology. Thus, Negishi et al. [8] reported that AhR ligation by an antiallergic compound modulates the in vivo Th1/Th2 cytokine balance toward Th1 cell dominance and enhances the production of IFN-γ. Moreover, 6-formylindolo[3,2-b]carbazole, proposed to be an endogenous AhR ligand present in humans [9], promotes the expansion of IL-17-producing T cells (Th17) [10–12]. In addition, a recent study showed that activation of AhR by dietary ligands is involved in mucosal immune responses, by directly upregulating IL-22 [13]. Altogether, these studies implicate the AhR as a central player in T-cell-mediated immune responses. In support of its putative functions in T cells, high expression of AhR has been described in Th17 cells and in some Treg cells, including Foxp3negative type 1 Treg cells [14, 15], whereas AhR levels remain lower in Th1 and Th2 cells. However, the molecular mechanisms controlling AhR expression in T cells are unknown. In this context, we have hypothesized that there is a link between activation of T cells and AhR expression. Indeed, T-cell-enriched lymphocyte cultures exhibited significant inducible CYP1A1-mediated Ah hydroxylase activity only upon preliminary T-cell activation by mitogenic lectins [16], supporting the idea that AhR is present and functional only in activated T cells and not in resting counterparts. The present study was designed to gain insight into this hypothesis. We report that activation of human T cells triggers an early and functional upregulation of AhR expression, which occurs before the start of T-cell proliferation and that is involved in IL-22 production, establishing AhR as a major player in T-cell physiology.

Results Stimulation of human T cells leads to a specific and early induction of AhR expression Human CD3+ T cells purified from healthy blood donors were activated with anti-CD3 and anti-CD28 Abs, which mimic physio C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Cellular immune response

logical T-cell activation, and the timecourse of AhR mRNA expression was analyzed using RT-qPCR. Activated human T cells exhibited a rapid and robust increase in AhR mRNA expression at 2 h after stimulation (Fig. 1A). The increase in AhR mRNA expression reached a maximal level at 4 h, started to decline at 8 h, and returned to basal level after 72–120 h (Fig. 1A). A similar, although delayed, increase in cellular AhR protein was detected at 8 h, and reached a maximal level at 24–72 h after CD3/CD28mediated activation (Fig. 1B). Next, we compared the kinetics of AhR mRNA upregulation with the upregulation of other T-cell activation markers in response to CD3/CD28-mediated activation. mRNA expression of the early activation genes CD40L, CD69, and TNF-α showed similar kinetics to that of AhR mRNA over the same time course of T-cell activation, whereas the late activation genes CTLA-4 and CD25 mRNA were induced later, indicating that AhR behaves as an early activated gene (Fig. 1C). The increase in CD69, CD40L, CTLA-4, and CD25 mRNA expression was associated with an enhanced cell surface expression of the corresponding proteins (Fig. 1D). In contrast to AhR mRNA, mRNA expression of other transcription factors, such as AhR nuclear translocator, and the ligand-activated transcription factors pregnane X receptor, and glucocorticoid receptor, were not upregulated in activated human T cells over a 4, 8, and 24 h period (Fig. 1E), indicating that the observed increase in AhR mRNA expression is not the result of unspecific induction of transcription factor mRNAs after T-cell activation. In addition to CD3/CD28 stimulation, the use of other T-cell activation stimuli, such as the lectin PHA, a combination of the PKC activator PMA and the Ca2+ ionophore ionomycin, and allogeneic DC, triggered increased AhR mRNA expression (Fig. 1F), demonstrating that induction of AhR expression occurs regardless of the nature of the T-cell activation signal. To determine whether the increase in AhR expression is restricted to certain T-cell subtypes, we investigated the effect of CD3/CD28-mediated activation on AhR expression in various T-cell populations, including CD4+ and CD8+ T cells, na¨ıve and memory CD4+ T cells, and several CD4+ Th-cell subsets such as Th1, Th2, and Th17. Upon activation, both CD4+ and CD8+ T cells exhibited a marked increase in AhR mRNA levels at 4 h after CD3/CD28 stimulation, followed by an increase in cellular AhR protein (Fig. 2A). Similarly, human CD4+ na¨ıve CD45RA+ and memory CD45RO+ T cells from the same donor exhibited upregulated AhR mRNA and protein levels after a 4- and 48-h activation, respectively (Fig. 2B). Next, we investigated whether the activation-mediated increase in AhR expression occurs in various Th cells subsets, including functional Th1, Th2, and Th17 cells (Supporting Information Fig. 1). Upon activation, the AhR mRNA and protein levels increased in all of the CD4+ T-cell subsets studied to the same extent as in Th0 cells (Fig. 2C), with the exception of AhR expression in Th17 cells, which was higher (Fig. 2C and D) as previously reported [17], indicating that the initial upregulation of AhR is not driven by cytokines controlling T-cell polarization. www.eji-journal.eu

1331

1332

Laurie Prigent et al.

Eur. J. Immunol. 2014. 44: 1330–1340

Figure 1. AhR expression in activated human T cells. CD3+ T cells were either unstimulated (UNS) or stimulated with (A–E) anti-CD3 and anti-CD28 Abs for the indicated lengths of time or with (F) PHA, PMA/ionomycin, or allogeneic DC for 24 h. (A, F) AhR, CD40L, CD69, TNF-α, CTLA-4, and (C) CD25 and (E) ARNT, pregnane X receptor, and glucocorticoid receptor mRNA expression was analyzed using RT-qPCR. Data are expressed relative to mRNA expression levels in unstimulated T cells, arbitrarily set at 1 unit, and are shown as mean + SEM of duplicate samples pooled from four independent experiments. (B) Expression of AhR protein was analyzed by Western blotting. Actin level was used as a loading control. Data are from one experiment representative of three independent experiments performed. (D) Cell surface expression of CD69, CD40L, CTLA-4, and CD25 was measured by flow cytometry. Data are expressed relative to the MFI of unstimulated T cells, arbitrarily set at 1 unit, and are shown as mean ± SEM of four samples pooled from independent experiments. *p < 0.05 when compared with unstimulated T cells (ANOVA followed by the Dunnett’s multirange test).

Stimulation of human T cells induces functional AhR activation We then performed experiments to determine the functional consequence of increased AhR expression in CD3/CD28-activated T cells. The cellular distribution of AhR in unstimulated and  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

CD3/CD28-activated T cells was examined by Western blotting and immunofluorescence labeling. Consistent with previous experiments using whole cell extracts (Fig. 1B), increased levels of AhR protein were observed in the cytoplasm of activated T cells (Fig. 3A). In addition, there was an increase in nuclear AhR expression in activated T cells when compared with their www.eji-journal.eu

Eur. J. Immunol. 2014. 44: 1330–1340

Cellular immune response

AhR upregulation in response to T-cell activation involves the NF-κB signaling pathway

Figure 2. AhR induction in various CD3/CD28-activated human T-cell populations. (A) CD4+ and CD8+ , (B) na¨ıve and memory CD4+ , and (C) CD4+ T cells were either unstimulated (UNS) or stimulated with antiCD3 and anti-CD28 Abs for the indicated lengths of time (A, B) and in the absence (Th0) or presence of Th1-, Th2-, and Th17-polarizing cytokines (Th1, Th2, and Th17) for 120 h (C), and in the presence of Th17-polarizing cytokines for various lengths of time (D). AhR mRNA expression was analyzed using RT-qPCR. Data are expressed relative to the AhR mRNA expression levels in unstimulated T cells, arbitrarily set at 1 unit, and are shown as mean ± SEM of duplicate samples pooled from five (A, C) and four (B, D) independent experiments. *p < 0.05 when compared with unstimulated T cells (ANOVA followed by the Dunnett’s multirange test). Expression of AhR protein was analyzed by Western blotting. Actin level was used as a loading control. Data are from one experiment representative of (A) four, (B) three, and (C) two independent experiments performed.

unstimulated counterparts (Fig. 3A). Similar results were obtained using immunolocalization assays (Fig. 3B). To determine whether the detected AhR nuclear translocation is associated with a transcriptionally active form of AhR, we analyzed mRNA expression of AhR target genes, such as CYP1A1, CYP1B1, and AhRR. mRNA levels of these different AhR target genes were increased by T-cell activation (Fig. 3C) and the addition of exogenous AhR ligands, such as dioxin and BaP, further increased CYP1A1 and CYP1B1 mRNA expression in activated human T cells (Fig. 3D).  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

To investigate the molecular mechanisms leading to AhR upregulation in response to CD3/CD28-mediated T-cell activation, the effects of the transcription inhibitor actinomycin D (ActD) and the translation inhibitor cycloheximide (CHX) on AhR mRNA induction were analyzed. ActD fully suppressed the increase in AhR mRNA expression in activated T cells, whereas CHX did not significantly alter AhR mRNA induction (Fig. 4A), indicating that the increase in mRNA levels occurs mainly at the transcriptional level and requires new RNA but not new protein synthesis. Next, we investigated various transcription factor signaling pathways that are activated upon T-cell activation. CD3/CD28mediated T-cell activation triggers a complex set of signaling pathways, causing the activation of well-characterized transcription factors such as NFAT and NF-κB (Supporting information Fig. 2A) to enhance IL-2 gene expression and increase T-cell proliferation. Since CD3/CD28-mediated T-cell activation after TCR engagement is cyclosporine A (CsA) sensitive [18] (Supporting information Fig. 2B), we examined the ability of this potent immunosuppressor inhibiting T-cell proliferation through preventing activation of NFAT to affect the expression of AhR in activated T cells. As expected, CsA significantly suppressed CD3/CD28mediated IL-2 gene expression, whereas it did not have a significant effect on PMA/CD28-induced IL-2 mRNA expression (Supporting information Fig. 2B). Interestingly, CsA did not modify the upregulation of AhR gene expression in either CD3/CD28or PMA/CD28-activated T cells (Supporting information Fig. 2C). Likewise, CD3/CD28-mediated upregulation of AhR protein expression was unaffected by CsA (Supporting information Fig. 2C). Altogether, these data suggest that the induction of AhR expression occurs independently of NFAT activation. Considering that the use of a combination of PMA/ionomycin (which bypasses TCR engagement) enhanced AhR expression, we analyzed the effect of PMA or ionomycin alone on AhR expression in T cells. Treatment with PMA alone induced AhR mRNA expression, whereas ionomycin alone did not significantly increase AhR expression (Fig. 4B), supporting the involvement of PMA-activated PKC in AhR induction in activated T cells. The involvement of the PKC pathway in the induction of AhR expression was confirmed by treating activated T cells with several pharmacological PKC inhibitors. Treatment with the pan-PKC inhibitor GFX109203 or the isoform-selective PKC inhibitors G¨ o6976 and rottlerin significantly inhibited AhR expression, whereas treatment with KN62, an inhibitor of Ca2+ -dependent pathways, or PD98059, a MEK inhibitor, did not (Fig. 4C). The NF-κB inhibitors, Bay 11-7082 and pyrrolidine dithiocarbamate, also significantly reduced AhR mRNA expression in activated T cells (Fig. 4C), suggesting that the NF-κB signaling pathway, activated by PKC in T cells (Supporting information Fig. 2A), participates in the induction of AhR expression. Since we identified three NF-κB putative binding sites located at positions −438, −461, and −479 on the AhR promoter (data not shown), we hypothesized that NF-κB may play a direct role in CD3/CD28-mediated induction of AhR expression. www.eji-journal.eu

1333

1334

Laurie Prigent et al.

Eur. J. Immunol. 2014. 44: 1330–1340

Figure 3. AhR functional activation in CD3/CD28-activated human T cells. (A–C) CD3+ T cells were either unstimulated or stimulated with anti-CD3 and anti-CD28 Abs and (D) either untreated (UNT) or treated with 10 nM dioxin or 2 μM BaP for 24 h in the absence or presence of anti-CD3 and anti-CD28 Abs. (A) Expression of AhR protein in cytosolic and nuclear extracts was analyzed by Western blotting. HSC70 and ARNT levels were used as a loading control. (B) AhR immunolocalization was analyzed by fluorescence microscopy (magnification ×40). (A, B) Data are from one experiment representative of two independent experiments performed. (C) AhRR and (C, D) CYP1A1, and CYP1B1 mRNA expression was analyzed using RT-qPCR. Data are expressed relative to mRNA expression levels in unstimulated T cells, arbitrarily set at 1 unit, and are shown as mean ± SEM of duplicate samples pooled from (C) five and (D) six independent experiments. *p < 0.05 when (C) compared with unstimulated T cells (ANOVA followed by the Dunnett’s multirange test) and (D) untreated CD3/CD28-activated T cells (ANOVA followed by the Tukey– Kramer multiple comparison test).

Stimulation of human T cells led to the activation of NF-κB signaling characterized by the phosphorylation of I-κBα, the translocation of the p65 subunit of NF-κB from the cytosol to the nucleus and its binding to NF-κB consensus sites (Supporting information Fig. 3A, B, D, and E). We also observed an increase in TNF-α and CD69 mRNA levels, which are classically induced by NF-κB and suppressed by the NF-κB inhibitor Bay 11-7082 (Supporting information Fig. 3C). In addition, EMSA has revealed an increase in the intensity of the complexes formed between the T-cell nuclear protein extracts and radiolabeled NF-κB-438 and NF-κB-479 probes after a 2-h PMA/ionomycin-mediated activation; however, the formation of such complexes appears unaffected by addition of excess unlabeled NF-κB consensus oligonucleotide probe (Supporting information Fig. 3F).

Functional activation of AhR in response to T-cell activation is involved in IL-22 secretion To address the physiological role of AhR functional activation in stimulated human T cells, the effect of the potent AhR antagonist CH-223191 was investigated during several T-cell activa-

 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

tion events. CH-223191 did not significantly affect CD69 or CD25 cell surface expression in activated human T cells (Fig. 5A). In addition, treatment with CH-223191 affected neither CD3/CD28induced T-cell proliferation assessed by 3 H-methylthymidine incorporation nor CD3/CD28-mediated IL-2 production measured by ELISA, suggesting that AhR signaling has no effect on T-cell proliferation (Fig. 5B and C). Interestingly, CH-223191 strongly inhibited IL-22 production by activated human T cells, whereas it did not significantly alter IL-17 production upon activation (Fig. 5C). To further characterize the contribution of AhR in these T-cell activation and proliferation events, we knocked down its expression by transient transfection of siRNA into human T cells. SiRNA-mediated AhR knockdown efficiency was validated at mRNA level by the significant decrease of AhR and its reference target gene CYP1B1 in siAhR-transfected CD3/CD28-activated T cells as compared with their non-targeting (siCTR) siRNA counterparts (Fig. 5D). This siRNA-mediated downregulation of AhR failed to alter CD25, IL-2, and IL-17 mRNA expression but significantly inhibited IL-22 mRNA expression (Fig. 5D). Altogether, these data indicate that signals from AhR are required for the expression and production of IL-22 during T-cell activation. Exposure to cigarette smoke extracts, which contain well-established

www.eji-journal.eu

Eur. J. Immunol. 2014. 44: 1330–1340

Cellular immune response

Figure 4. Effects of PKC and NF-κB signaling pathways on T-cell activation-mediated AhR mRNA induction in human T cells. CD3+ T cells were (A) either untreated (UNT) or pretreated with 5 μg/mL actinomycin D (ActD) or 5 μg/mL cycloheximide (CHX) in the absence or presence of anti-CD3 and anti-CD28 Abs for 4 h, (C) with various inhibitors of T-cell activation signaling pathways for 1 h before CD3/CD28-mediated activation for 4 h, and (B) either unstimulated (UNS) or stimulated with PMA or ionomycin for 4 h. AhR mRNA expression was analyzed using RTqPCR. Data are expressed relative to the AhR mRNA expression levels in unstimulated T cells, arbitrarily set at 1 unit, and are shown as mean ± SEM of duplicate samples pooled from six independent experiments. *p < 0.05 when compared with (B) unstimulated T cells (ANOVA followed by the Dunnett’s multirange test) and (A, C) untreated CD3/CD28-activated T cells (ANOVA followed by the Tukey–Kramer multiple comparison test).

Figure 5. Effects of CD3/CD28-mediated AhR induction in IL-22 production by human T cells. (A–C) CD3+ T cells were either untreated (UNT) or pretreated with 3 μM CH-223191 for 1 h before CD3/CD28mediated activation for (A, C) 48, (B) 72, and (C) 120 h. (A) Cell surface expression of CD69 and CD25 was measured by flow cytometry. Data are expressed as percentage of positive cells and shown as mean + SEM of five samples pooled from independent experiments. (B) After 72 h, 3 H-methylthymidine incorporation into DNA was determined during the last 18 h of culture. Data are expressed as cpm/μg protein and shown as mean + SEM of triplicate samples pooled from six independent experiments. (C) IL-2, IL-17, and IL-22 concentrations were measured by ELISA after 48 (IL-2) and 120 h (IL-17 and IL-22). Data are expressed as nanogram per milliliter and shown as mean + SEM of duplicate samples pooled from three (IL-2) and six (IL-17 and IL-22) independent experiments. (D) CD3+ T cells were transfected with AhR-specific (siAhR) or nontargeting (siCTR) siRNA and after 48 h were stimulated with anti-CD3 and anti-CD28 Abs for 30 h. mRNA expression of AhR, CYP1B1, CD25, IL-2, IL-17, and IL-22 was analyzed using RT-qPCR. Data are expressed relative to mRNA expression levels in siCTR-transfected CD3/CD28-activated T cells, arbitrarily set at 1 unit, and are shown as mean + SEM of duplicate samples pooled from five independent experiments. *p < 0.05 when compared with (C) untreated (paired Student’s t-test) and (D) siCTR-transfected (unpaired Student’s t-test) CD3/CD28-activated T cells.

agonists of AhR such as polycyclic aromatic hydrocarbons, leads to increased production of IL-22 in primary T lymphocytes [19]. Therefore, we examined the effects of T-cell activation on IL-22 production by using T cells isolated from healthy nonsmokers, past and current smokers with normal lung function, or chronic

obstructive pulmonary disease (COPD) patients with altered lung function [20]. IL-22 production was higher in activated T cells isolated from current smokers than in those of healthy nonsmokers or past smokers with or without COPD (Fig. 6A). Since no association was found between COPD and IL-22 secretion and considering the

 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.eji-journal.eu

1335

1336

Laurie Prigent et al.

effect of current smoking on IL-22 production by activated T cells, the effect of the AhR antagonist CH-223191 was analyzed in all current smokers, past smokers and nonsmokers. CH-223191 significantly inhibited IL-22 production by activated T cells isolated from nonsmokers, past smokers, and current smokers, whereas it did not significantly alter IL-17 production (Fig. 6B), indicating that IL-22 production in these study subjects depended on T-cell activation-mediated upregulation of AhR.

Discussion The results presented in this paper indicate that AhR expression in human T cells is significantly increased by cellular activation. To the best of our knowledge [21], this is the first study to analyze the cellular and molecular mechanisms controlling AhR expression in human T cells. Our data clearly show a marked and specific increase in AhR levels following a physiologically relevant T-cell stimulation approach using anti-CD3 and anti-CD28 Abs. Other types of T-cell activation stimuli such as PHA or PMA/ionomycin also induced AhR expression, indicating a general upregulation of AhR expression by T-cell activation, whatever the nature of the activating signal. Moreover, AhR upregulation occurs in various T lymphocyte subtypes and is not restricted to Th17 cells, although Th17 cells exhibit higher AhR expression than other T-cell subtypes, as previously reported [17]. Interestingly, an increase in AhR expression has also been reported in activated human B lymphocytes [22], as well as in stimulated murine splenic T lymphocytes [23], suggesting a critical role for AhR in the physiology of activated lymphocytes. In our study, the increase in AhR mRNA expression after T-cell activation occurred rapidly and transiently and was dependent on new RNA synthesis, but independent of new protein synthesis, as previously reported for several early T-cell activation genes such as TNF-α, CD40L, and c-myc [24]. These data identify AhR as an immediate-early activation gene. Among the signaling pathways involved in regulation of immediate-early activation genes in T cells, the key transcription factor NFAT is unlikely to play a major role in AhR upregulation, owing to the absence of NFAT putative binding sites in the promoter region of AhR (data not shown) and the lack of effects of the calcineurin/NFAT inhibitor CsA on AhR mRNA induction. On the other hand, the use of NF-κB inhibitors counteracted the upregulation of AhR, suggesting that the molecular mechanisms responsible for T-cell activation-mediated AhR mRNA induction are associated with signaling events related to the NF-κB signaling pathway. One NF-κB site identified on the AhR promoter has been recently shown to be responsible for mediating the induction of AhR expression by LPS in U937-derived DC [25], but the exact role of the NF-κB signaling in the upregulation of AhR expression in activated T cells remains to be determined. The increased expression of AhR in activated T cells has functional consequences such as its nuclear translocation and induction of CYP1A1 and CYP1B1 mRNA expression in the absence of exogenous ligands. Crawford et al. [26] reported similar findings, showing that leukocyte activation by PMA/ionomycin induces AhR  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Eur. J. Immunol. 2014. 44: 1330–1340

upregulation, DNA binding, and increased CYP1A1 expression in the absence of exogenous ligands. It is tempting to speculate that the increase in CYP1A1 expression is caused by the binding of an endogenous ligand to the AhR, as previously reported [27]. Another possible explanation for the induction of CYP1A1 in activated T cells could be that excess AhR protein behaves as a constitutive, ligand-independent, and ligand-insensitive DNA-binding factor, as previously described [28], to activate basal expression of AhR gene targets in the absence of ligands, and exert a physiological effect on activated human T cells. The early and functional induction of AhR expression in response to T-cell activation described here raises the question of why T-cell stimulation leads to transcription and functional activation of AhR. The AhR is best known for its important physiological role in controlling the expression of detoxification-mediating genes in a ligand-dependent manner. We hypothesize that the constitutive activation of AhR upon T-cell stimulation may increase the ability of T cells to metabolize AhR exogenous ligands such as BaP to protect activated proliferating lymphocytes from such environmental contaminants. The marked induction of CYP1A1 and CYP1B1 mRNA expression in CD3/CD28-activated T cells exposed to BaP observed in our study supports this hypothesis. In addition to the historical link between AhR and environmental contaminants, a major link was described recently between the activity of AhR and the regulation of gut immunity via IL-22 production [29, 30]. In our study, modulation of AhR signaling by siRNA-mediated knockdown and treatment with the antagonist CH-223191 counteracted IL-22 expression and production by activated T cells, without significantly affecting those of IL-17. Likewise, CD4+ T cells from AhR-deficient mice fail to produce IL-22 after exposure to AhR ligands, while they still develop normal Th17 cell responses [11], thus reinforcing the specific link between AhR and IL-22. Recently, Torii et al. demonstrated that addition of tobacco smoke extract to CD4+ memory T cells obtained from patients with psoriasis increases IL-22 expression [19], and another study found a significant correlation between current cigarette smoking and the proportion of IL-22+ cells in the memory T-cell population of COPD patients [20]. Consistent with these studies, we found that CD3/CD28-activated T cells obtained from healthy current smokers have increased IL-22 secretion compared with those of nonsmokers or past smokers. Moreover, the AhR antagonist CH-223191 counteracted such production of IL-22, indicating that the activation-induced AhR expression and function allows T cells to respond to environmental contaminants, such as AhR agonists present in tobacco smoke, by producing IL-22. Finally, the efficient mobilization of AhR in activated T cells may serve as a transcriptional regulator, dictating T-cell subset dominance, and exhibiting immunomodulatory effects depending on the environment during an immune response [11, 14, 15, 31]. In conclusion, our main finding is that activation of human T cells triggers a marked and early upregulation of AhR expression, occurring regardless of the T-cell subtype, and regulating IL-22 production. Our findings support the concept that AhR is a major player in T lymphocyte physiology and a putative target for immune regulation. www.eji-journal.eu

Eur. J. Immunol. 2014. 44: 1330–1340

Cellular immune response

Figure 6. Effect of current smoking on IL-22 production by CD3/CD28-activated human T cells. CD3+ T cells obtained from healthy nonsmokers (n = 8), past smokers (n = 4), or current smokers (n = 7) with normal lung function and COPD patients who were past (n = 5) or current (n = 4) smokers were (A) stimulated with anti-CD3 and anti-CD28 Abs and (B) either untreated (UNT) or pretreated with 3 μM CH-223191 for 1 h before CD3/CD28-mediated activation for 120 h. IL-22 and IL-17 concentration was measured by ELISA. Data are shown as (A) dot plots with median values or as (B) mean + SEM of duplicate samples pooled from at least four independent experiments. *p < 0.05 when compared with (A) the different groups (ANOVA followed by the Tukey– Kramer multiple comparison test) and (B) untreated CD3/CD28-activated T cells (paired Student’s t-test).

Materials and methods Chemicals and reagents BaP, PMA, ionomycin, ActD, CHX, the MEK inhibitor PD98059, and the NF-κB inhibitor pyrrolidine dithiocarbamate were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other chemical inhibitors were purchased from Calbiochem (La Jolla, CA, USA). Dioxin was obtained from Cambridge Isotopes Laboratories (Cambridge, MA, USA). The rabbit polyclonal anti-AhR Ab was supplied by Biomol Research Laboratories (Plymouth, PA, USA). Human recombinant cytokines (IL-1β, IL-2, IL-4, IL-6, IL-12, IL-23, and TGF-β) and neutralizing Abs against human IL-4 and IFN-γ were from R&D Systems (Abington, UK).

Cell isolation and activation PBMCs were obtained from buffy coats (Etablissement Franc¸ais du Sang, Rennes, France) by Ficoll (Life Technologies, Cergy Pontoise, France) gradient centrifugation. After separation of monocytes by a 1-h adhesion step, CD3+ , CD4+ , and CD8+ T cells were purified from nonadherent cells by negative selection R CD3, CD4, and CD8 T cell kits (Life Technolousing Dynabeads gies). For some experiments, na¨ıve and memory CD4+ T cells were  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

stained with FITC-conjugated anti-CD45RO and anti-CD45RA Abs and FACS sorted on a FACS Aria cytometer (BD Biosciences, San Jose, CA, USA). T cells were cultured in RPMI medium (Eurobio, Les Ulis, France) supplemented with 20 IU/mL penicillin, 20 μg/mL streptomycin, and 10% decomplemented FBS R T-Expander (Life Technologies), and stimulated with Dynabeads beads coated with anti-CD3 and anti-CD28 Abs (Life Technologies) at a 1:1 cell/bead ratio in the absence or presence of the different chemicals. Chemicals were used as stock solutions in DMSO. The final concentration of DMSO in culture medium was always < 0.2% v/v and control cultures received the same dose of vehicle as treated cultures. For polarization experiments, CD4+ T cells stimulated with anti-CD3 and anti-CD28 Abs were treated for 120 h with no cytokines (Th0) or with appropriate cytokines and neutralizing Abs. For Th1-cell polarization, cells were treated with IL-12 (10 ng/mL) and an anti-IL-4 Ab (10 μg/mL). For Th2-cell polarization, cells were treated with IL-4 (30 ng/mL) and an anti-IFN-γ Ab (10 μg/mL). For Th17-cell polarization, cells were treated with IL-1β (10 ng/mL), IL-6 (10 ng/mL), IL-23 (20 ng/mL), and TGF-β (2 ng/mL), and Abs against IFN-γ (10 μg/mL) and IL-4 (10 μg/mL). The stimulation of T cells with allogeneic monocyte-derived DC in MLR was performed as previously described [32] with allogeneic T cells at a ratio of 1:5 DC/T cells for 24 h. www.eji-journal.eu

1337

1338

Laurie Prigent et al.

Eur. J. Immunol. 2014. 44: 1330–1340

Table 1. Characteristics of study population.

Clinical characteristics

Number of subjects Age (years)a) Sex ratio (M/F) Smoking (pack years) BMI FEV1/FVC (%) FEV1 (% pred)

Nonsmokers

8 56.3 ± 1.3b) * 4/4 0 22 ± 1 79 ± 2***, **** 105 ± 6***, ****

Past smokers

Current smokers

Normal lung function

COPD

All

Normal lung function

COPD

All

4 53.3 ± 1.4* 1/3 18 ± 3** 26 ± 3 82 ± 2***, **** 105 ± 8***, ****

5 70.0 ± 2.2 1/4 32 ± 7 24 ± 1 59 ± 4 46 ± 2

9 62.6 ± 3.2 2/7 24 ± 4 25 ± 1 69 ± 5 72 ± 11

7 53.9 ± 4.5* 2/5 23 ± 4** 24 ± 2 83 ± 3***, **** 110 ± 7***, ****

4 61.8 ± 4.4 2/2 50 ± 7 28 ± 3 47 ± 7 48 ± 4

11 56.7 ± 3.7 4 36 ± 4 25 ± 2 70 ± 4 87 ± 10

Values are mean ± SEM. *p < 0.05 when compared with past smokers with COPD; **p < 0.05 when compared with current smokers with COPD; ***p < 0.05 when compared with current smokers with COPD; ****p < 0.05 when compared with past smokers with COPD (ANOVA followed by the Tukey–Kramer multiple comparison test). M/F: male/female; BMI: body mass index; FEV1: forced expiratory volume in 1 s; % pred: % predicted; FVC: forced vital capacity.

a)

b)

Study subjects The study population consisted of eight healthy nonsmoker volunteers, 11 past or current smokers with normal lung function, and nine past or current smokers with COPD. The clinical characteristics of the three subject groups are shown in Table 1. The study was approved by the Medical Ethical Committee of the Rennes University Hospital, France (Ethics No. 12-08). After obtaining written informed consent from each subject, peripheral blood was collected in heparin tubes and CD3+ T cells were purified and stimulated with anti-CD3 and anti-CD28 Abs for 120 h.

Proliferation assay T-cell proliferation was measured by 3 H-methyl-thymidine (Amersham Biosciences, Buck, UK) incorporation as previously described [33].

Proteins were separated on a polyacrylamide gel and electrophoretically transferred onto nitrocellulose membranes (Millipore, Guyancourt, France). After blocking, membranes were incubated with primary Abs and incubated with appropriate HRP-conjugated secondary Abs (Dako A/S, Glostrup, Denmark). Immunolabeled proteins were visualized by chemiluminescence.

Flow cytometry immunolabeling assay After blocking with 30 μg human polyclonal IgG (R&D systems) for 30 min on ice, T cells were labeled with FITC- or PE-conjugated Abs (BD Biosciences) for 40 min on ice in PBS containing 1% BSA and 0.1% NaN3 . Isotype control labeling was performed in parallel. After washing, cells were analyzed by flow cytometry using a FC500 flow cytometer (Beckman Coulter, Villepinte, France) as previously described [35].

Immunofluorescence RT-qPCR Total RNA was isolated from cells using the TRIzol method (Life Technologies) and then reverse-transcribed into cDNA using the RT Applied Biosystems kit (Foster City, CA, USA). qPCR assays were performed using gene-specific primers from Qiagen (Courtaboeuf, France) and the fluorescent dye SYBR Green methodology, as previously described [34].

T cells fixed on PolysineTM slides (Thermo Fisher Scientific, Braunschweig, Germany) with 4% paraformaldehyde for 10 min at 4°C were incubated with primary Abs for 2 h at 4°C. After washR 488 goat anti-rabbit IgG (Life Technologies) ing, Alexa Fluor was used as the secondary Ab. Nuclei were counterstained with 0.5 μg/mL DAPI for 10 min. Slides were viewed using an automated microscope Leica DMRXA2.

Cytokine measurements Preparation of cell lysates and Western blotting Total cellular protein extracts were prepared by lysis of T cells as previously described [34]. Cytosolic and nuclear extracts were preR extraction kit (Clontech, CA, USA). pared using the Transfactor  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The concentrations of IL-2, IFN-γ, TNF-α, IL-5, IL-13, IL-17, and IL-22 cytokines in supernatants of T-cell cultures were quantified by ELISA using specific Duoset ELISA development system kits (R&D Systems). www.eji-journal.eu

Eur. J. Immunol. 2014. 44: 1330–1340

Measurement of NF-κB DNA binding DNA binding of p65 NF-κB was analyzed using the ELISA-based TransAM NF-κB kit (Active Motif, Rixensart, Belgium) as previously described [36].

Cellular immune response

References 1 Barouki, R., Coumoul, X. and Fernandez-Salguero, P. M., The aryl hydrocarbon receptor, more than a xenobiotic-interacting protein. FEBS Lett. 2007. 581: 3608–3615. 2 Poland, A. and Kende, A., 2,3,7,8-Tetrachlorodibenzo-p-dioxin: environmental contaminant and molecular probe. Fed. Proc. 1976. 35: 2404–2411. 3 Hattemer-Frey, H. A. and Travis, C. C., Benzo-a-pyrene: environmental

EMSA

partitioning and human exposure. Toxicol. Ind. Health 1991. 7: 141–157. 4 Sorensen, M., Autrup, H., Moller, P., Hertel, O., Jensen, S. S., Vinzents, P.,

EMSA was performed with nuclear extracts prepared from unstimulated or PMA/ionomycin-activated (2 h) T cells, as previously described [35].

Knudsen, L. E. et al., Linking exposure to environmental pollutants with biological effects. Mutat. Res. 2003. 544: 255–271. 5 Nebert, D. W., Dalton, T. P., Okey, A. B. and Gonzalez, F. J., Role of aryl hydrocarbon receptor-mediated induction of the CYP1 enzymes in environmental toxicity and cancer. J. Biol. Chem. 2004. 279: 23847–23850.

RNA interference

6 Fernandez-Salguero, P., Pineau, T., Hilbert, D. M., McPhail, T., Lee, S. S., Kimura, S., Nebert, D. W. et al., Immune system impairment and hepatic

T cells were transiently transfected with 30 pmol AhR-specific or nontargeting siRNA (smartpool, Dharmacon, Chicago, IL, USA) using the nucleofection technology (program E0-115, Amaxa Biosystems, Gaithersburg, MD, USA). After transfection, T cells were cultured for 48 h in culture medium prior to activation with anti-CD3 and anti-CD28 Abs for 30 h.

fibrosis in mice lacking the dioxin-binding Ah receptor. Science 1995. 268: 722–726. 7 Vorderstrasse, B. A., Steppan, L. B., Silverstone, A. E. and Kerkvliet, N. I., Aryl hydrocarbon receptor-deficient mice generate normal immune responses to model antigens and are resistant to TCDD-induced immune suppression. Toxicol. Appl. Pharmacol. 2001. 171: 157–164. 8 Negishi, T., Kato, Y., Ooneda, O., Mimura, J., Takada, T., Mochizuki, H., Yamamoto, M., et al., Effects of aryl hydrocarbon receptor signaling on the modulation of TH1/TH2 balance. J. Immunol. 2005. 175: 7348–7356.

Statistical analysis

9 Wincent, E., Amini, N., Luecke, S., Glatt, H., Bergman, J., Crescenzi, C., Rannug, A. et al., The suggested physiologic aryl hydrocarbon

Data are expressed as mean ± SEM. Statistical significance of the differences was assessed using GraphPad Prism (GraphPad software, INC., La Jolla, CA, USA) by paired or unpaired Student’s t-test, or ANOVA followed by the Dunnett’s multirange test or the Tukey–Kramer test when multiple comparisons were studied.

receptor activator and cytochrome P4501 substrate 6-formylindolo(3,2b)carbazole is present in humans. J. Biol. Chem. 2009. 284: 2690–2696. 10 Quintana, F. J. and Weiner, H. L., Environmental control of Th17 differentiation. Eur. J. Immunol. 2009. 39: 655–657. 11 Veldhoen, M., Hirota, K., Westendorf, A. M., Buer, J., Dumoutier, L., Renauld, J. C. and Stockinger, B., The aryl hydrocarbon receptor links TH17-cell-mediated autoimmunity to environmental toxins. Nature 2008. 453: 106–109. 12 Kimura, A., Naka, T., Nohara, K., Fujii-Kuriyama, Y. and Kishimoto, T., Aryl hydrocarbon receptor regulates Stat1 activation and participates in the development of Th17 cells. Proc. Natl. Acad. Sci. USA 2008. 105: 9721–

Acknowledgements: We thank Dr. St´ephanie Guillot for performing the lung function tests, and Dr. Graziella Brinchault, Dr. C´ecile Rochefort-Morel, and Ga¨elle Prodhomme (Respiratory and Lung Function Departments, Rennes University Hospital, France) for recruiting the study subjects and obtaining the blood samples. We would like to thank Dr. Marie-Laure Island for expert techR nical advice on EMSA. We are also grateful to the Biogenouest SynNanoVect platform (Synthetic NanoVectors) for its technical support (www.synnanovect.ueb.ue), the cytometry platform of Biosit (Laurent Deleurme), University of Rennes 1 (France), and Dr. Patricia Ame-Thomas for helpful discussion on flow cytometry analysis. This work was supported by grants from INERIS/antiopes programme post-Grenelle (O.F.).

9726. 13 Monteleone, I., Rizzo, A., Sarra, M., Sica, G., Sileri, P., Biancone, L., MacDonald, T. T. et al., Aryl hydrocarbon receptor-induced signals upregulate IL-22 production and inhibit inflammation in the gastrointestinal tract. Gastroenterology 2011. 141: 237–248. 14 Gandhi, R., Kumar, D., Burns, E. J., Nadeau, M., Dake, B., Laroni, A., Kozoriz, D. et al., Activation of the aryl hydrocarbon receptor induces human type 1 regulatory T cell-like and Foxp3(+) regulatory T cells. Nat. Immunol. 2010. 11: 846–853. 15 Apetoh, L., Quintana, F. J., Pot, C., Joller, N., Xiao, S., Kumar, D., Burns, E. J. et al., The aryl hydrocarbon receptor interacts with c-Maf to promote the differentiation of type 1 regulatory T cells induced by IL-27. Nat. Immunol. 2010. 11: 854–861. 16 Busbee, D. L., Shaw, C. R. and Cantrell, E. T., Aryl hydrocarbon hydroxylase induction in human leukocytes. Science 1972. 178: 315–316. 17 Trifari, S., Kaplan, C. D., Tran, E. H., Crellin, N. K. and Spits, H., Identification of a human helper T cell population that has abundant production

Conflict of interest: The authors declare no financial or commercial conflict of interest.  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

of interleukin 22 and is distinct from T(H)17, T(H)1 and T(H)2 cells. Nat. Immunol. 2009. 10: 864–871.

www.eji-journal.eu

1339

1340

Laurie Prigent et al.

Eur. J. Immunol. 2014. 44: 1330–1340

18 June, C. H., Ledbetter, J. A., Lindsten, T. and Thompson, C. B., Evidence

30 Qiu, J., Heller, J. J., Guo, X., Chen, Z. M., Fish, K., Fu, Y. X. and Zhou, L., The

for the involvement of three distinct signals in the induction of IL-2 gene

aryl hydrocarbon receptor regulates gut immunity through modulation

expression in human T lymphocytes. J. Immunol. 1989. 143: 153–161.

of innate lymphoid cells. Immunity 2012. 36: 92–104.

19 Torii, K., Saito, C., Furuhashi, T., Nishioka, A., Shintani, Y., Kawashima,

31 Quintana, F. J., Basso, A. S., Iglesias, A. H., Korn, T., Farez, M. F.,

K., Kato, H. et al., Tobacco smoke is related to Th17 generation with

Bettelli, E., Caccamo, M. et al., Control of T(reg) and T(H)17 cell

clinical implications for psoriasis patients. Exp. Dermatol. 2011. 20:

differentiation by the aryl hydrocarbon receptor. Nature 2008. 453:

371–373.

65–71.

20 Paats, M. S., Bergen, I. M., Hoogsteden, H. C., van der Eerden, M. M.

32 Laupeze, B., Amiot, L., Sparfel, L., Le Ferrec, E., Fauchet, R. and Fardel, O.,

and Hendriks, R. W., Systemic CD4+ and CD8+ T-cell cytokine profiles

Polycyclic aromatic hydrocarbons affect functional differentiation and

correlate with GOLD stage in stable COPD. Eur. Respir. J. 2012. 40: 330–337.

maturation of human monocyte-derived dendritic cells. J. Immunol. 2002.

21 Esser, C., Biology and function of the aryl hydrocarbon receptor: report of an international and interdisciplinary conference. Arch. Toxicol. 2012. 86: 1323–1329. 22 Allan, L. L. and Sherr, D. H., Constitutive activation and environmental chemical induction of the aryl hydrocarbon receptor/transcription factor in activated human B lymphocytes. Mol. Pharmacol. 2005. 67: 1740–1750. 23 Lawrence, B. P., Leid, M. and Kerkvliet, N. I., Distribution and behavior of the Ah receptor in murine T lymphocytes. Toxicol. Appl. Pharmacol. 1996. 138: 275–284. 24 Ford, G. S., Barnhart, B., Shone, S. and Covey, L. R., Regulation of CD154 (CD40 ligand) mRNA stability during T-cell activation. J. Immunol. 1999. 162: 4037–4044. 25 Vogel, C. F., Kahn, E. M., Leung, P. S., Gershwin, M. E., Chang, W. L., Wu, D., Haarmann-Steemmann, T. et al., Cross-talk between aryl hydrocarbon receptor and the inflammatory response: a role for NF-kB. J. Biol. Chem. 2013. 289: 1866–1875. 26 Crawford, R. B., Holsapple, M. P. and Kaminski, N. E., Leukocyte activa-

168: 2652–2658. 33 Sparfel, L., Payen, L., Gilot, D., Sidaway, J., Morel, F., Guillouzo, A. and Fardel, O., Pregnane X receptor-dependent and -independent effects of 2-acetylaminofluorene on cytochrome P450 3A23 expression and liver cell proliferation. Biochem. Biophys. Res. Commun. 2003. 300: 278–284. 34 Morzadec, C., Macoch, M., Robineau, M., Sparfel, L., Fardel, O. and Vernhet, L., Inorganic arsenic represses interleukin-17A expression in human activated Th17 lymphocytes. Toxicol. Appl. Pharmacol. 2012. 262: 217–222. 35 Pinel-Marie, M. L., Louarn, L., Desmots, S., Fardel, O. and Sparfel, L., Aryl hydrocarbon receptor-dependent induction of the IgA receptor FcαRI by the environmental contaminant benzo(a)pyrene in human macrophage. Toxicology 2011. 290: 89–95. 36 N’Diaye, M., Le Ferrec, E., Kronenberg, F., Dieplinger, H., Le Vee, M. and Fardel, O., TNFalpha- and NF-kappaB-dependent induction of the chemokine CCL1 in human macrophages exposed to the atherogenic lipoprotein(a). Life Sci. 2009. 84: 451–457.

tion induces aryl hydrocarbon receptor up-regulation, DNA binding, and increased Cyp1a1 expression in the absence of exogenous ligand. Mol. Pharmacol. 1997. 52: 921–927. 27 Veldhoen, M., Hirota, K., Christensen, J., O’Garra, A. and Stockinger, B., Natural agonists for aryl hydrocarbon receptor in culture medium are

Abbreviations: ActD: actinomycin D · AhR: aryl hydrocarbon receptor · BaP: benzo[a]pyrene · CHX: cycloheximide · CsA: cyclosporine A · COPD: chronic obstructive pulmonary disease · CYP: cytochrome P-450

essential for optimal differentiation of Th17 T cells. J. Exp. Med. 2009. 206: 43–49. 28 Pongratz, I., Mason, G. G. and Poellinger, L., Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding

Full correspondence: Dr. Lydie Sparfel, UMR INSERM U1085, IRSET, ´ Universite´ de Rennes 1, 2 Avenue du Pr Leon Bernard, 35043 Rennes, France Fax: +33-2-23-23-47 94 e-mail: [email protected]

activity and repression of intrinsic DNA binding activity. J. Biol. Chem. 1992. 267: 13728–13734. 29 Lee, J.S., Cella, M., McDonald, K. G., Garlanda, C., Kennedy, J. C., Nukaya, M., Mantovani, A. et al., AHR drives the development of gut ILC22 cells and postnatal lymphoid tissues via pathways dependent on and independent of Notch. Nat. Immunol. 2012. 13: 144–151.

 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Received: 22/7/2013 Revised: 14/1/2014 Accepted: 24/1/2014 Accepted article online: 4/2/2014

www.eji-journal.eu

The aryl hydrocarbon receptor is functionally upregulated early in the course of human T-cell activation.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates immunosuppression caused by a variety of environmental co...
459KB Sizes 0 Downloads 2 Views