Activation of OX40 Signal Transduction Pathways Leads to Tumor Necrosis Factor Receptor-associated Factor (TRAF) 2- and TRAF5-mediated NF-κB Activation*

We investigated the intracellular signaling of OX40, a member of the tumor necrosis factor receptor family. Activation of NF-κB in OX40-transfected HSB-2 cells was detected by electrophoretic mobility shift assay within 30 min after the binding of the ligand gp34. In vitro binding experiments showed that tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF2, TRAF3, and TRAF5 but not TRAF4 associated with glutathioneS-transferase-OX40 fusion protein. The cotransfection experiments using human embryo kidney cell derived HEK 293T cells showed that TRAF2, TRAF3, and TRAF5 associated with OX40 in vivo. Studies with OX40 deletion mutants demonstrated that the cytoplasmic portion consisting of amino acid sequence 256–263 (GGSFRTPI) was required for the association with TRAFs and NF-κB activation. The introduction of the dominant negative mutants of TRAF2 and TRAF5 into HSB-2-OX40 cells suppressed NF-κB activation in a dose-dependent manner. In addition, the introduction of TRAF3 together with the dominant negative mutants of TRAF2 or TRAF5 further reduced NF-κB activation. These results indicate that the NF-κB activation resulting from OX40 stimulation is mediated by both TRAF2 and TRAF5, and is likely to be negatively modulated by TRAF3.

Human OX40 is a 50-kDa cell surface glycoprotein expressed primarily on activated CD4 ϩ T cells and some human T cell leukemia virus type I (HTLV-I) 1 -infected T cell lines, but not on resting peripheral T cells, peripheral B cells, or thymocytes. OX40 was originally described as a cell surface antigen on the activated rat T cells (1). Molecular cloning of its cDNA (1)(2)(3)(4) revealed that OX40 is a member of the nerve growth factor receptor/tumor necrosis factor receptor (NGF-R/TNF-R) superfamily which is now known to include low affinity nerve growth factor receptor (p75 NGF-R), tumor necrosis factor receptors (p50/55 TNF-R1 and p75/80 TNF-R2), lymphotoxin-␤ receptor, Fas antigen (CD95/APO-1), CD40, CD30, CD27, and 4-1 BB (5,6). All the members of this superfamily share a characteristic repeating cysteine-rich motif in the extracellular domain, which is believed to be related to their ability to interact with the TNF-related ligands. The diverse cellular responses such as cell growth, differentiation, and programmed cell death (apoptosis) are triggered by the interaction between the members of the NGF-R/TNF-R superfamily and their ligands.
The ligand for human OX40 was also cloned and identified as previously reported gp34, a cell surface protein expressed on HTLV-I-infected T cell lines and subsequently demonstrated to be induced by transactivator p40 tax of HTLV-I (7-9). As expected, the deduced amino acid sequence of gp34 revealed that it is a member of the TNF family. Gp34 has been reported to be expressed on some HTLV-I-infected cell lines such as Hut 102 and MT-2 (10), human umbilical vein endothelial cells (11), and stimulated B lymphoblastoid cell line MSAB (12).
Since its first description, OX40 has been known to transmit costimulatory signals to T cells. Recent studies with human T cells have confirmed this finding and showed that the binding of gp34 to OX40 results in enhanced T cell proliferation and induction of interleukin-2 and -4 production in the presence of anti-CD3 or anti-T cell receptor-␣␤ antibody (7,8). We recently reported that the OX40/gp34 system directly mediates the adhesion of activated or HTLV-I-transformed T cells to vascular endothelial cells (11,13). Furthermore, we examined the role of the OX40/gp34 system in the development of angitis related diseases such as systemic lupus erythematosus and erythema nodosum (14). Although these observations have served to delineate OX40 as a multifunctional cell surface molecule, its physiological as well as pathophysiological significance in viral infection, inflammation (15), or malignant cell infiltration has been poorly defined. In particular, intracellular signaling of OX40 has not been described to date.
In the present study, we examined the intracellular events of OX40 signaling using a unique coculture system of OX40transfected T cells and gp34-transfected adherent cells. We here demonstrate that the OX40 stimulation leads to TRAF-mediated NF-B activation. Based on the experimental results, a possible physiological and pathophysiological significance of the OX40 signaling in activated T cells is discussed.

MATERIALS AND METHODS
Preparation of Plasmid Constructs-Based on the published cDNA sequence of human gp34, cDNA of the entire coding region of gp34 was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) method. The PCR products were ligated into an expression vector pMKIT Neo (a gift of Dr. K. Maruyama, Tokyo Medical and Dental University) to construct pMKIT Neo-gp34. The preparation of an expression vector, pMKIT Neo-OX40, was described previously (11). The constructs for OX40 cytoplasmic deletion mutants were generated by PCR method using TAG (stop codon)-tagged oligonucleotides as the primer and pMKIT Neo-OX40 as the template. The PCR products were ligated into pMKIT Neo to construct pMKIT Neo-OX40-del 1, -del 2, -del 3, -del 4, -del 5, and -del 6. The partial DNA sequences were determined for all the OX40 deletion mutants to confirm the constructs.
Based on the published cDNA sequences of murine TRAF1, TRAF2, TRAF5, and human TRAF3 and TRAF4, the cDNAs of TRAFs were obtained by RT-PCR. The oligonucleotides covering the entire coding regions of TRAFs and cDNAs from murine cell line DA-1 (for TRAF1, TRAF2, and TRAF5) or phytohemagglutinin-stimulated human peripheral blood mononuclear cells (for TRAF3 and TRAF4) were used as the primers and the templates, respectively. The PCR products were integrated into the expression vector pcDNA3 (Invitrogen, San Diego, CA) or HA-tagged expression vector pCMV4-3 HA ϩ (pCMV-HA, a gift of Dr. W. C. Greene, Gladstone Institute of Virology and Immunology, University of California, San Francisco) to construct pcDNA3-TRAFs or pCMV-HA-TRAFs. The cDNAs of truncated TRAF2 (TRAF2 DN) and truncated TRAF5 (TRAF5 DN) were generated by PCR with the primers covering the amino acid sequence (amino acids 256 -501) of TRAF2 (21) and (amino acids 233-558) of TRAF5 (35), respectively, and then integrated into pcDNA3.
Preparation of Soluble gp34 -A construct for soluble gp34 was designed by fusing the extracellular portion of gp34 (nucleotide sequence, 187-585) to the signal sequence of OX40 (nucleotide sequence, 6 -116) at the SmaI site. The fused fragments were ligated into the expression vector pME18S (11) (a gift of Dr. K. Maruyama). Four g of pME18S-soluble gp34 were transfected into COS-7 cells (1 ϫ 10 7 cells) by the DEAE-dextran method (39). The transfected cells were cultured with Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 10% fetal calf serum (Bio Whittaker, Verviers, Belgium) for 24 h and with 1% fetal calf serum for another 48 h. The culture supernatants were collected and concentrated 10-fold with Centriprep 10 (Amicon Inc., Beverly, MA) prior to the assay. The supernatant of COS-7 cells transfected with pME18S-soluble gp34 (soluble gp34-sup) was added to the cell culture at 25% v/v for each assay. The supernatant of COS-7 cells transfected with empty vector (mock-sup) was prepared by the same method as soluble gp34-sup and concentrated 10-fold prior to the assay.
Cells and Culture Conditions-Human T cell line HSB-2 and murine epithelial cell line MMCE were cultured in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 60 mM tobramycin, and 2 mM L-glutamine. Human embryo kidney cell-derived cell line HEK 293T and COS-7 were cultured with Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.
Electrophoretic Mobility Shift Assay (EMSA)-Nuclear extracts from HSB-2-OX40 cells or HSB-2-mock cells (2 ϫ 10 6 cells) cocultured with either MMCE-gp34 cells or MMCE-mock cells, or cultured with either soluble gp34-sup or mock-sup for the indicated periods were prepared as described previously (40). The nuclear extracts from HSB-2-OX40 cells preincubated (at 37°C for 30 min) with either anti-OX40 monoclonal antibody (50 g/ml) (11) or anti-interleukin-2 receptor ␣ chain antibody (anti-Tac, control antibody, 50 g/ml) prior to coculture with MMCE-gp34 cells were also prepared. Eight g of nuclear extracts were mixed with 32 P-labeled B oligonucleotide containing a binding site for NF-B/c-Rel homodimeric and heterodimeric complexes (5Ј-AGTT-GAGGGGACTTTCCCAGGC-3Ј) (Santa Cruz Biotechnology, Santa Cruz, CA) or 32 P-labeled mutant B oligonucleotide (5Ј-AGTTGAGGC-GACTTTCCCAGGC-3Ј) (Santa Cruz). The binding assay was performed as described previously with a slight modification (41). The composition of the induced NF-B complex was examined by super shift assay with anti-NF-B p50 subunit antibody or anti-NF-B p65 subunit antibody (Upstate Biotechnology Inc., Lake Placid, NY). Eight g of nuclear extracts in 10 l of nuclear extract buffer (40) was incubated with 100 ng of anti-NF-B antibodies at room temperature for 40 min prior to the binding assay.
Coimmunoprecipitation and in Vivo Binding Assay-Two g of pCMV-HA-TRAF1, -TRAF2, -TRAF3, -TRAF4, -TRAF5, or pCMV-HA were cotransfected with 2 g of pME18S-OX40 or pME18S into HEK 293T (2 ϫ 10 5 cells) by the calcium phosphate precipitation method (26). After 48 h of incubation, the cells were lysed in the Triton X lysing buffer (0.5% Triton X-100, 25 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM MgCl 2 , 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 g/ml aprotinin, 2 g/ml leupeptin, and 2 g/ml pepstatin A). The cell lysates were immunoprecipitated with protein A-Sepharose 4FF (Pharmacia Biotech Inc., Uppsala, Sweden) and anti-OX40 monoclonal antibody (11). The immunoprecipitates were analyzed by SDS-PAGE with 7.5% gel (ATTO, Tokyo, Japan) and subjected to immunoblotting (11) with anti-HA monoclonal antibody, 12CA5 (Boehringer Mannheim). HA-tagged TRAFs were visualized by ECL detection system (Amersham Life Science, Arlington Heights, IL). The same membrane was  (44). The total amount of pcDNA3 constructs was adjusted to 3 g by adding an empty vector. After 28 h of incubation the cells were lysed in 250 l of reporter lysis buffer (Toyo Ink Co., Tokyo Japan). Twenty l of cell extract from each sample were fractionated to measure the luciferase activity in accordance with the manufacturer's protocol (Toyo Ink) using a luminometer (Bio-Orbit Oy, Turku, Finland). Forty l of cell extract were fractionated to measure ␤-galactosidase activity as an internal control (27). The luminescence values were normalized by the individual ␤-galactosidase activity. In the experiments of TRAF2-and TRAF5-mediated NF-B activation, pcDNA3-TRAF2 DN, -TRAF5 DN, -TRAF3, or pcDNA3 was cotransfected with 500 ng of B-luc and 250 ng of pCSK-LacZ into HSB-2-OX40 cells or HSB-2 mock cells (1 ϫ 10 6 cells) by the DEAEdextran method. The total amount of pcDNA3 constructs was adjusted to 3 g by adding an empty vector. After 24 h of incubation, the transfected cells were cocultured with either MMCE-gp34 cells or MMCE-mock cells for 24 h and then harvested to measure luciferase activity and ␤-galactosidase activity as described above.

NF-B Is Activated by OX40
Stimulation-Activation of NF-B in HSB-2-OX40 cells was detected by EMSA when the cells were cocultured with MMCE-gp34 cells but not with MMCE-mock cells (Fig. 1A). The activation was detected from 30 min up to 6 h (the end of culture period, data not shown) after ligand stimulation and blocked clearly by preincubation of HSB-2-OX40 cells with anti-OX40 antibody. The supershift of the band with anti-NF-B p50 subunit antibody or anti-NF-B p65 subunit antibody indicated the involvement of NF-B, consisting of p50 and p65 subunits in OX40-mediated activation. Similar results were obtained when the HSB-2-OX40 cells were incubated with soluble gp34-sup, but not with mock-sup (Fig.  1B). Furthermore, the studies with HSB-2-OX40 deletion mutants demonstrated that NF-B activation was detected in HSB-2-OX40-del 5 (amino acids 1-263) cells and -OX40-del 6 (amino acids 1-271) cells, but not HSB-2-OX40-del 4 (amino acids 1-255) cells after ligand stimulation. These results indicated that the cytoplasmic portion of OX40 consisting of the amino acid sequence 256 -263 (GGSFRTPI) was required for the activation of NF-B (Fig. 1, C and D). TRAF 1, TRAF2, TRAF3, and TRAF5 but Not TRAF 4 Associate with OX40 in Vitro-It has been reported that TRAFs associate with the receptors of several members of the TNF-R family and initiate the signal transduction upon the ligand stimulation. We examined the association of TRAFs with OX40 using HA-tagged-TRAFs and GST-OX40 fusion protein. As shown in Fig. 2A, HA-TRAF1, -TRAF2, -TRAF3, and -TRAF5 but not HA-TRAF4 associated with GST-OX40. HA-TRAF1, -TRAF2, -TRAF3, -TRAF4, and -TRAF5 were successfully expressed in HEK293 T cells and immunoprecipitated with anti-HA antibody (data not shown). Furthermore, the studies with GST-OX40 deletion mutants demonstrated that TRAF1, TRAF2, TRAF3, and TRAF5 associated with GST-OX40-del 5 (amino acids 241-263) and GST-OX40-del 6 (amino acids 241-271) (data not shown), but not with GST-OX40-del 3 (amino acids 241-249) (data not shown) or GST-OX40-del 4 (amino acids 241-255) in vitro (Fig. 2B). In other words, the cytoplasmic portion of OX40 consisting of the amino acid sequence 256 -263 (GGSFRTPI) was required for the association with TRAF1, TRAF2, TRAF3, and TRAF5 in vitro.
TRAF2, TRAF3, and TRAF5 Associate with OX40 in Vivo-We next examined the association of TRAFs with OX40 in vivo. HA-TRAF2, HA-TRAF3, and HA-TRAF5 were coimmunoprecipitated with anti-OX40 antibody when coexpressed with OX40 in HEK 293T cells, indicating that these TRAFs can associate with OX40 in vivo (Fig. 3). HA-TRAF1 and HA-TRAF4 were successfully expressed in HEK 293T cells and immunoprecipitated with anti-HA antibody. However, the association of HA-TRAF1 or HA-TRAF4 with OX40 in vivo was not detected under this condition (data not shown). An expression vector encoding OX40, pME18S-OX40, or pME18S was  cotransfected into HEK 293T cells with  pCMV-HA-TRAF2, -TRAF3, -TRAF5, or pCMV-HA. After 48 h of incubation, cell lysates were prepared and subjected to immunoprecipitation (IP) with anti-OX40 monoclonal antibody, IgG subclass-matched control antibody (C), or anti-HA polyclonal antibodies. The samples were analyzed by SDS-PAGE with 7.5% gel, followed by immunoblotting with anti-HA monoclonal antibody. The same membrane was reused for the detection of OX40 by immunoblotting. The bands near 51 kDa and below 34 kDa are mouse IgG heavy chain and light chain, respectively. The association of OX40 with TRAF2, TRAF3, and TRAF5 is shown in lanes 1-6, lanes 7-12, and lanes 13-18, respectively.

FIG. 3. Association of TRAFs with OX40 in vivo.
TRAF2 and TRAF5 Mediate NF-B Activation in OX40 Signaling, while TRAF3 Negatively Modulates NF-B Activation-To evaluate the ability of various TRAFs to mediate NF-B activation in HSB-2-OX40 cells, pcDNA3-TRAFs were transfected with the luciferase reporter plasmid B-luc into HSB-2-OX40 cells. The luciferase assay of the transfected cell lysates demonstrated that TRAF2 and TRAF5 but not TRAF1, TRAF3, or TRAF4 were able to mediate NF-B activation when overexpressed in HSB-2-OX40 cells (Fig. 4A). Since TRAF2, TRAF3, and TRAF5 were found to associate with OX40 in vivo, we examined the effects of TRAF3 on NF-B activation mediated by TRAF2 or TRAF5 in HSB-2-OX40 cells. As shown in Fig. 4A, the introduction of TRAF3 reduced the levels of NF-B activation by overexpressed TRAF2 or TRAF5. Based on this experiment, we further examined the roles of TRAF2, TRAF5, and TRAF3 in NF-B activation resulting from OX40 stimulation by introducing the dominant negative forms of TRAF2 (TRAF2 DN), TRAF5 (TRAF5 DN), or wild type TRAF3 into HSB-2-OX40 cells. The transfected HSB-2-OX40 cells were stimulated by MMCE-gp34 cells. As shown in Fig. 4B, the introduction of TRAF2 DN or TRAF5 DN suppressed the luciferase activity in a dose dependent manner. Furthermore, the introduction of both TRAF2 DN and TRAF5 DN suppressed the luciferase activity markedly to the lowest level. The introduction of TRAF3 together with TRAF2 DN or TRAF5 DN reduced NF-B activation further, which suggests that TRAF3 modulates NF-B activation negatively in OX40 signaling. DISCUSSION In the present study, we demonstrated that TRAF2-and TRAF5-mediated NF-B activation was induced by OX40 stimulation. We have had difficulties in the study of human OX40 signaling, since neither of our two anti-OX40 monoclonal antibodies (11) could trigger OX40 signaling even when crosslinked with the second antibody. We, therefore, employed a unique coculture system of OX40-transfected HSB-2 cells and human gp34-transfected MMCE cells. The separation of HSB-2-OX40 cells from MMCE-gp34 cells was easy and the contamination of MMCE-gp34 cells in the harvested cells was estimated to be less than 0.5% by RT-PCR method using murine specific primers. We also employed the culture supernatants of COS-7 cells transfected with the soluble gp34-construct in most of the assays to confirm the data obtained from the coculture system. Although the signals triggered by soluble gp34 were somewhat weaker than those by membrane-bound gp34, the experiments with soluble gp34 gave essentially the same results.
The studies with OX40 deletion mutants demonstrated that the cytoplasmic portion of OX40 consisting of the amino acid sequence 256 -263 (GGSFRTPI) was required for association with TRAFs and NF-B activation. It is notable that the potential phosphorylation site for protein kinase C is included in this portion of 8 amino acid residues (4). In addition, our preliminary studies showed the induction of c-jun mRNA by OX40 stimulation, for which the same intracytoplasmic portion GGSFRTPI was required (data not shown). Taken together, we consider that this portion of 8 amino acid residues constitutes a part of the crucial domain that initiates multiple signal transductions upon OX40 stimulation.
In most of the members of the TNF-R family, TRAF2 and/or TRAF5 are responsible for the activation of NF-B, while the function of TRAF1 (31), TRAF3, or TRAF4 (47) has not been clearly understood. We demonstrated that both TRAF2 and TRAF5 mediated NF-B activation in OX40 signaling, whereas TRAF3 exerted suppressive effects on NF-B activation as previously reported in CD30 signaling (31). Further studies will be needed to elucidate the precise role of TRAF3 in OX40 signaling.
TRAF-mediated NF-B activation, indeed, has furnished a clue to the understanding of the signaling in the several members of the TNF-R family. However, studies of the downstream events after NF-B activation as well as other signaling pathways would be one of the key issues to be addressed to give a satisfactory explanation of the diverse cellular responses triggered by the stimulation of the members of the TNF-R family.
Recently several groups reported that the activation of NF-B blocked apoptosis (48 -50), which may help to understand the role of the OX40/gp34 system in vivo. The conspicuous feature of the OX40/gp34 system among the TNF-R/TNF family is its ability to mediate adhesion between activated or HTLV-Itransformed T cells and endothelial cells. It is, therefore, possible that the OX40 signaling in T cells is triggered by the interaction with endothelial cells of the tissues where activated T cells expressing OX40 are infiltrating. OX40-mediated NF-B activation in T cells may serve to protect them from apoptosis, which results in the amplification and prolongation of the immune responses, or in the case of adult T cell leukemia, prolonged survival of leukemic cells in the infiltrated tissues.