Proc. Natl. Acad. Sci. USA Vol. 96, pp. 9803–9808, August 1999 Immunology Targeted disruption of Traf5 gene causes defects in CD40- and CD27-mediated lymphocyte activation HIROYASU NAKANO*†‡,SACHIKO SAKON*†,HARUHIKO KOSEKI†§,TOSHITADA TAKEMORI¶,KURISU TADA*, ࿣ MITSURU MATSUMOTO ,EIKO MUNECHIKA*, TSUYOSHI SAKAI**, TAKUJI SHIRASAWA**, HISAYA AKIBA*†, TETSUJI KOBATA*†,SYBIL M. SANTEE††,CARL F. WARE††,PAUL D. RENNERT‡‡,MASARU TANIGUCHI†§§, HIDEO YAGITA*†, AND KO OKUMURA*† *Department of Immunology, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan; †Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, 2-3 Surugadai, Kanda, Chiyoda-ku, Tokyo 101-0062, Japan; Departments of §Molecular Embryology and §§Molecular Immunology, Graduate School of Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-0856, Japan; ¶Department of Immunology, ࿣ National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-0052, Japan; Division of Informative Cytology, Institute for Enzyme Research, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan; **Department of Molecular Genetics, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan; ††Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121; and ‡‡Department of Immunology and Inflammation, Biogen, Inc., Cambridge, MA 02142 Edited by Elliott D. Kieff, Harvard University, Boston, MA, and approved June 23, 1999 (received for review February 4, 1999) ABSTRACT TRAF5 [tumor necrosis factor (TNF) recep- family, MAP kinase͞ERK kinase kinase 1 (MEKK1) and tor-associated factor 5] is implicated in NF-␬B and c-Jun apoptosis signal-regulating kinase 1 (ASK1) (16, 18). These NH2-terminal kinase͞stress-activated protein kinase activa- results indicated that the two signaling pathways to activation tion by members of the TNF receptor superfamily, including of NF-␬B and JNK͞SAPK diverge downstream of TRAFs. CD27, CD30, CD40, and lymphotoxin-␤ receptor. To investi- Although these pathways have been investigated extensively, gate the functional role of TRAF5 in vivo, we generated other signaling cascades potentially mediated by TRAFs have TRAF5-deficient mice by gene targeting. Activation of either been largely unknown. Moreover, although both TRAF2 and NF-␬B or c-Jun NH2-terminal kinase͞stress-activated protein TRAF5 are recruited to CD27, CD30, CD40, OX40, lympho- kinase by tumor necrosis factor, CD27, and CD40 was not toxin-␤ receptor (LT-␤R), herpesvirus entry mediator, and ؊͞؊ ؊͞؊ abrogated in traf5 mice. However, traf5 B cells showed receptor activator of NF-␬B, it remains to be determined defects in proliferation and up-regulation of various surface whether TRAF2 and TRAF5 have individually specific func- molecules, including CD23, CD54, CD80, CD86, and Fas in tions or act redundantly in transmitting signals via these response to CD40 stimulation. Moreover, in vitro Ig produc- receptors (3). ؊͞؊ tion of traf5 B cells stimulated with anti-CD40 plus IL-4 Recent generation of TRAF2-deficient mice and transgenic was reduced substantially. CD27-mediated costimulatory sig- mice expressing a dominant negative form of TRAF2 ؊͞؊ nal also was impaired in traf5 T cells. Collectively, these (TRAF2-DN) revealed that TRAF2 is requisite for JNK͞ results demonstrate that TRAF5 is involved in CD40- and SAPK activation but not for NF-␬B activation by TNF (19, 20). CD27-mediated signaling. Whereas TRAF2- and TRAF3-deficient mice die earlier (19, 21), TRAF3-deficient mice showed impaired T-dependent Tumor necrosis factor receptor (TNFR) superfamily members immune response (21). Collectively, these results suggested transmit signals regulating proliferation, differentiation, and that each TRAF could act redundantly or specifically in apoptosis in various types of cells (1, 2). TNFR-associated particular signaling cascades. factors (TRAFs) emerged as a novel family of downstream To examine the functional role of TRAF5 in vivo,we mediators on the signal-transduction pathway of the TNFR generated TRAF5-deficient mice by gene targeting. Whereas superfamily (3). To date, six members of the TRAF family NF-␬BorJNK͞SAPK activation by CD27 or CD40 was not have been identified (4–12). All TRAFs, except for TRAF1, abrogated, we found that CD27- and CD40-mediated lym- Ϫ͞Ϫ are composed of N-terminal zinc RING finger and multiple phocyte activation was substantially impaired in traf5 zinc fingers, coiled-coil, and C-terminal receptor-binding lymphocytes. (TRAF) domains. With the exception of TRAF4, all the other TRAFs have been shown to interact directly with some MATERIALS AND METHODS members of the TNFR superfamily lacking death domains (3). TRAF2 also interacts indirectly with death domain receptors Cells. Murine mastcytoma P815, murine CD70-transfected via the adapter molecules, TRADD and RIP (13). Overex- P815 (CD70-P815), and murine CD80-transfected P815 pression of TRAF2, -5, and -6 activates NF-␬B, and truncated (CD80-P815) have been described (22) and were maintained TRAF2, -5, and –6, which lack Zn-binding domains, act as a in RPMI 1640 medium containing 10% FCS. dominant negative inhibitor in receptor-mediated NF-␬B ac- Generation of traf5؊͞؊ Mice by Gene Targeting. A genomic tivation (9, 10, 14), suggesting that these TRAFs are common DNA clone for traf5 was isolated by screening a 129͞Sv͞J mediators for NF-␬B activation by TNFR superfamily mem- mouse genomic DNA library with cDNA encoding a RING bers. It has been shown that TRAF2, -5, and -6 recruit finger domain of TRAF5 as a probe. The cDNA encoding the NF-␬B-inducing kinase (NIK), which, in turn, activates I␬B RING finger domain contained two separate exons, which kinases (15–17). TRAF2, -5, and -6 also participate in the were designated exon I and exon II. The targeting construct ͞ activation of c-Jun NH2-terminal kinase (JNK) stress- (T5-KO) was made by replacing the exon II encoding a activated protein kinase (SAPK), which is mediated by two members of the MAP kinase kinase kinase (MAPKKK) This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: TNFR, tumor necrosis factor receptor; TRAF, TNFR- The publication costs of this article were defrayed in part by page charge associated factor; JNK, c-Jun NH2-terminal kinase; SAPK, stress- activated protein kinase; LT-␤R, lymphotoxin-␤ receptor; ES, embry- payment. This article must therefore be hereby marked ‘‘advertisement’’ in onic stem; LPS, lipopolysaccharide. accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡To whom reprint requests should be addressed. E-mail: hnakano@ PNAS is available online at www.pnas.org. med.juntendo.ac.jp. 9803 Downloaded by guest on September 26, 2021 9804 Immunology: Nakano et al. Proc. Natl. Acad. Sci. USA 96 (1999) C-terminal half of the RING finger domain and surrounding anti-CD23, anti-CD54, anti-CD80, anti-CD86, or anti-Fas introns with a pMC1neo gene cassette (Stratagene) in the mAb and analyzed by flow cytometry. reverse orientation to the endogenous traf5 gene (Fig. 1A). Electrophoretic Mobility-Shift Assay (EMSA). EMSA was Linealized T5-KO then was transfected into embryonic stem performed essentially as described (9). Briefly, 3 ϫ 107 thy- (ES) cells (R1 cells) by electroporation. Neomycin-resistant mocytes were incubated with agonistic anti-CD27 mAb (10 ES clones were selected by G418 and GANC as described (23). ␮g͞ml; PharMingen) on ice for 30 min. Then, the cells were Homologous recombinants were identified by Southern blot- washed with ice-cold PBS and crosslinked with prewarmed ting. Genomic DNA was isolated and digested with EcoRI or goat anti-hamster Igs (100 ␮g͞ml; Cappel) at 37°C for 15 min. BamHI, and Southern blots were hybridized with the 3Ј For CD40 or LPS stimulation, 2 ϫ 107 splenocytes were flanking probe (probe A) as shown in Fig. 1A. Germ-line incubated with anti-CD40 mAb (10 ␮g͞ml) at 37°C for 15 min chimeras were generated by the aggregation method as de- or LPS (5 ␮g͞ml) for 30 min. Then, the nuclear extracts were scribed (24). The resulting male chimeras were backcrossed prepared. The nuclear extracts were subjected to EMSA. with C57BL͞6J females, and germ-line transmission in F1 Reactions were subjected to 6% PAGE and analyzed on a Fuji traf5ϩ͞Ϫ mice was verified by Southern blot analysis. Geno- BAS2000 image analyzer. typing of the F2 mice was performed by PCR analysis of tail In Vitro Kinase Assay. In vitro kinase assay was performed ϫ 6 genomic DNA. The PCR primers (P1, 5Ј-GGG TCA TGC essentially as described (27). Briefly, 5 10 thymocytes were ␮ ͞ CAC TTG TTC GA-3Ј, and P2, 5Ј-ACC CAC ACG AGG incubated with agonistic anti-CD27 mAb (10 g ml) on ice for AAG GTC TGA-3Ј; see Fig. 1A) were designed to encompass 30 min. Then, the cells were washed with ice-cold PBS and ␮ ͞ the 0.7-kb fragment containing exon II and surrounding crosslinked with prewarmed goat anti-hamster Igs (100 g ml) ϫ introns, which was replaced by pMC1neo, resulting in a 1.2-kb at 37°C for 15 min. For CD40 stimulation, purified B cells (5 6 ␮ ͞ fragment in the targeted allele. 10 ) were incubated with anti-CD40 (10 g ml) at 37°C for 15 Western Blot Analysis. Total lung lysates were prepared min. The cells were lysed in the lysis buffer containing 1 mM from traf5ϩ͞ϩ, traf5ϩ͞Ϫ, and traf5Ϫ͞Ϫ littermates by homoge- NaF and 0.1 mM Na3VO4, incubated with rabbit polyclonal nization in a lysis buffer containing 20 mM Hepes (pH 7.2), 150 anti-JNK antibody (Santa Cruz Biotechnology), and precipi- mM NaCl, 0.5% Triton-X100, 2 mM EDTA, 1 ␮g͞ml aproti- tated with protein G-Sepharose beads (Amersham Pharma- nin, 1 ␮g͞ml leupeptin, 1 mM PMSF, and 1 ␮g͞ml pepstatin. cia). The immunoprecipitates then were subjected to in vitro TRAF2 and TRAF5 were affinity-purified from the lung kinase assay by using GST-c-Jun (1–79) (a gift from E.