Activation of Human T Lymphocytes Is Inhibited by Peroxisome Proliferator-activated Receptor γ (PPARγ) Agonists PPARγ CO-ASSOCIATION WITH TRANSCRIPTION FACTOR NFAT

T lymphocyte activation is highlighted by the induction of interleukin-2 (IL-2) gene expression, which governs much of the early lymphocyte proliferation responses. Peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. PPARγ mRNA expression was found in human peripheral blood T lymphocytes, raising the possibility of PPARγ involvement in the regulation of T cell function. Here we show that PPARγ ligands, troglitazone and 15-deoxy-Δ12,14 prostaglandin J2, but not PPARα agonist Wy14643, inhibited IL-2 production and phytohemagglutinin-inducible proliferation in human peripheral blood T-cells in a dose-dependent manner. This inhibitory effect on IL-2 was restricted to the PPARγ2-expressing, not the PPARγ-lacking, subpopulation of transfected Jurkat cells. The activated PPARγ physically associates with transcriptional factor NFAT regulating the IL-2 promoter, blocking NFAT DNA binding and transcriptional activity. This interaction with T-cell-specific transcription factors indicates an important immunomodulatory role for PPARγ in T lymphocytes and could suggest a previously unrecognized clinical potential for PPARγ ligands as immunotherapeutic drugs to treat T-cell-mediated diseases by targeting IL-2 gene expression.

signaling cascade, resulting in cell proliferation and secretion of cytokines that enhance the immune response (1,2). Interleukin-2 (IL-2) 1 plays a key role in controlling T-cell proliferation, and consequently, numerous studies have focused on understanding IL-2 gene regulation (2). Peroxisome proliferatoractivated receptors (PPARs) are transcription factors and members of the nuclear receptor superfamily (3). To date, three different PPARs, named PPAR␣, PPAR␦, and PPAR␥, have been identified in mammalian cells. PPAR␥ is primarily involved in the regulation of genes related to lipid metabolism and plays a role in adipocyte differentiation (4,5). However, in higher organisms, the widespread tissue distribution of PPAR␥ (6) suggests an involvement of the nuclear receptor in multiple biological processes. Recent findings have indicated that PPAR␥ is a negative regulator of macrophage activation and inhibits production of monocyte inflammatory cytokines (7,8). It is well known that the activation of T lymphocytes is much different from monocytes because it proceeds by the initial activation of transcription factors that ultimately fulfill the biological amplification of the immune response. Although PPAR␥ mRNA is expressed in human peripheral blood T lymphocytes (9), nothing is known about the physiologic regulation of PPAR␥ in T-cell activation. Here we demonstrate that the activated PPAR␥ physically associates with the T-cell specific transcriptional factor NFAT and blocks NFAT DNA binding and transcriptional activity. Ultimately, the transcription and production of the vital T-cell cytokine IL-2 is blocked.

EXPERIMENTAL PROCEDURES
Cell Culture-Fresh human peripheral blood T lymphocytes obtained from normal healthy donors (10) and Jurkat T cells were maintained as described (9,10).
IL-2 Measured by ELISA-T cells were grown to approximately 2.5 ϫ 10 6 cells/ml and treated with PHA/PMA in the presence or absence of the different ligands. Cell supernatants were collected and assayed for human IL-2 using Endogen kits (Wolburn).
Ribonuclease Protection Assays-Total RNA was isolated using TriZOL (Life Technologies, Inc.) and a RNase protection assay was performed as described (11). 20 g of RNA was hybridized with 33 Plabeled probes corresponding to IL-2 (Pharmingen).
Transient Transfection-Transient transfections of Jurkat T cells were performed using DEAE-dextran methods (11). After 24 h of transfection, cells were treated with or without different ligands for an additional 24 h in the presence or absence of PHA/PMA.
Flow Cytometry and Sterile Cell Sorting-Cell sorting for green fluorescent protein (GFP) and propidium iodide fluorescence were performed (12) using a FACStar Plus (Becton Dickinson, San Jose, CA) * This project has been funded in whole or in part with Federal funds from the NCI, National Institutes of Health, under Contract NO1-CO-56000 and sponsored in part by the NCI, Department of Health and Human Services, under a contract with ABL. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ʈ Luciferase Assays-The transfected cells were pelleted, lysed, and measured by a luminometer (Monolight 3010, Pharmingen) according to the manufacturer's instruction.

RESULTS AND DISCUSSION
To determine whether PPAR␥ interferes with T lymphocyte activation, we investigated the effect of the PPAR␥ ligands on PHA-mediated T-cell proliferation and IL-2 production. Human peripheral blood T cells were stimulated by PHA and cultured with various concentrations of different ligands for 72 h. As shown in Fig. 1A, the specific PPAR␥ agonist of the thiazolidinedione family, troglitazone (13) , or dimethyl sulfoxide. Total RNA was isolated and subjected to analysis by RNase protection assay for expression of human IL-2 or L32 control genes using 33 P-labeled probe.

FIG. 2. Troglitazone and 15d-PGJ 2 inhibit transcription and production of IL-2 in transfected Jurkat T cells in a PPAR␥-dependent manner.
A, Jurkat cells were transfected with either control vector (pSG5) or PPAR␥2 wild type expression vector (pSG5hPPAR␥2) and co-transfected with pEGFPC1 plasmid. Transfected target cells were sorted by FACS and then assayed for IL-2 production. B, upper panel, Jurkat cells were co-transfected with a PPAR␥-expression plasmid (ϩPPAR␥2) or control DNA (ϪPPAR␥2) and a PPRE reporter gene (AOx-TK). Cells were treated with 15d-PGJ 2 or troglitazone for an additional 24 h. The luciferase activity was determined. Lower panel, Jurkat cells were co-transfected with an IL-2 promoter-luciferase reporter plasmid and a PPAR␥ expression plasmid (ϩPPAR␥) or control DNA (ϪPPAR␥). Cells were treated with ligands, stimulated by PHA/ PMA, and collected for analysis of reporter gene activity 24 h later. (14), which is a natural PPAR␥ ligand. By contrast, the PPAR␣specific ligand Wy14643 did not inhibit PHA-mediated T-cell proliferation. Furthermore, PHA/PMA-induced IL-2 synthesis assayed by ELISA was also restricted by the co-addition of troglitazone or 15d-PGJ 2 in a dose-dependent manner (Fig. 1B).
Because the induction of IL-2 gene expression is a pivotal event in early T lymphocyte activation, we next performed RNase protection assays of human peripheral blood T cells stimulated with PHA/PMA in the presence or absence of troglitazone or 15d-PGJ 2 . Total RNA was isolated and probed for IL-2 mRNA. As shown in Fig. 1C, IL-2 mRNA steady-state levels were detected as early as 6 h and strongly increased after 12 h of PHA/PMA stimulation. Treatment with troglitazone or 15d-PGJ 2 totally abrogated expression of IL-2 mRNA. The quantification of decreased IL-2 mRNA levels was performed by normalizing densitometric traces against L32 mRNA. The IL-2 transcript was reduced by greater than 90% as compared with controls, suggesting that PPAR␥ ligands blocked transcription of the IL-2 gene.
Because transfection efficiency of circulating human T cells is very poor, we chose to use the human T cell line Jurkat that expresses little detectable PPAR␥ mRNA (9) as a model system to directly assess the actual role of PPAR␥ expression in the PPAR␥ ligand-induced inhibitory effects on T lymphocyte activation. The Jurkat cells were transfected with PPAR␥2 wild type expression vector (pSG5hPPAR␥2) (15) or control pSG5 plasmid. For this study, we used GFP as a cotransfected biomarker to simplify the selection of transfected target cells (13) by FACS sorting and assay for IL-2 production. As shown in Fig. 2A, PPAR␥ ligands troglitazone and 15d-PGJ 2 inhibited IL-2 production only in the PPAR␥2-expressing, but not in PPAR␥2-nonexpressing, subpopulation of transfected Jurkat cells. Interestingly, Wy14643 did not also show this inhibitory effect. Thus, from the data presented, it is clear that such inhibitory effects on T-cell activation are mediated by PPAR␥.
To verify that the human PPAR␥2 cDNA is capable of activating gene transcription through a PPRE in human T cells, we scription factors NFAT (A) and Sp-1 (B) induced by PHA/PMA in human peripheral blood T cells as demonstrated by EMSA. Cells were treated with dimethyl sulfoxide-control or troglitazone or 15d-PGJ 2 and incubated with medium (Ϫ) or PHA/PMA (ϩ) for 2 h at 37°C. Nuclear extracts (5 g) were incubated with a 32 P-labeled probe. Arrow indicates migrational location of each DNA complex. Excess amounts of unlabeled oligonucleotides containing wild type (wt) or mutated (mt) NFAT sites were used as competitors to test for specificity of DNA complex. Antibodies against NFAT supershifted the protein-DNA complex. C, Jurkat cells were co-transfected with a reporter construct directed by the NFAT distal site of the IL-2 promoter and either a PPAR␥-wild type (ϩPPAR␥) or control DNA plasmid (ϪPPAR␥). Cells were treated with different ligands, stimulated by PHA/PMA as shown, and collected for analysis of reporter gene activity 24 h later. introduced a PPAR␥2 expression vector (pSG5hPPAR␥2) together with a PPRE-driven luciferase reporter construct (AOx-TK) (14) into Jurkat T cells. Fig. 2B (upper panel) shows that activation of a PPAR-dependent promoter required co-expression of PPAR␥ in Jurkat T cells. Upon addition of the PPAR␥ ligand troglitazone or 15d-PGJ 2 , luciferase activity was significantly increased corresponding to overexpression of PPAR␥. To determine whether changes in IL-2 mRNA levels can be ascribed, at least in part, to differences in promoter activity, Jurkat T cells were co-transfected with IL-2 luciferase reporter constructs and a PPAR␥ expression plasmid. PMA/PHA treatment resulted in a marked increase in IL-2 promoter activity. Lack of PPAR␥ expression rendered the Jurkat cells unresponsive to the inhibitory effects of troglitazone and 15d-PGJ 2 on PHA/PMA-induced IL-2 promoter activation (Fig. 2B, lower  panel). However, this effect was restored by transfection of PPAR␥. These data suggest that the inhibitory effects of troglitazone and 15d-PGJ 2 on IL-2 promoter activity are directly dependent on the expression and activation of PPAR␥.
The transcription factor NFAT plays an essential role in IL-2 gene expression. NF-ATc1 is also involved in the proliferation of peripheral T lymphocytes (16). Therefore, we evaluated transcriptional activity and DNA binding of NFAT to determine whether NFAT might be a target for negative regulation of T-cell activation by PPAR␥ ligands. As shown in Fig. 3A, the specific binding of NFAT probe corresponding to the human IL-2 promoter is strongly induced by PHA/PMA, whereas equivalent nuclear extracts from troglitazone-or 15d-PGJ 2 -treated cells displayed diminished binding capacity to the 32 P-radiolabeled probes. Moreover, the inhibitory effect of PPAR␥ ligands on NF-B and AP-1 DNA-binding activities was also observed in T cells activated by PHA/PMA (data not shown). In contrast, the DNA binding of Sp-1 (17) was unaffected by troglitazone and 15d-PGJ 2 (Fig. 3B). The EMSA data obtained correlated with the effect of PPAR␥ ligands on the transcriptional activity analyzed by a reporter construct directed by the NFAT distal site of the IL-2 promoter. The transcriptional activation of the above reporter construct was abrogated by troglitazone or 15d-PGJ 2 in the presence of PPAR␥ overexpression (Fig. 3C). Thus, one conceivable explanation for the repression of IL-2 transcription in troglitazone-or 15d-PGJ 2 -treated cells could be due to selective disruption of the transcriptional regulation of the IL-2 promoter.
Next we focused our attention on how functional co-association of PPAR␥ with transcription factors NFAT might mediate T-cell suppression. We tested for complex formation between PPAR␥ and NFAT in a co-immunoprecipitation experiment. Fresh human peripheral blood T cells were induced by PMA/PHA and treated with troglitazone or 15d-PGJ 2 . Cell extracts were prepared and immunoprecipitated with a PPAR␥-specific antibody; immunoprecipitates were developed on Western blots with an NFATc1-specific antibody. As shown in Fig. 4, The NFATc1 complex can be co-precipitated with PPAR␥ in cells induced by PMA/PHA and troglitazone or 15d-PGJ 2 . Furthermore, the addition of anti-PPAR␥ antibody induced high affinity binding of extracts to the NFAT probes as determined by EMSA (data not shown), demonstrating that removal of PPAR␥ with this antiserum increases the target specificity of NFAT. These data indicate that a direct physical protein-protein interaction occurs between nuclear receptor PPAR␥ and transcription factors NFAT.
Recent studies have provided insights into the mechanisms by which steroid nuclear receptors regulate cytokine genes (18). Corticosteroids may bind directly as a dimer to regulatory sequences (GRE) in target gene promoters and subsequently affect activation of transcription. Dexamethasone has also been shown to inhibit the IL-2 promoter by interference with AP-1. The current model of dexamethasone inhibition has demon-strated a physical interaction between corticosteroid receptor and NF-B. In the case of PPAR␥, a non-steroid nuclear receptor, it has been reported that PPAR␥ agonists specifically inhibit expression of tumor necrosis factor ␣ through inhibition of tumor necrosis factor ␣ promoter activity in monocytes (9). However, no physical co-precipitation of PPAR␥ with other transcription factors was shown. The data presented here demonstrate that PPAR␥ ligands suppress transactivation of IL-2 via downregulation of the IL-2 promoter in a PPAR␥-dependent manner. The transcriptional regulation of the IL-2 gene has been analyzed extensively at the level of the IL-2 promoter. cis-Acting elements for several transcription factors have been identified within this regulatory region. The factors that bind to these motifs include NFAT, AP-1, and NF-B proteins. Also, a binding site for Sp-1 has been identified immediately upstream of the distal NFAT site. Our data show that PPAR␥ ligands selectively block DNA binding and transcriptional activation of transcription factors such as NFAT regulating the IL-2 promoter in T cells.
From our data, PPAR␥ down-regulates the IL-2 promoter by a novel mechanism that involves protein-protein interactions. As shown in Fig. 4, formation of a molecular complex between PPAR␥ and NFAT may occur in T cells, which could block PHA/PMA-induced DNA binding and transactivation. Although we have focused on the interaction between NFAT and PPAR␥, we cannot exclude the possibility that PPAR␥ interaction with AP-1 or NF-B may also play an important role in blocking IL-2 gene transcription. However, activation and function of NFAT is an absolute requirement for IL-2 transcription.
Taken together, these data provided the evidence that the inhibitory role of PPAR␥ in the immunomodulation of T lymphocytes is based on functional interaction between PPAR␥ and T-cell-specific transcription factors. Such a model could highlight the importance of transcriptional cross-talk in T-cell biology and may lead to a new molecular class of immunomodulators. PPAR␥ ligands may be of therapeutic value in human T-cell-mediated diseases by targeting IL-2 gene expression.