Identification of a ligand for glucocorticoid-induced tumor necrosis factor receptor constitutively expressed in dendritic cells

BBRC Biochemical and Biophysical Research Communications 310 (2003) 433–438 www.elsevier.com/locate/ybbrc

Identification of a ligand for glucocorticoid-induced tumor necrosis factor receptor constitutively expressed in dendritic cellsq Kang-Yeol Yu,a Han Soo Kim,b Si Young Song,b Sung-Shik Min,a Jae Jun Jeong,a and Byung-S. Youna,* a

KOMED Institute for Life Science, Graduate School of Biotechnology, Korea University, Rm 640, Anam-dong, Sungbuk-ku, Seoul, South Korea b Brain Korea 21 Project for Medical Science, Department of Internal Medicine, Yonsei University College of Medicine, Shinchon-dong, Seodaemun-ku, Seoul, South Korea Received 22 August 2003

Abstract Glucocorticoid-induced tumor necrosis receptor (GITR) has been implicated in regulation of T cell suppression by CD25þ CD4þ regulatory T cells (Tregs). We isolated a cDNA encoding GITR ligand (GITRL) from mouse endothelioma cells. When stably expressed in HEK293 cells, its specific interaction with GITR was confirmed by flow cytometry with the use of GITR-Fc. The interaction was greatly diminished by the addition of soluble GITRL. Consistent with this, soluble GITRL bound to the cell surface of the GITR-expressing HEK293 cells. Coexpression of GITR with GITRL or stimulation of the GITR-expressing cells with soluble GITRL led to activation of NF-jB, which was significantly reduced by anti-GITR. More importantly, GITRL was expressed by both immature and mature dendritic cells, suggesting that the interaction between GITR and GITRL may contribute to immune regulation of Tregs by dendritic cells. This isolated TNFRL represents a bona fide GITRL whose presence has been elusive until this time. Ó 2003 Elsevier Inc. All rights reserved.

The members of the tumor necrosis factor (TNF) family are involved in modulating diverse biological activities such as regulation of cell proliferation, differentiation, cell survival, cell death, cytokine production, lymphocyte co-stimulation, and isotype switching [1]. When recognized by their respective ligands, these receptors transduce signals for heterogeneous functions. Ligands in this family are type II transmembrane proteins and share a conserved TNF sequence in their extracellular domains. Most members of this receptor family represent type I transmembrane proteins characterized by cystein-rich motifs in their extracellular domains [2]. While some receptors contain a conserved “death domain” which is associated with the activation of apoptotic signaling pathways, other members contain low sequence identity in the intracellular domains and q The nucleotide sequence encoding GITRL has been deposited in the GenBank database under GenBank Accession No. AY359852. * Corresponding author. Fax: +82-2-926-1670. E-mail address: [email protected] (B.-S. Youn).

0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.09.024

stimulate the transcription factors NF-jB and AP-1 [2,3]. The glucocorticoid-induced TNFR family-related gene (GITR, TNFRSF18) was cloned into a glucocorticoid-treated murine T cell hybridoma. GITR is expressed in T cells from thymus, spleen, and lymph nodes, and its expression is induced with T cell activation [4]. GITR is structurally similar to the cytoplasmic domains of murine and human CD27, 4-1BB, and OX40 among TNFRSF members and mediates intracellular signaling by recruiting TRAF molecules to the cytoplasmic tails. These molecules are highly expressed after lymphocyte or T cell activation. Similar to CD27, 41BB, and OX40, GITR is also involved in the regulation of TCR/CD3-driven T cell activation and death [4,5]. Recent evidence has implicated suppressor T cells in transplantation tolerance and the prevention of autoimmune disease. One such regulatory T cell population is the CD25þ CD4þ subset, called regulatory T cells (Treg). Thymic-derived regulatory T cells have been shown to be critical in the regulation of self-reactive T

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cells in the periphery [6–8]. GITR is predominantly expressed as a 70-kDa homodimeric glycoprotein on CD25þ CD4þ regulatory T cells in the thymus and periphery. Signaling through GITR in CD25þ CD4þ cell abrogates their suppressive function and breaks immunological self-tolerance [9]. Stimulation of GITR with a specific mAb abrogated CD25þ CD4þ T cell mediated suppression in vitro and in vivo, which induced autoimmunity [9,10]. The human form of GITRL (AITRL) has been identified by two independent studies [5,11]. However, murine GITRL has been unidentified for a number of years. By searching an EST and mouse genome database, this new member of the TNFR superfamily was identified, named GITRL, and characterized as a ligand for GITR. We report the initial characterization of GITRL which may act as a T cell mediated immune regulator.

Materials and methods Cells. EOMA cells, a mouse endothelioma cell line, and RAW264.7 cells, a murine macrophage cell line, were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 lg/ml streptomycin. Animals. Six to eight-week-old female C57B/6 mice were purchased from Charles River Laboratory, USA. Mice were housed in a SPF barrier area of the Department of Laboratory Animal Medicine, Yonsei University College of Medicine, in accordance with the guidelines and regulations for use and care of animals in Yonsei University. BM-derived dendritic cell preparation. Dendritic cells (DCs) were prepared as described previously [12] with modifications. Briefly, bone marrow cells were harvested by flushing the marrow cavities of femurs and tibiae under aseptic conditions, and single cell suspensions were seeded into six-well plates at 1
106 cells/ml in RPMI-1640 supplemented with penicillin–streptomycin, 5
105 M of 2-ME and 10% FCS, GM-CSF (100 U/ml, PeproTech), IL-4 (100 U/ml, PeproTech), and Flt-3 ligand (10 ng/ml, R&D systems, Minneapolis, MN). Cytokines were replenished on days 2, 4, and 6. On day 7 of the in vitro culture, non-adherent and loosely adherent cells were collected (iDC). Part of the iDCs were matured with LPS (1 lg/ml, Sigma Chem., St. Louis, MO) in the presence of GM-CSF and IL-4 for an additional 48 h (mDC). For staining, DCs were first incubated with normal mouse serum for 15 min at 4 °C. After washing, cells were stained with antibodies against MHC class II (I-A/I-E), CD11c, CD86, rat polyclonal anti-mouse GITRL antibody or matching control antibody for 15 min. Samples were analyzed by FACScalibur (Becton–Dickinson). Identification and cloning of GITRL cDNA. A mouse EST database and mouse genome database were screened for sequence homology with the cystein-rich motif of AITRL, using the tblastn algorithms. A partial amino-terminal sequence of GITRL was obtained from a mouse EST and an overlapped partial carboxyl-terminal sequence of GITRL was identified from mouse genomic resources (NCBI). A fulllength GITRL cDNA clone encoding an intact amino-terminal signal peptide was obtained from a mouse endothelial cell line, EOMA and selected for further investigation. The complete cDNA sequence of both strands of this clone was determined and its homology to the TNFR family was confirmed. Expression plasmids and recombinant proteins. Full-length GITRL was cloned into the mammalian expression vector pCEP4 (Invitrogen, Carlsbad, CA) and stably transfected into HEK293-EBNA cells to

generate the surface protein. The extracellular domain of GITRL genes was amplified by PCR using a set of forward and reverse primers, and then cloned with a C-terminus hexa-histidine tag into the bacterial expression vector pET21a (Novagen, Madison, WI). Soluble His-tagged GITRL protein was produced in Escherichia coli strain BL21 and purified according to the manufacturer’s protocol. Endotoxins were removed from the purified GITRL protein with a polymyxin B column. Full-length GITR/pCEP4 was cloned and stably transfected into HEK293-EBNA cells. GITR-Fc fusion genes were cloned into pCEP4 with human IgG1-Fc conjugated at the COOHterminus of the extracellular domain of GITR and stably transfected into HEK293-EBNA cells to generate recombinant GITR-Fc protein. Serum free culture media from these GITR-Fc/pCEP4 transfected cells were passed through a protein G–Sepharose column. The column eluents were fractionated by SDS–PAGE and stained with Coomassie to determine greater than 95% purity. Antibodies. For anti-GITRL polyclonal antibody production, Wistar rats were intraperitoneally immunized three times at two week intervals with soluble His-tagged GITRL and then sacrificed for collecting antiserum. Flow cytometric analysis. For cell-binding assays, full-length GITRL expressing HEK293-EBNA was harvested with 1 mM EDTA in PBS and then incubated with GITR-Fc for 20 min on ice. Cells were washed and stained with FITC-conjugated goat anti-human IgG for Fc fusion protein binding. His-tagged GITRL binding in full-length GITR expressing HEK293-EBNA cells was consecutively stained with anti-(His) 6 and FITC-conjugated goat anti-mouse IgG. Fluorescence was analyzed by FACScalibur (Becton–Dickinson, San Diego, CA). To detect cell-surface GITRL expression, RAW264.7, EOMA, and bone marrow-derived dendritic cells were stained with rat anti-GITRL antibody and FITC conjugated goat anti-rat IgG. Luciferase assay. HEK293-EBNA cells were transfected by FuGENE-6 (Roche, Basel, Switzerland) with the (NF-jB)3 -Luc reporter construct and constructs encoding Renilla luciferase cDNA and the indicated DNAs according to manufacturer’s recommendations. After 24 h, cells were treated with the indicated reagents overnight. Cells were harvested and luciferase activities were quantitated using the dual luciferase system (Promega, Madison, WI).

Results and discussion Isolation of a mouse TNFRL resembling activationinduced tumor necrosis factor ligand The mouse genome sequence was searched for homology with the amino acid sequence encoding the open reading frame of activation-induced tumor necrosis factor ligand (AITRL), a human TNFRL. An exon sequence with a striking homology to the carboxyl-terminal region of AITRL was retrieved from chromosome 1 supercontig NW-000158.1 in the mouse genome sequence. Both sequences were found in the chromosomal syntenic regions and the occurrence of adjacent genes was also similar, suggesting that these genes may be orthologues. A partial amino-terminal sequence was identified by searching the mouse EST database whose accession number is BY327839 (gi26518410). Fulllength sequence was retrieved by PCR from a mouse endothelioma, EOMA, cDNA library. The open reading frame of GITRL consisted of 173 amino acids and was predicted to be a type II membrane protein. There was

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one potential N-linked glycosylation site (Asn-74) in GITRL. As shown in Fig. 1, a significant amino acid homology was found between GITRL and AITRL among the TNFL supergene family members. The amino acid identity and syntenic chromosomal location strongly suggest that the TNFRL identified may encode a natural ligand for GITR. Identification of glucocorticoid-induced tumor necrosis factor ligand

Fig. 1. Aligned amino acid sequence of human AITRL with mouse GITRL. Identical amino acid residues are marked in box. Transmembrane domain is underlined. The potential N-glycosylation sites are marked by an asterisk. The amino acid sequence of GITRL was aligned with AITRL on the basis of sequence homology. The nucleotide sequence encoding GITRL has been deposited in the GenBank whose Accession No. is AY359852.

To ascertain whether the isolated TNTRL is a functional ligand for GITR, the gene was stably expressed in HEK293 cells. Polyclonal antibodies against GITR and the TNFRL were generated from rat and rabbit, respectively. Uniform expression of TNFRL was observed by flow cytometry with the polyclonal antibody (Fig. 2A). In Western blots (see the inset of Fig. 2A) two

Fig. 2. Identification of the natural ligand for GITR. HEK293-EBNA cells were transfected with pCEP4 control vector (shaded area) or pCEP4/encoding full-length GITRL cDNA (solid lines in A, B, and C) or pCEP4/encoding full-length GITR cDNA (solid lines in D, E). GITRL-expressed cells were incubated with anti-GITRL (1 lg) (A), GITR-Fc (1 lg) (B), GITR-Fc (1 lg), and GITRL-His (3 lg) (C). (A) GITRL-expression was confirmed by flow cytometry analysis and Western blot with anti-GITRL antibody. Cells were stained with anti-human IgG-FITC for GITR-Fc binding. GITR-expressed cells were incubated with anti-GITR (1 lg) (D) and GITRL-His (1 lg) (E). (D) GITR-expression was confirmed by flow cytometry and Western blot with anti-GITR antibody. Cells were stained with anti-poly (His)-FITC for GITRL-His and analyzed for binding by flow cytometry.

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Fig. 3. GITRL induced GITR-mediated NF-jB activation. (A) HEK293-EBNA cells were transiently transfected with an (NF-jB)3 -Luc reporter construct and with constructs encoding Renilla luciferase cDNA and the indicated amounts of an empty vector (pCEP4), GITR/pCEP4, and/or GITRL/pCEP4 plasmids. Dual luciferase activities were measured 24 h after transfection. (B) GITR-expressed HEK293-EBNA cells were transiently transfected with a (NF-jB)3 -Luc reporter gene and the Renilla luciferase construct. After 24 h, cells were treated with indicated reagents overnight. For experiments presented in (A) and (B), the error bars represent means  SD of duplicate samples. All experiments were repeated three times.

major protein bands were observed around 25–28 kDa, which may have been created by different glycosylation states. GITR-Fc bound to the stable transfectant, but this binding was significantly diminished by the addition of the recombinant soluble form in TNFRL-expressing cells (Figs. 2B and C), suggesting that TNFRL is a ligand for GITR. Mouse 4-1BB-Fc did not bind to the cells (data not shown). Therefore, the protein was named GITRL. Soluble GITRL (sGITRL) was expressed in the His-tagged form and purified. GITR was stably expressed in HEK293 cells whose expression was uniformly detected with flow cytometry and a single 45 kDa protein band was detected by Western blot (Fig. 2D). When sGITRL was used with flow cytometry of stable GITR transfectants (Fig. 2D), a significant shift was observed (Fig. 2E). Taken together, these data suggest that TNFRL derived from EOMA is a GITRL whose presence has long been suspected. GITRL is a functional ligand for GITR A salient feature of signaling through TNFR is induction of NF-jB activation [3,13]. It has been shown that overexpression of GITR activates NF-jB and the association of the intracellular domain of GITR with an adaptor protein called Siva induces apoptosis [14]. When coexpressed with GITR, GITRL dramatically induced NF-j activity by 5-fold (Fig. 3A) in comparison with GITR alone, whereas GITRL itself did not induce NF-jB activation. Moreover, stimulation of GITR expressing HEK293 cells with sGITRL also activated NFjB albeit weakly, which was then dampened further by anti-GITR (Fig. 3). It is likely that the preexisting

activation of NF-jB in the GITR stable transfectants accounts for the weak activation produced by sGITRL, or sGITRL may have a suboptimal confirmation compared with membrane bound GITRL, such that when engaged with GITR its agonistic potential becomes weak. Taken together, these data suggest that the ligand identified in this study is a functional ligand for GITR. Expression of GITRL in dendritic cells It has been reported that stimulation of a mouse monocytic cell line, RAW264.7 with the recombinant extracellular domain of GITR or GITR-Fc led to an increase in both COX-2 activity and MMP9 expression. Levels of these proteins were also upregulated by LPS, suggesting that GITRL may be expressed in monocyte, macrophage or DCs and may cause signaling by itself [15,16]. Consistent with this, significant expression of GITRL was observed by flow cytometry with the use of anti-GITRL (Fig. 4A) or GITR-Fc (data not shown). The expression was notably upregulated by LPS. Immature DCs (iDCs) were generated from mouse bone marrow cells with the addition of GM-CSF, IL-1, and Flt-3 ligand and further differentiated from mature DCs (mDCs) with LPS. GITRL was similarly expressed in both iDCs and mDCs (Fig. 4C). Interestingly, modest expression of GITRL was detected in EOMA and was also upregulated by LPS (Fig. 4B). Typically, two different GITRLþ populations (low and high expressers) were noted. Observation of GITRL expression in endothelial cells is interesting because the interaction between GITR in T cells and GITR in endothelial cells may affect T cell functions, such as adhesion or alteration of expression of

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Fig. 4. GITRL expression in RAW264.7, EOMA, and dendritic cells. (A) GITRL expression was up-regulated with LPS stimulation in RAW264.7 cells. LPS stimulated RAW264.7 cells were stained with anti-GITRL and analyzed for expression by flow cytometry. (B) EOMA, mouse endothelioma cell line, constitutively expresses GITRL. (C) The expressions of CD86, MHC class II (I-A/I-E), and GITRL were analyzed on day 7 BMDC generated from C57BL/6 mice. Part of iDCs were matured with LPS in the presence of GM-CSF and IL-4 for additional 48 h (mDC). Cells were stained with antibodies against MHC class II (I-A/I-E), CD11c, CD86, rat polyclonal anti-mouse GITRL antibody or matching control antibody.

other costimulatory molecules, and endothelial cell functions such as proliferation or migration. Thus, it is very interesting to see whether pro-inflammatory cytokines, chemokines, endothelins or other biological response modifiers such as lipids or icosanoids may modulate expression of GITRL in endothelial cells. To our knowledge, ours is the first data showing that DCs constitutively express GITRL. Yamazaki et al. [17] recently showed that Tregs could proliferate by antigenprocessing DCs in an IL-2-dependent manner, but complete blockage of proliferation was not possible by using

anti-CD25 or by depleting CD80 or CD86 expression; this suggests that other costimulators may contribute to proliferation. GITRL may be a likely candidate for producing this residual proliferative capability observed in DCs. Acknowledgments B.-S.Y. is supported by a grant (FPR02A5-44-120) of 21C Frontier Functional Proteomics Project from Korean Ministry of Science and Technology.

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