Characterization of murine CD70 by molecular cloning and mAb.

International Immunology, Vol. 10, No. 4, pp. 517–526

© 1998 Oxford University Press

Characterization of murine CD70 by molecular cloning and mAb Hideo Oshima1,2, Hiroyasu Nakano2,3, Chiyoko Nohara2, Tetsuji Kobata2,3, Atsuo Nakajima4, Nancy A. Jenkins5, Debra J. Gilbert5, Neal G. Copeland5, Tetsuichiro Muto1, Hideo Yagita2,3 and Ko Okumura2,3 1Department of First Surgery, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan 2Department of Immunology, Juntendo University School of Medicine, Tokyo 113, Japan 3CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology

Corporation (JST), Kawaguchi 332, Japan 4Department of Joint Disease and Rheumatism, Nippon Medical School, Tokyo 113, Japan 5Mammalian Genetics Laboratory, ABL-Basic Research Program, NCI–Frederick Cancer Research and Development Center, Frederick, MD 21702, USA

Keywords: CD27, CD70, T cell co-stimulation

Abstract CD27, a member of the tumor necrosis factor (TNF) receptor family, has been implicated in T cell activation, T cell development and T-dependent antibody production by B cells. Its ligand CD70 has been identified only in humans, and, thus, physiological and pathological roles of the CD70–CD27 interaction remain to be determined in an experimental animal system. In the present study, we identified murine (m) CD70 by molecular cloning, and characterized its expression and function by generating an anti-mCD70 mAb. The mCD70 cDNA encoded a type II transmembrane glycoprotein of the TNF family, having 56.5% identity to the human CD70 amino acid sequence. The mCd70 gene was assigned in the central region of chromosome 17. To explore the expression and function of mCD70, we generated cDNA transfectants and anti-mCD70 mAb (FR70), which inhibited binding of a murine CD27–Fc fusion protein (mCD27–Ig) to mCD70 transfectants. FR70, as well as mCD27–Ig, immunoprecipitated a 30–33 kDa surface protein from A20 and mCD70-P815 cells but not from P815 cells. The mCD70 transfectants exhibited a potent co-stimulatory activity for antiCD3-stimulated T cell proliferation, which was blocked by FR70 far more efficiently than mCD27–Ig. FR70 also abrogated the CD28-independent co-stimulatory activity of A20 cells. The expression of mCD70 was detected on splenic T cells after stimulation with anti-CD3 and anti-CD28 mAb, and on splenic B cells after stimulation with anti-CD40 mAb. Cross-linking of surface Ig by anti-IgM mAb did not induce the mCD70 expression but enhanced the anti-CD40-induced mCD70 expression on splenic B cells. These results suggest a contribution of CD70 to murine T–B cognate interaction as proposed in the human system. FR70 will be useful for further investigating the physiological and pathological roles of the CD70–CD27 interaction in T cell development, T-dependent antibody production and various disease models in the murine system. Introduction CD27 is a member of the tumor necrosis factor (TNF) receptor family, and is expressed on the majority of mature T cells, medullary thymocytes, a subset of B cells and NK cells in humans (1–9). Its ligand has been identified to be CD70, a type II transmembrane glycoprotein of the TNF family, which was initially characterized as a marker of Reed-Sternberg cells in Hodgkin’s disease and non-Hodgkin’s lymphomas (10). Human (h) CD70 is not expressed on resting T and B

cells but can be induced on these cells after cellular activation in vitro (11–14). The function of these molecules has been extensively studied in the human system. In earlier studies, we and others have demonstrated that the CD70–CD27 interaction plays an important role in T cell co-stimulation, NK cell activation and T cell-dependent B cell activation, especially in the development of Ig-producing plasma cells (11,15–24). How-

Correspondence to: H. Yagita, Department of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan Transmitting editor: M. Miyasaka

Received 13 December 1997, accepted 6 January 1998

518 Characterization of murine CD70

Fig. 1. Alignment of murine and human CD70 amino acid sequences. Identical residues are indicated by asterisks. The predicted transmembrane domains are underlined and potential N-linked glycosylation sites are overlined. The mCD70 cDNA sequence has been deposited in the DDBJ/GenBank/EMBL Data Bank (accession no. U78091).

ever, these observations were obtained from in vitro studies and a physiological importance of the CD70–CD27 interaction in vivo remains obscure. In contrast to the human system, the murine system is superior for investigating the roles of the CD70–CD27 interaction in vivo. However, such studies in mice have been hampered by the lack of specific reagents. Although the cDNA for murine (m) CD27 has been isolated and an anti-mCD27 mAb has been recently developed (25,26), mCD70 has not been identified yet. In this study, we identified mCD70 by cDNA cloning and established a functional blocking mAb against mCD70, which will be useful for interrupting the CD70–CD27 interaction in vivo. Our initial studies with this mAb clearly indicated a potent co-stimulatory activity of mCD70 on T cell proliferation, and the expression of mCD70 on activated T and B cells, suggesting its contribution to T–B interactions that lead to antibody production. Methods

Animals Six-week-old female DBA/2 and BALB/c mice (both H-2d) and F344/DuCrj rats were purchased from Charles River Japan (Atsugi, Japan).

Cell lines A murine B cell lymphoma A20 (H-2d), a murine myeloma P3U1 (P3X63Ag8U.1), a murine mastcytoma P815 (H-2d) and a hamster kidney cell line BHK21 were purchased from ATCC (Rockville, MD). These cells were cultured in RPMI 1640 medium containing 10% FCS, 10 mM HEPES, 0.25 µg/ ml gentamycin, 2-mercaptoethanol and 2 mM L-glutamine (culture medium).

Antibodies and reagents Hybridomas producing mAb against I-Ad (M5/114), CD24 (J11d), CD3 (2C11), CD4 (RL172), CD8 (3.155), Thy-1.2 (J1j) and surface IgM (Bet-2) were obtained from ATCC. FITC-

conjugated goat anti-human and anti-rat IgG antibodies were purchased from Caltag (South San Francisco, CA). AntiCD16/32 mAb (2.4G2), biotinylated anti-CD80 (1G10), antiCD86 (GL1), anti-CD27 (LG.3A10) mAb and control rat IgG2b, FITC-conjugated anti-CD3 (2C11) and anti-B220 (RA3-6B2) mAb, phycoerythrin (PE)-labeled streptavidin, and recombinant IL-4 were purchased from PharMingen (San Diego, CA). Anti-CD40 (HM40–3), anti-CD80 (RM80) and anti-CD86 (PO3) mAb were prepared as described previously (27,28). AntiCD28 mAb (PV-1) was kindly provided by R. Abe (Science University of Tokyo, Noda, Japan) (29). Lipopolysaccharide (LPS) and G418 were purchased from Sigma (St Louis, MO).

Flow cytometry Cells (53105) were first preincubated with anti-CD16/32 mAb to avoid non-specific binding of antibodies via FcγR and then incubated with a saturating amount of fusion protein (described below) or biotinylated mAb. After washing with PBS twice, FITC-conjugated goat anti-human IgG antibody for fusion protein or PE-labeled streptavidin for biotinylated mAb were added to the cells. After washing with PBS twice, stained cells (live-gated on the basis of forward and side scatter profiles) were analyzed on a FACScan (Becton Dickinson, San Jose, CA) and data were processed by using the CellQuest program (Becton Dickinson). To verify the purity of splenic T and B cells, cells were directly stained with FITCconjugated anti-CD3 or anti-B220 mAb.

Construction of mCD27–Ig fusion protein To make a fusion protein consisting of the extracellular domain of mCD27 (amino acids 1–156) (25) and the Fc portion of human IgG1 (mCD27–Ig), a RT-PCR was performed using total RNA from BALB/c splenocytes. 59-TCACTCGAGGGGCAGGAGCTATGGCATGG-39 and 59-TCAGGATCCCACAGGGGTCTGATGA-39 were used as 59 and 39 primers respectively. The PCR product was subcloned into the XhoI and BamHI sites of pBSK which contained the Fc portion of human IgG1 (kindly provided by B. Seed, Harvard Medical School, Boston).

Characterization of murine CD70 519 corresponding to the extracellular domain of hCD70 (amino acids 50–193) (kindly provided by C. Morimoto, University of Tokyo, Tokyo) (20) as a probe. The probe was labeled with [α-32P]dCTP using random primed labeling kit (Amersham, Buckinghamshire, UK) and hybridized in 43SSC/0.5% SDS, 23Denhard’s solution and 0.1 mg/ml salmon sperm DNA at 65°C. The blots were washed to a final stringency of 0.53SSC/ 0.1% SDS at 65°C. Several overlapping clones were sequenced on both strands using a series of synthetic oligonucleotide primers. The mCD70 cDNA sequence has been deposited in the DDBJ/GenBank/EMBL Data Bank (accession no. U78091). The mCD70 cDNA was subcloned into pMKITneo, resulting in pMKITneo/mCD70. P815 or BHK21 cells were transfected with pMKITneo/mCD70 by electroporation as previously described (30). After selection with 1 mg/ml G418 and limiting dilution, stable transfectants, designated mCD70-P815 or mCD70-BHK21, were selected by staining with mCD27–Ig.

Chromosomal mapping

Fig. 2. Mapping of Cd70 in the central region of mouse chromosome 17. Cd70 was placed on mouse chromosome 17 by interspecific backcross analysis. The segregation patterns of Cd70 and flanking genes in 165 backcross animals that were typed for all loci are shown at the top of the figure. For individual pairs of loci, .165 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J3M. spretus) F1 parent. The shaded boxes represent the presence of a C57BL/6J allele and white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 17 linkage map showing the location of Cd70 in relation to linked ones is shown at the bottom. Recombination distances between loci in cM are shown to the left of the chromosome and the positions of loci in human chromosomes, where known, are shown to the right. References for the human map positions of loci cited in the study can be obtained from GDB (Genome Data Base), a computerized database of human linkage information maintained by the William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).

The insert coding mCD27–Ig was subcloned into a mammalian expression vector pMKITneo (kindly provided by K. Maruyama, Tokyo Medical Dental University, Tokyo), resulted in pMKITneo/mCD27–Ig. P3U1 stably transfected with pMKITneo/mCD27–Ig was generated by electroporation and G418 selection. mCD27–Ig fusion protein was purified by Protein G-affinity chromatography (Pharmacia, Uppsala, Sweden) from the culture supernatant of a high-producing clone.

cDNA cloning and transfection An A20 cDNA library in λZAP (Stratagene, La Jolla, CA) was screened with a 0.6 kb XhoI–NotI fragment of hCD70 cDNA

Interspecific backcross progenies were generated by mating (C57BL/6J3Mus spretus) F1 females and C57BL/6J males as described (31). A total of 205 N2 mice were used to map the Cd70 locus. DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer and hybridization were performed essentially as described (32). All blots were prepared with Hybond-N1 nylon membrane (Amersham). The probe, a 0.7 kb EcoRI–XhoI fragment of mouse cDNA, was labeled with [α-32P]dCTP using a nick translation labeling kit (Boehringer Mannheim, Mannheim, Germany) and washing was done to a final stringency of 0.8x SSCP/0.1% SDS at 65°C. Fragments of 3.2 and 2.6 kb were detected in TaqI-digested C57BL/6J DNA, and fragments of 3.7 and 2.5 kb were detected in TaqI-digested M. spretus DNA. The presence or absence of the 3.7 and 2.5 kb TaqI M. spretus-specific fragments, which co-segregated, was followed in backcross mice. A description of the probes and restriction fragment length polymorphisms for the loci linked to Cd70 including Tcfeb, Vav, Fert1 and Lamal has been reported previously (33,34). Recombination distances were calculated using Map Manager, version 2.6.5. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.

Generation of anti-mCD70 mAb A F344/DuCrj rat was immunized with A20 cells 3 times at 10 day intervals. Three days after final immunization, the splenocytes were fused with P3U1 cells as described (30). After HAT selection, one hybridoma producing mAb FR70 (rat IgG2b, κ) was identified by its strong reactivity with mCD70BHK21 cells but not with the parental BHK21 cells and cloned by limiting dilution. FR70 was purified from ascites by standard procedures with caprylic acid and purity was verified by SDS– PAGE analysis.

Immunoprecipitation A20, P815 and mCD70-P815 cells (53106) were surface labeled with 100 µg/ml S-NHS-biotin (pH 8.0) (Pierce, Rockford, IL), and lysed in 1% NP-40 lysis buffer (50 mM HEPES, pH 7.4, 250 mM NaCl, 10% glycerol, 2 mM EDTA,


Fig. 3. FR70 recognizes mCD70. (A) Reactivity with mCD70 transfectants. BHK21 and mCD70-BHK21 cells were stained with FR70, followed by FITC-conjugated goat anti-rat IgG. (B) Immunoprecipitation with FR70 and mCD27–Ig. A20, P815 and mCD70-P815 cells were surface labeled with biotin, solubilized in 1% NP-40 lysis buffer and immunoprecipitated with Protein G–Sepharose precoated with FR70, control rat IgG (rIgG), mCD27–Ig or control human IgG (hIgG). Antigens were eluted in sample buffer containing 2-mercaptoethanol, separated on 12% SDS–PAGE gels and detected by HRP-labeled streptavidin and chemiluminescence. The positions of molecular weight markers are indicated at the left (kD). (C) Inhibition of mCD27–Ig binding to mCD70 by FR70. mCD70-BHK21 cells were stained with FR70 (plain line), mCD27–Ig (broken line) or with mCD27–Ig after preincubation with FR70 (bold line), followed by FITC-conjugated goat anti-human IgG.

1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ ml pepstatin A and 2 µg/ml leupeptin). The lysates were first precleared with control rat or human IgG and Protein G– Sepharose, and then were immunoprecipitated with FR70 or mCD27–Ig and Protein G–Sepharose. The beads were washed with the lysis buffer and eluted with 1% SDS sample buffer containing 2-mercaptoethanol. Immunoprecipitates were separated on a 12% SDS–PAGE gel, transferred onto a nylon membrane, and detected by horseradish peroxidase (HRP)-labeled streptavidin and chemiluminescence using the ECL system (Amersham).

Preparation of splenic T and B cells Splenic T cells from DBA/2 or BALB/c mice were purified as previously described (35). For preparing small resting B cells, freshly isolated splenocytes from DBA/2 mice were treated with a mixture of hybridoma supernatants containing antiThy-1.2, anti-CD4 and anti-CD8 mAb, and Low-Tox rabbit complement (Cedarlane, Hornby, Ontario, Canada). After Percoll (Pharmacia) gradient centrifugation, small B cells were collected from the 60/70% interface. Purity of each population was .95% CD31 or B2201, as determined by flow cytometry. In some experiments, purified T cells (53106/ml) were stimu-

Characterization of murine CD70 521 Results

Cloning of mCD70 cDNA In order to identify mCD70, we first examined the expression of CD27 ligand on various mouse cell lines. For this purpose, a recombinant soluble form of mCD27 (mCD27–Ig) was generated for flow cytometric analysis. We detected a high level of mCD27–Ig binding to several B lymphoma cell lines, especially A20 (data not shown). Then, we isolated the mCD70 cDNA from an A20 cDNA library by cross-hybridization with an hCD70 cDNA probe. Nucleotide sequence of the isolated murine cDNA showed the highest homology to hCD70 cDNA (11), indicating that it encodes the murine homolog of hCD70. The mCD70 cDNA encodes a type II transmembrane protein of 195 amino acids with a predicted mol. wt of 21 kDa. Comparison of mCD70 and hCD70 amino acid sequences is shown in Fig. 1. mCD70 has 63.5 and 56.5% identity to hCD70 at nucleotide and amino acid levels respectively. mCD70 mature protein is predicted to consist of a cytoplasmic domain of 21 amino acids, a hydrophobic transmembrane domain of 18 amino acids and an extracellular domain of 156 amino acids with three potential N-glycosylation sites.

Chromosomal mapping of mCd70

Fig. 4. Co-stimulatory activity of mCD70 transfectants on T cell proliferation. (A) Splenic T cells from DBA/2 mice were cultured for the indicated periods with mitomycin C-treated P815, mCD70-P815 or mCD80-P815 cells in the presence of 1 µg/ml anti-CD3 mAb in 96-well round-bottomed microplates. (B) Splenic T cells were cultured for 48 h with mitomycin C-treated mCD70-P815 in the presence of serially diluted FR70 or mCD27–Ig and 1 µg/ml anti-CD3 mAb. The cultures were pulsed with 0.5 µCi/well [3H]thymidine during the last 6 h. Data are expressed as the mean 6 SD of triplicate samples. Similar results were obtained from three independent experiments. No inhibition was observed with control IgGs (not shown).

lated with immobilized anti-CD3 mAb (10 µg/ml) and soluble anti-CD28 mAb (10 µg/ml) for 24–120 h. Purified B cells (53106/ml) were stimulated with anti-CD40 mAb (10 µg/ml) and/or anti–IgM mAb (10 µg/ml), or LPS (10 µg/ml) in the presence of 10 U/ml IL-4 for 24–120 h.

T cell proliferation assay Purified T cells (13105/well) were co-cultured with mitomycin C-treated P815, mCD70-P815 or A20 cells (13104/well) in the presence of anti-CD3 mAb (1 µg/ml) in 96-well roundbottomed microplates for 24–96 h. The cultures were pulsed with 0.5 µCi/well of [3H]thymidine (Amersham) for the last 6 h and harvested using a Micro 96 Harvester (Skatron, Lier, Norway). Incorporated radioactivity was measured in a micro β-counter (Micro Beta Plus; Wallac, Turku, Finland).

The chromosomal location of the mCd70 gene was determined by interspecific backcross analysis using progeny derived from matings of [(C57BL/6J3M. spretus) F13C57BL/6J] mice. This interspecific backcross mapping panel has been typed for .2500 loci that are well distributed among all the autosomes as well as the X chromosome (31). C57BL/6J and M. spretus DNAs were digested with several enzymes and analyzed by Southern blot hybridization for informative restriction fragment length polymorphisms using the mCD70 cDNA probe. The 3.7 and 2.5 kb TaqI M. spretus restriction fragment length polymorphisms were used to follow the segregation of the Cd70 locus in backcross mice. The mapping results indicated that Cd70 is located in the central region of mouse chromosome 17 linked to Tcfeb, Vav, Fert1 and Lamal (Fig. 2). Although 165 mice were analyzed for every marker and are shown in the segregation analysis, up to 193 mice were typed for some pairs of markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the additional data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order is: centromere–Tcfeb–14/188–Cd70–0/193–Vav–3/188– Fert1–7/170–Lamal. The recombination frequencies (expressed as genetic distances in cM 6 SEM) are: Tcfeb–7.5 6 1.9– [Cd70, Vav]–1.6 6 0.9–Fert1–4.7 6 1.5–Lamal. No recombinants were detected between Cd70 and Vav in 193 animals typed in common, suggesting that the two loci are within 1.6 cM of each other (upper 95% confidence limit). The central region of mouse chromosome 17 shares regions of homology with human chromosomes 6p, 19p, 5p and 18p (Fig. 2). Indeed, hCD70 has been mapped on chromosome 19p (11).

Establishment of anti-mCD70 mAb To further characterize the expression and function of mCD70, we generated stable transfectants and anti-mCD70 mAb. The mCD70 cDNA was subcloned into a mammalian expression

522 Characterization of murine CD70 vector and stably transfected into BHK21 and P815 cells. Cell surface expression of mCD70 was first verified by staining with mCD27–Ig. To generate an anti-mCD70 mAb, we immunized a F344/DuCrj rat with A20 cells, fused the splenocytes with P3U1 myeloma cells and screened the hybridomas for producing mAb that specifically reacted with the mCD70 transfectants. As represented in Fig. 3(A), one mAb, designated FR70, that bound to mCD70-BHK21 cells but not to the parental BHK21 cells was obtained. To characterize the antigen recognized by FR70, A20, mCD70-P815 and P815 cells were surface labeled with biotin and their cell lysates were immunoprecipitated with FR70 or mCD27–Ig. Then, the precipitates were analyzed by SDS– PAGE under reducing conditions. FR70 specifically precipitated a 30–33 kDa polypeptide from the lysates of A20 and mCD70-P815 but not from P815, as did mCD27–Ig (Fig. 3B). We next examined whether FR70 recognized the binding site of CD70 to CD27. As shown in Fig. 3(C), the binding of mCD27–Ig to mCD70-BHK21 cells was almost completely inhibited by preincubation of the cells with FR70. This indicated that FR70 can interrupt the interaction between mCD70 and mCD27.

Co-stimulatory activity of mCD70 on T cell proliferation It has been demonstrated that the engagement of CD27 by CD70 or anti-CD27 mAb provides a co-stimulatory signal for T cell proliferation in humans (10,16–18) and that an antimCD27 mAb acted co-stimulatory for murine T cell proliferation (26). We then examined the co-stimulatory effect of mCD70 on the proliferation of anti-CD3-stimulated T cells. Purified splenic T cells from DBA/2 mice were cultured with P815 or mCD70-P815 cells in the presence of a suboptimal dose of anti-CD3 mAb. A mCD80 transfectant (mCD80-P815), which has been demonstrated to have a potent co-stimulatory activity (35), was also included in this experiment for comparison (Fig. 4A). When T cells were cultured with mCD70-P815 cells, a substantial level of T cell proliferation was observed with a peak response at 48 h, corresponding to ~65% of the proliferative response induced by mCD80-P815 cells. As shown in Fig. 4(B), the proliferative response co-stimulated by mCD70-P815 cells was inhibited by either FR70 or mCD27– Ig in a dose-dependent manner. An almost complete inhibition was achieved by 0.01 µg/ml FR70 or 50 µg/ml mCD27–Ig, indicating a remarkable high efficiency of FR70 to inhibit the co-stimulatory function of mCD70. We next examined the blocking effect of FR70 on the proliferative response of anti-CD3-stimulated T cells when costimulated by A20 cells, which constitutively express CD70 and CD86 (Fig. 5A). While either FR70 or anti-CD80/CD86 mAb alone partially inhibited the proliferation to ~50% of the control, an almost complete inhibition was achieved by the combination of these mAb (Fig. 5B). These results indicated that the CD70-mediated co-stimulation is responsible for the CD80/86-independent co-stimulatory activity of A20 cells.

Expression of CD70 on activated T and B cells Previous studies have demonstrated that hCD70 is not expressed on freshly isolated lymphocytes but induced following in vitro activation (11–13). We first examined the expression of mCD70 and mCD27 on splenic T cells after activation by

Fig. 5. Co-stimulatory activity of mCD70 on A20 cells. (A) A20 cells were stained with biotinylated FR70, anti-CD80 or anti-CD86 mAb, followed by PE-labeled streptavidin. (B) Splenic T cells from BALB/c mice were cultured with mitomycin C-treated A20 cells in the presence of 1 µg/ml anti-CD3 mAb in 96-well round-bottomed microplates. Control IgG, FR70 and/or anti-CD80/CD86 mAb were added to the cultures at a final dose of 10 µg/ml each. The cells were harvested at 72 h after 8 h pulse with 0.5 µCi/well [3H]thymidine and the incorporation was determined. The actual incorporation of the control was 14,000 c.p.m. Data are expressed as the mean 6 SD of triplicate samples. Similar results were obtained from three independent experiments.

flow cytometry utilizing FR70 (Fig. 6A). Purified splenic T cells (.95% CD31) were stimulated for 24–120 h with immobilized anti-CD3 mAb and soluble anti-CD28 mAb. While CD70 expression was not observed on resting T cells, it appeared at 48 h after stimulation and reached a peak at 96 h . In contrast, CD27 was expressed on almost all resting T cells and its level was up-regulated by the stimulation. We next examined the expression of mCD70 and mCD27 on activated B cells. It has been reported that CD40-mediated stimulation induced the expression of CD70 on human B cells (36). Thus, purified splenic B cells (.95% B2201) were stimulated with anti-CD40 mAb plus IL-4 for 24–120 h. As shown in Fig. 6(B), mCD70 expression was not significantly observed on resting B cells but it appeared at 24 h after the stimulation and reached a peak at 96 h. In contrast, only a marginal expression of mCD27 was induced on B cells after the CD40 and IL-4 stimulation. We also examined mCD70 expression on B cells after stimulation with anti-IgM mAb and/or anti-CD40, or LPS in the presence of IL-4 (Fig. 6C). The B cells cultured with IL-4 alone did not express mCD70 (data not shown). Although the stimulation with anti-IgM mAb alone did not significantly induce mCD70 expression, its combination with anti-CD40

Characterization of murine CD70 523

Fig. 6. Expression of mCD70 and mCD27 on activated T and B cells. (A) Splenic T cells were stimulated with immobilized anti-CD3 mAb and soluble anti-CD28 mAb in 24-well microplates and harvested at the indicated periods. (B) Splenic B cells were stimulated with anti-CD40 mAb plus IL-4 in 24-well microplates and harvested at the indicated periods. (C) Splenic B cells were stimulated with anti-IgM mAb, anti-CD40 mAb, anti-IgM plus anti-CD40 mAb or LPS in the presence of IL-4 for 96 h. Cells were first blocked with anti-CD16/32 mAb and stained with biotinylated FR70, anti-CD27 mAb or control rat IgG2b, followed by PE-labeled streptavidin. The bold lines represent staining with the indicated mAb and the plain lines represent background staining with control rat IgG2b. Similar results were obtained from two independent experiments.

524 Characterization of murine CD70 mAb markedly up-regulated mCD70 expression as compared to anti-CD40 mAb alone. Stimulation with LPS also induced mCD70 expression but its level was lower than that induced by anti-CD40 mAb. These results suggest that the CD40mediated signal is primarily responsible for the CD70 induction in murine B cells but antigen-specific B cells have an advantage of enhancement by surface Ig-mediated signal. Discussion In the present study, we identified mCD70 by cDNA cloning. Overall homology between mCD70 and hCD70 is 57% at the amino acid level, which is comparable to the 65% homology between mCD27 and hCD27 (25). mCD70 and hCD70 show the highest homology in the extracellular domain (63%), while the intracellular and transmembrane domains are less conserved. We found mCd70 was located in the central region of chromosome 17, where 4-1BB ligand (Cd137) has been also mapped (37). Other TNF family members, TNF-α, and lymphotoxin α and β are also located in the distant H-2 region of chromosome 17 (38–40). Recently, Tesselaar et al. have also reported the cloning of mCD70 cDNA (41) and the amino acid sequence was completely identical to ours. We then characterized the expression and function of mCD70 by generating cDNA transfectants and anti-mCD70 mAb (FR70) that can block the interaction with mCD27. The co-stimulatory effect on T cell proliferation is one of the prominent functions of the CD70–CD27 interaction in humans (11,15–18). The co-stimulatory effect of mCD70-P815 cells on the proliferation of anti-CD3-stimulated T cells was 60–70% as potent as that of mCD80-P815 cells. The proliferation costimulated by mCD70-P815 was almost completely blocked by ~5000-fold lower concentration of FR70 than mCD27–Ig, indicating a high efficiency of FR70 as a functional blocking mAb. T cell co-stimulatory activity of A20 cells, which constitutively express both CD70 and CD86 at a comparable level, was partially inhibited by either anti-CD70 mAb or anti-CD80/ CD86 mAb, but almost completely blocked by the mixture of them. This indicated that mCD70–CD27 interaction does play a substantial role in the CD28-independent pathway of T cell co-stimulation. In this respect, it is interesting to note that CD40-activated murine B cells have been reported to acquire a CD28-independent co-stimulatory activity (42). As discussed below, activation via CD40 efficiently induces the CD70 expression on murine B cells, which may be responsible for the CD28-independent co-stimulatory activity. This possibility will be verified in the future study. We further examined the expression of mCD70 on murine T and B cells. In general, mCD70 showed a similar expression pattern in murine lymphocytes to that in humans. mCD70 was not expressed on resting T cells, but it was induced on activated T cells, peaking 3–4 days after the stimulation with anti-CD3 and anti-CD28 mAb. Although the kinetics of mCD70 expression on murine T cells showed a similar pattern to that of hCD70, the population of CD701 T cells was ,5% at the peak, while 25% of activated T cells expressed CD70 in humans (43). Our previous studies demonstrated that hCD70 expression on activated T cells was restricted to CD45RO1 memory CD4 T cells (43) and that CD27 was preferentially expressed on CD45RA1 naive CD4 T cells (44). Since murine

splenic T cells used in this study were obtained from mice under a specific pathogen-free condition and most of them strongly expressed CD27 on the surface, they were suppose to be naive T cells. Therefore, the observed difference in CD70 expression in murine and human T cells may reflect a difference in the stage of differentiation, rather than speciesspecific features. Alternatively, it is interesting to note that IL12 has been reported to strongly enhance CD70 expression on human T cells (45). In our present experiments, we used highly purified T cells from which IL-12-producing macrophages and dendritic cells were depleted. This lack of IL-12 might be responsible for the low induction of CD70 observed. The effect of various cytokines on CD70 expression in murine T cells will be determined in the future study. It has been reported that stimulation via surface IgM or CD40 induced CD70 expression on human B cells (36). In mice, stimulation with anti-IgM mAb alone was insufficient to induce CD70 expression on small resting B cells. In contrast, CD70 expression was obviously induced by anti-CD40 mAb stimulation alone and it was further up-regulated by the combination with anti-IgM mAb. As discussed above for T cells, murine resting B cells are possibly naive B cells that might be relatively inactive to induce CD70 expression by cross-linking of surface IgM alone as compared to human tonsillar B cells. Effects of T cell-derived cytokines also remain to be determined. However, our present observation suggests that antigen-specific B cells, which have been primed by surface Ig-mediated signaling, have an advantage over nonspecific B cells for the CD40-mediated CD70 induction, which appears to be relevant to an antigen-specific T–B cognate interaction. We and others have previously demonstrated that hCD70– CD27 interaction plays a critical role in regulating T celldependent B cell activation and Ig synthesis in vitro (19,20,23,24). In addition, it has been recently reported that mCD27 may contribute to the early T cell development in the thymus (46). Given the ability of FR70 to efficiently interfere with the mCD70 functions, the murine system will give us a good opportunity to understand physiological and pathological roles of the CD70–CD27 interaction in vivo. We are now investigating the effects of FR70 on T cell development and T-dependent antibody production in vivo, transplantation and various autoimmune disease models, which will clarify the above issues.

Acknowledgements We greatly thank K. Takeda, H. Matsuda, H. Akiba and M. Barnstead for excellent technical assistance. We also thank C. Morimoto, K. Maruyama and R. Abe for reagents. This research was supported by grants from the Ministry of Education, Science, Sports and Culture and Ministry of Health, Japan and, in part, by the National Institute, DHHS, under contact with ABL.

Abbreviations h HRP LPS m

human horseradish peroxidase lipopolysaccharide murine

Characterization of murine CD70 525 PE TNF

phycoerythrin tumor necrosis factor

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