Cells by OX40 Ligand and CD70 on Activated B CD28-Independent

CD28-Independent Costimulation of T Cells by OX40 Ligand and CD70 on Activated B Cells1

Hisaya Akiba,*† Hideo Oshima,*‡ Kazuyoshi Takeda,*† Machiko Atsuta,*† Hiroyasu Nakano,*† Atsuo Nakajima,§ Chiyoko Nohara,* Hideo Yagita,*† and Ko Okumura2*†

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti- CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28؊ T cells from CD28-deﬁcient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells. The Journal of Immunology, 1999, 162: 7058–7066.

n humoral immune responses, the cognate interaction be- superfamily, can provide a costimulatory signal to T cells, result- tween Ag-specific T cells and B cells is critical not only for ing in increased proliferation and cytokine production (10–12). B cell activation but also for T cell activation. Optimal acti- OX40 was originally identified as a cell surface Ag on activated I ϩ vation of Ag-specific T cells requires engagement of the TCR with CD4 T cells in rats (13). cDNA cloning of rat, mouse, and human Ag/MHC and a costimulatory signal provided by APC (1, 2). It has OX40 showed that it belongs to the TNF receptor superfamily been shown that CD28 on T cells and its ligands (CD80 and CD86) (14–16). It has been reported that expression of OX40 is restricted on APC play a major role in providing a costimulatory signal for to activated T cells in rats, humans, and mice (13, 15, 17). On the T cells (3–5). However, CD28-deficient mice are not fully defec- other hand, expression of OX40L has been found on mouse acti- by guest on October 1, 2021. Copyright 1999 Pageant Media Ltd. tive in humoral immune responses (6, 7). It has also been demon- vated B cells (15, 18), human dendritic cells (DC) (19), human strated that activated B cells exhibited a potent costimulatory ac- vascular endothelial cells (20), and human and rat HTLV-1-trans- ϩ tivity for proliferation of CD4 T cells from CD28-deficient mice formed T cells (10, 21). Recent studies demonstrated that ligation (8). These results suggested the existence of a CD28-independent of OX40L on human DC enhanced their maturation and produc- costimulatory pathway. tion of cytokines (19) and that blockade of OX40L during naive We previously demonstrated that CD27, which is a member of T-DC interaction suppressed the development of IL-4-producing T the TNF receptor superfamily and is constitutively expressed on cells (22), suggesting that OX40-OX40L may play an important most T cells, can transmit a CD28-independent costimulatory sig- role in T-DC interaction. In addition, OX40L expressed on acti- nal upon interaction with its ligand CD70 expressed on a mouse B

http://classic.jimmunol.org vated B cells has been shown to transmit a signal that enhances the lymphoma cell line (9). In addition, we and others have shown that proliferation of and Ig secretion by B cells (18). This suggested OX40 ligand (OX40L),3 which is another member of the TNF that the cross-linking of OX40L on B cells by OX40 on T cells might deliver a B cell differentiation signal, and thus, the OX40- OX40L system might play a unique role in T-B interaction. To *Department of Immunology, Juntendo University School of Medicine, Tokyo, Ja- support this idea, it has been reported that administration of an pan; †CREST (Core Research for Evolutional Science and Technology) of Japan ‡ anti-OX40 Ab inhibited the T cell-dependent humoral immune Downloaded from Science and Technology Corporation, Tokyo, Japan; Department of Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan; and §Department of Joint Disease response in mice, possibly by interrupting the OX40-OX40L and Rheumatism, Nippon Medical School, Tokyo, Japan interaction (23). Received for publication February 9, 1999. Accepted for publication March 31, 1999. To further characterize the role of OX40/OX40L in T-B cell The costs of publication of this article were defrayed in part by the payment of page interaction, we established a functional blocking mAb against charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mouse OX40L, which will be useful for interrupting the OX40- 1 This work was supported in part by grants from the Science and Technology Agen- OX40L interaction in vivo. Our present study formally deﬁned the cy; the Ministry of Education, Science, and Culture; and the Ministry of Health, expression of OX40 and OX40L on activated T and B cells. We Japan. found that the expression of OX40 on activated T cells is CD28 2 Address correspondence and reprint requests to Dr. Ko Okumura, Department of independent, and the CD28-independent costimulatory activity of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail address: [email protected] surface Ig- and CD40-activated B cells is coordinately mediated by 3 Abbreviations used in this paper: OX40L, OX40 ligand; DC, dendritic cells; OX40L and CD70. The physiological relevance of these ﬁndings HTLV-1, human T cell leukemia virus type I; CD40L, CD40 ligand. is discussed.

Materials and Methods 123–142 of murine OX40L cDNA (11) tagged with an XhoI site and 5Ј- Ј Animals and cell lines GACGCGGCCGCGTCCACCCTCAGAGTGGAA-3 corresponding to nucleotides 721–739 tagged with a NotI site were used as 5Ј and 3Ј primers, Six- to seven-week-old male DBA/2 and C57BL/6 mice and 6-wk-old respectively. The PCR product was cloned into pMKITneo vector, pro- female SD rats were purchased from Charles River Japan (Atsugi, Japan). vided by Dr. K. Maruyama (Tokyo Medical and Dental University, Tokyo, Six- to seven-week-old C57BL/6 CD28-deficient (Ϫ/Ϫ) mice were pur- Japan), and transfected into L5178Y, NRK-52E, and P815 cells by elec- chased from The Jackson Laboratory (Bar Harbor, ME). Murine mastcy- troporation. Mock-transfected cells were also produced by electroporation toma P815, murine T lymphoma L5178Y, murine B lymphomas A20.2J of the pMKITneo vector without an insert. After selection by 1 mg/ml of and 2PK-3, and murine myeloma P3U1 (P3 x 63Ag8U.1) were purchased geneticin, transfectants stably expressing OX40L were identified by stain- from American Type Culture Collection (Manassas, VA). The hybridomas ing with OX40-Ig. producing mAbs against MHC class II (M5/114), heat stable Ag (J11d), B220 (RA3-3A1), CD8 (3.155), and Thy1.2 (J1j) were also obtained from Generation of anti-mouse OX40L mAb American Type Culture Collection. The hybridoma OX86 producing anti- An SD rat was immunized with OX40L-transfected NRK cells three times mouse OX40 mAb (24) was obtained from European Collection of Cell at 7-day intervals. Three days after final immunization, the splenocytes Cultures (Wiltshire, U.K.). The hybridoma producing mAb against CD4 were fused with P3U1 cells as previously described (31). After hypoxan- (RL172) was provided by Dr. T. Tanaka (Tokyo Metropolitan Institute of thine-aminopterin-thymidine selection, one hybridoma producing mAb Medical Science, Tokyo, Japan). A murine B lymphoma cell line BCL1- RM134L (rat IgG2b, ␬) was identified by its strong reactivity with OX40L- B20 (25) was provided by Dr. K. Takatsu (Institute of Medical Science, transfected L5178Y cells, but not with mock-transfected L5178Y cells, and University of Tokyo, Tokyo, Japan). A murine T lymphoma cell line was cloned by limiting dilution. RM134L was purified from ascites by MBL2 (26) was provided by Dr. T. Nishimura (Tokai University, Kana- standard procedures with caprylic acid, and purity was verified by SDS- gawa, Japan). These cells were cultured in RPMI 1640 medium contain- PAGE analysis. ing 10% FCS, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mg/ml penicillin and streptomycin, and 50 ␮M 2-ME. Preparation and culture of splenic T cells and B cells A normal rat kidney cell line, NRK-52E (27), was provided by Dr. T. Otsuka (Institute of Cytosignal Research, Tokyo, Japan) and was Splenic T and B cells were purified as previously described (9). Briefly, cultured in DMEM containing 10% FCS, 10 mM HEPES, 4 mM L-glu- splenic T cells were purified by passage through nylon wool column tamine, 1 mM sodium pyruvate, 0.1 mg/ml penicillin and streptomycin, (Wako, Osaka, Japan) and treatment with a mixture of hybridoma super- and 50 ␮M 2-ME. natants (anti-MHC class II, anti-heat stable Ag, and anti-B220) and low-tox rabbit complement (Cedarlane, Hornby, Canada). For preparing small rest- Antibodies ing B cells, splenocytes were treated with a mixture of hybridoma supernatants (anti-Thy-1.2, anti-CD4, and anti-CD8) and low-tox rabbit PE-conjugated goat anti-human and anti-rat IgG Abs were purchased from complement. After Percoll (Pharmacia, Piscataway, NJ) gradient centrifu- Caltag (South San Francisco, CA). Purified anti-CD16/32 (2.4G2), CD3 gation, small B cells were collected from the 60/70% interface. The purity (145-2C11) mAbs, biotinylated anti-CD80 (16-10A1), CD86 (GL1), CD69 of the T and B cell population was Ͼ95% CD3ϩ and Ͼ95% B220ϩ, re- (H1.2F3) mAbs, FITC-conjugated anti-CD3 (145-2C11), CD4 (RM4-4), spectively, as determined by flow cytometry. In some experiments purified CD8 (53-6.7), B220 (RA3-6B2), CD11b (M1/70) mAbs, PE-conjugated T cells (3 ϫ 106/ml) were stimulated with immobilized anti-CD3 mAb (10 anti-CD25 (3C7), control rat IgG2b, and PE-labeled streptavidin were pur- ␮g/ml) with or without soluble anti-CD28 mAb (5 ␮g/ml) for 24–120 h. chased from PharMingen (San Diego, CA). Anti-CD40 (HM40–3), anti- Purified B cells (3 ϫ 106/ml) were stimulated with anti-IgM Ab (10 ␮g/ml) CD80 (RM80), anti-CD86 (PO3), and anti-CD70 (FR70) mAbs were pre- and/or anti-CD40 mAb (5 ␮g/ml) for 24–96 h. pared as described previously (9, 28, 29). Anti-CD28 mAb (PV-1) (30) was provided by Dr. R. Abe (Science University of Tokyo, Chiba, Japan). Goat Flow cytometric analysis anti-mouse IgM F(abЈ) Ab was purchased from Jackson ImmunoResearch 2 Cells (5 ϫ 105) were first preincubated with anti-CD16/32 mAb to avoid by guest on October 1, 2021. Copyright 1999 Pageant Media Ltd. Laboratories (West Grove, PA). A fusion protein consisting of the extra- ␥ cellular portion of murine OX40 and the Fc portion of human IgG1 (OX40- nonspecific binding of Abs to Fc R and then incubated with a saturating Ig) was prepared as described previously (10). amount of OX40-Ig, FITC- or PE-labeled mAb, or biotinylated mAb. After washing with PBS twice, the cells were incubated with PE-conjugated goat Preparation of mouse OX40L transfectants anti-human IgG Ab for OX40-Ig or PE-labeled streptavidin for biotinylated mAb. After washing with PBS twice, the stained cells (live-gated on the A cDNA fragment encoding the entire open reading frame of mouse basis of forward and side scatter profiles and propidium iodide exclusion) OX40L was prepared by RT-PCR from BCL1-B20 mRNA. 5Ј-GACCTC were analyzed on a FACScan (Becton Dickinson, San Jose, CA), and data GAGATGGAAGGGGAAGGGGTTCAA-3Ј corresponding to nucleotides were processed using the CellQuest program (Becton Dickinson). http://classic.jimmunol.org Downloaded from

FIGURE 1. Characterization of RM134L mAb. A, Reactivity of RM134L to mouse OX40L transfectants and cell lines. NRK-52E- and L5178Y-derived transfectants (OX40L/NRK, OX40L/L5178Y, mock/NRK, and mock/L5178Y), a T lymphoma cell line (MBL2), and B lymphoma cell lines (BCL1-B20, A20.2J, and 2PK-3) were stained with biotinylated RM134L followed by PE-labeled streptavidin. The shaded histograms indicate staining with RM134L, and the blank histograms indicate background staining with control IgG. B, RM134L inhibits OX40-Ig binding to OX40L transfectants. OX40L/L5178Y cells were pretreated with or without RM134L and then stained with OX40-Ig followed by PE-labeled goat anti-human IgG Ab. The control histogram represents background staining with RM134L and PE-labeled anti-human IgG without OX40-Ig. 7060 T CELL COSTIMULATION BY ACTIVATED B CELLS

OptEIA Mouse IL-2 Set (PharMingen) according to the manufacturer’s instruction. Results Establishment of anti-mouse OX40L mAb To isolate murine OX40L cDNA, we first tested the reactivities of various mouse cell lines with OX40-Ig. A B lymphoma cell line, BCL1-B20, exhibited high reactivity with OX40-Ig (data not shown). Then, OX40L cDNA was cloned into a mammalian expression vector from RNA of BCL1-B20 cells by RT-PCR and transfected into NRK-52E, L5178Y, and P815 cells. Stable expression of OX40L on the cDNA transfectants (OX40L/NRK, OX40L/L5178Y, and OX40L/P815), but not on mock-transfected cells (mock/NRK, mock/L5178Y, and mock/P815), was verified by binding of OX40-Ig (data not shown). We then immunized an SD rat with OX40L/NRK cells and fused the splenocytes with P3U1 myeloma cells. The hybridomas producing anti-OX40L mAb were screened by specific reactivity to OX40L/L5178Y cells. As represented in Fig. 1A, one mAb, designated RM134L, was obtained, which bound to OX40L/L5178Y and OX40L/NRK cells but not to mock/L5178Y or mock/NRK cells. A number of mouse cell lines were also tested for OX40L expression by staining with RM134L and Northern blot analysis. BCL1-B20 and T lymphoma cell line MBL2, which expressed OX40L mRNA as estimated by Northern blot analysis (not shown), were reactive with RM134L (Fig. 1A), but the other T (WR19L, YAC-1, and BW5147) or B (A20.2J, 2PK-3, CH1, and WEHI231) lymphoma cell lines, which did not express OX40L mRNA, were not reactive with RM134L (Fig. 1A and data not shown). Macrophage cell lines (J774.1 and P388D1) also did not express OX40L mRNA or react with RM134L (not shown). These results indicated a strict coincidence of OX40L mRNA expression and RM134L reactivity. Moreover, preincubation with RM134L blocked OX40-Ig binding to OX40L/ L5178Y cells (Fig. 1B), further substantiating that RM134L is spe-

T cell costimulatory activity of mouse OX40L and its blockade by RM134L FIGURE 2. Costimulation of T cell proliferation by mouse OX40L transfectants and its blockade by RM134L. A, Expression of OX40L on It has been shown that OX40L provided a costimulatory signal for OX40L- or mock-transfected P815 cells. The cells were stained with bio- T cell proliferation in mice, humans, and rats (10–12). To examine tinylated RM134L followed by PE-labeled streptavidin. The shaded histo- whether RM134L can block the costimulatory activity of mouse grams indicate staining with RM134L, and the blank histograms indicate OX40L, splenic T cells were cocultured with OX40L/P815 or background staining with control IgG. B, Kinetics of T cell proliferation mock/P815 cells in the presence of anti-CD3 mAb and RM134L, ϫ 5 http://classic.jimmunol.org costimulated by OX40L/P815. Purified splenic T cells (1 10 /well) were and the proliferative responses were assessed. As shown in Fig. cocultured with irradiated OX40L/P815 or mock/P815 cells (2 ϫ 104/well) 2A, while OX40L/P815 cells expressed a high level of mouse in the presence of anti-CD3 mAb for the indicated periods. The proliferative response was assessed by pulsing the cultures with 0.5 ␮Ci/well OX40L, mock/P815 cells also expressed OX40L at a low level, [3H]thymidine for the last 6 h. Data are expressed as the mean Ϯ SD of which was verified by Northern blot analysis (not shown). As triplicate wells. C, RM134L inhibits the costimulation by OX40L/P815. shown in Fig. 2B, OX40L/P815 cells costimulated the proliferation Purified splenic T cells (1 ϫ 105/well) were cocultured with irradiated of anti-CD3-stimulated T cells far more efficiently than mock/P815 4 Downloaded from OX40L/P815 cells (2 ϫ 10 /well) in the presence of anti-CD3 mAb and the cells. The peak proliferative response on day 2 was blocked by indicated concentrations of RM134L or control IgG. The proliferative re- RM134L, but not by control IgG, in a dose-dependent manner sponse was assessed at 48 h as described above. (Fig. 2C). In addition, the proliferative response costimulated by mock/P815 cells was abrogated by RM134L (data not shown), indicating a substantial contribution of endogenously expressed T cell proliferation and IL-2 assay OX40L to the costimulatory activity of mock/P815 cells. These results indicated that mouse OX40L exhibits a potent costimula- ϫ 5 Purified splenic T cells (1 10 /well) were cocultured with irradiated (100 tory activity for anti-CD3-stimulated T cell proliferation, which Gy) transfectants (2 ϫ 104/well) or irradiated (20 Gy) preactivated B cells (5 ϫ 104/well) in the presence or the absence of anti-CD3 mAb (2C11, 1 can be blocked by RM134L. ␮g/ml) in 96-well round-bottomed culture plates at 37°C for 48 h. For estimating proliferative responses, the cultures were pulsed with 0.5 ␮Ci/ Expression of OX40L and OX40 on activated T cells 3 well of [ H]thymidine (DuPont-New England Nuclear, Boston, MA) for We next examined the expression of OX40L and OX40 on splenic the last 9 h and then harvested using a Micro 96 Harvester (Skatron, Lier, Norway). Incorporated radioactivity was measured in a micro beta counter T cells by flow cytometric analysis using RM134L and an anti- (Micro ␤ Plus, Wallac, Turku, Finland). To determine IL-2 production, mouse OX40 mAb (MRC OX86) (24). An anti-CD25 mAb was cell-free supernatants were collected at 48 h and subjected to ELISA using also included as an activation marker. Purified splenic T cells were The Journal of Immunology 7061 by guest on October 1, 2021. Copyright 1999 Pageant Media Ltd.

FIGURE 3. Expression of OX40 and OX40L on activated T cells. Splenic T cells (3 ϫ 106/ml) from C57BL/6 mice were stimulated with immobilized anti-CD3 mAb without (A) or with (B) soluble anti-CD28 mAb and were harvested at the indicated periods. Cells were stained with PE-labeled anti-CD25 mAb, biotinylated RM134L, anti-OX40 mAb, or control IgG followed by PE-labeled streptavidin. C, C57BL/6 splenocytes (3 ϫ 106/ml) were stimulated with immobilized anti-CD3 mAb for 48 h and double-stained with FITC-labeled anti-CD4 or CD8 mAb and biotinylated anti-OX40 mAb or control IgG ϫ 6 Ϫ Ϫ

http://classic.jimmunol.org followed by PE-labeled streptavidin. D, Splenic T cells (3 10 /ml) from CD28-deﬁcient ( / ) mice were stimulated with immobilized anti-CD3 mAb for 48 h and stained with biotinylated RM134L, anti-OX40 mAb, or control IgG followed by PE-labeled streptavidin. In A, B, and D, the shaded histograms indicate staining with the indicated mAb and the blank histograms indicate background staining with control IgG.

stimulated for 24–120 h with immobilized anti-CD3 mAb in the veriﬁed using CD28Ϫ T cells from CD28-deﬁcient mice. As presence or the absence of soluble anti-CD28 mAb. As shown in shown in Fig. 3D, a comparable level of OX40 expression was Ϫ

Downloaded from Fig. 3A, OX40 expression was detected on T cells after stimulation found on CD28 T cells to that on wild-type T cells. Collectively, with anti-CD3 mAb alone, which appeared at 24 h and reached a these results indicated that mouse OX40 is expressed on TCR/ peak at 72 h. The addition of anti-CD28 mAb signiﬁcantly in- CD3-stimulated CD4ϩ and CD8ϩ T cells in a CD28-independent creased and sustained the expression of OX40 on anti- manner and that OX40L is not expressed on normal mouse T cells. CD3-stimulated T cells (Fig. 3B). In contrast, OX40L was not found on T cells even when stimulated with both anti-CD3 and Expression of OX40L and OX40 on activated B cells anti-CD28 mAbs (Fig. 3B). In the mouse system, it has been re- We next examined the expression of OX40L and OX40 on splenic ported that OX40 was expressed on both activated CD4ϩ and B cells. Puriﬁed splenic B cells were stimulated with anti-IgM Ab, CD8ϩ T cells (24), while OX40 was expressed predominantly on anti-CD40 mAb, or both for 24–96 h and stained with RM134L or CD4ϩ T cells in the rat system (13). As shown in Fig. 3C,we anti-OX40 mAb. An anti-CD69 mAb was included as an activation found mouse OX40 expression on both CD4ϩ and CD8ϩ T cells marker. As shown in Fig. 4C, OX40L expression was detected on after stimulation with anti-CD3 mAb. B cells after stimulation with the combination of anti-IgM Ab and The above experiments suggested that the CD28-mediated co- anti-CD40 mAb, which appeared at 24 h and reached a peak at stimulation was facilitating, but not required, for the OX40 ex- 72 h. In contrast, stimulation with either anti-IgM Ab or anti-CD40 pression on anti-CD3-stimulated T cells. This idea was further mAb alone did not induce OX40L expression (Fig. 4, A and B). 7062 T CELL COSTIMULATION BY ACTIVATED B CELLS

FIGURE 4. Expression of OX40 and OX40L on activated B cells. Puriﬁed splenic B cells (3 ϫ 106/ml) from C57BL/6 mice were stimulated with anti-IgM Ab (A), anti-CD40 mAb (B), or anti-IgM Ab plus anti-CD40 mAb (C). Cell were harvested at the indicated periods and stained with biotinylated anti-CD69, RM134L, anti-OX40 mAb, or control IgG followed by PE-labeled streptavidin. The shaded histograms indicate staining with the indicated mAb, and the blank histograms indicate background staining with control IgG.

Unexpectedly, marginal expression of OX40 was reproducibly ited T cell proliferation, the combination of these two mAbs ex- found on anti-IgM-stimulated B cells, which was apparent at hibited a significant inhibitory effect ( p Ͻ 0.01) comparable to that 48–72 h (Fig. 4A). The combination with anti-CD40 mAb mark- of anti-CD80/86 mAbs (Fig. 5B). On the other hand, IL-2 produc- edly enhanced OX40 expression (Fig. 4C), whereas stimulation tion was strongly inhibited by anti-CD80/86 mAbs, and further with anti-CD40 mAb alone did not induce OX40 expression (Fig. inhibition was observed by their combination with anti-OX40L 4B). These results indicated that OX40L is specifically expressed and/or anti-CD70 mAb (Fig. 5C). Anti-OX40L or anti-CD70 mAb on surface Ig- and CD40-stimulated B cell and that either stimu- alone significantly inhibited IL-2 production ( p Ͻ 0.01), and fur- lation alone was not effective. Moreover, a novel expression of ther inhibition was observed by the mixture of these two mAbs

by guest on October 1, 2021. Copyright 1999 Pageant Media Ltd. OX40 on B cells was revealed. (Fig. 5C). These results indicated the substantial contributions of OX40L and CD70 to the costimulatory activity of anti-IgM- plus Involvement of CD80, CD86, CD70, and OX40L in T cell anti-CD40-stimulated B cells, which can mostly account for the costimulation by activated B cells CD80/86-independent part. The above observations of a potent T cell costimulatory activity of

OX40L and the expression of OX40L on anti-IgM- plus anti- Ϫ CD40-stimulated B cells suggested that OX40L might play a sub- Costimulation of CD28 T cells by OX40L and CD70 on stantial role in T cell costimulation by activated B cells. It has been activated B cells shown that surface Ig- and/or CD40-stimulated B cells expressed The above experiment implied that the OX40L and CD70 ex- http://classic.jimmunol.org the CD28 ligands CD80 and CD86 (32–35). We also previously pressed on activated B cells could costimulate T cells indepen- demonstrated that anti-IgM- plus anti-CD40-stimulated B cells ex- dently of the CD80/86 and CD28 interaction. To further address pressed CD70, which is another member of the TNF superfamily this idea, we examined the contributions of OX40L and CD70 to and provides a costimulatory signal via CD27 constitutively ex- costimulation of CD28Ϫ T cells from CD28-deﬁcient mice by pre- pressed on most T cells (9). Consistent with these observations, the activated B cells. As shown in Fig. 6A, the anti-IgM- plus anti- anti-IgM plus anti-CD40-stimulated B cells expressed CD80, CD40-stimulated B cells from CD28-deﬁcient mice expressed

Downloaded from CD86, CD70, and OX40L at high levels (Fig. 5A). We then ex- CD80, CD86, CD70, and OX40L at levels comparable to those in amined the relative contributions of CD80/86, CD70, and OX40L B cells from wild-type C57BL/6 mice (Fig. 5A). As shown in Fig. to T cell costimulation by anti-IgM- plus anti-CD40-stimulated B 6, B and C, when costimulated by these preactivated B cells, the cells. Puriﬁed splenic T cells were cocultured with preactivated B CD28Ϫ T cells exhibited 50–60% reduced proliferation and 60– cells in the presence of anti-CD3 mAb and combinations of anti- 70% reduced IL-2 production compared with wild-type T cells, CD80/86, anti-CD70, and/or anti-OX40L mAb for 48 h, and then which were almost comparable to those of wild-type T cells in the the proliferative response and IL-2 production were assessed. As presence of anti-CD80/86 mAbs (Fig. 5, B and C). As expected, shown in Fig. 5, B and C, the anti-IgM- plus anti-CD40-stimulated the addition of anti-CD80/86 mAbs showed no inhibitory effect. In B cells exhibited a potent costimulatory activity for anti- contrast, both T cell proliferation (Fig. 6B) and IL-2 production CD3-stimulated T cell proliferation and IL-2 production. Anti- (Fig. 6C) were signiﬁcantly inhibited by either anti-OX40L or anti- CD80/86 mAbs only partially inhibited T cell proliferation, and CD70 mAb alone. Notably, T cell proliferation and IL-2 produc- further inhibition was obtained by their combination with either tion were mostly inhibited by the mixture of these two mAbs (75.8 anti-OX40L or anti-CD70 mAb ( p Ͻ 0.05; Fig. 5B). The strongest and 70.6% inhibition, respectively). These results indicated that inhibition was obtained by the combination of all these mAbs. the CD28-independent T cell costimulatory activity of surface Ig- While anti-OX40L or anti-CD70 mAb alone only modestly inhib- and CD40-stimulated B cells are mostly mediated by OX40L and The Journal of Immunology 7063

CD70, although a minor contribution of some other molecule was also suggested. Discussion In this study we generated an mAb specific for murine OX40L and determined the expression of OX40L and OX40 on murine T and B lymphocytes. We also examined the T cell costimulatory function of OX40L expressed on activated B cells. Some novel findings were obtained, as follows. OX40 has been originally identified as an activation marker preferentially expressed on CD4ϩ T cells in the rat system (13). In contrast, a recent study demonstrated that OX40 was expressed on both CD4ϩ and CD8ϩ T cells after Con A stimulation of mouse splenocytes, suggesting a differential expression of OX40 on activated rat and mouse T cells (24). We confirmed this in the present study and observed that both CD4ϩ and CD8ϩ T cells expressed OX40 after anti-CD3 stimulation in the mouse system (Fig. 3C). Although the expression of OX40 on human T cells has not been well characterized, our preliminary results showed that OX40 is expressed on both CD4ϩ and CD8ϩ T cells after anti-CD3 stimulation of human peripheral blood lymphocytes (unpublished observations). Therefore, the rather restricted expression of OX40 on CD4ϩ T cells appears to be a unique feature of the rat system. We also examined the stimulatory requirement for OX40 expression on T cells, which has not been determined in previous studies. We found that OX40 can be expressed on anti-CD3-stimulated T cells without costimulation with anti-CD28 mAb (Fig. 3A) and also on CD28Ϫ T cells from CD28-deficient mice (Fig. 3D). This inde- pendence of OX40 expression from CD28 costimulation underlies the CD28-independent costimulatory activity of OX40L as represented in Fig. 6. A previous report (11) showed that OX40L was expressed on a murine T lymphoma cell line and anti-TCR-stimulated murine splenic T cells as estimated by OX40-Ig binding, whereas staining

by guest on October 1, 2021. Copyright 1999 Pageant Media Ltd. of the latter was marginal. We found a high expression of OX40L in a Moloney virus-induced T lymphoma cell line MBL2 (Fig. 1A), but not in the other T cell lines tested or in anti-CD3- or anti-CD3/ CD28-stimulated splenic T cells as estimated by staining with anti- OX40L mAb. Consistently, we could detect OX40L mRNA in MBL2, but not in anti-CD3- or anti-CD3/CD28-stimulated splenic T cells, by Northern blot analysis (data not shown). Therefore, OX40L appears not to be expressed on splenic T cells under these conditions, although it remains to be determined whether OX40L http://classic.jimmunol.org can be expressed on T cells under a particular condition. In this respect, it is noteworthy that OX40L is frequently expressed on HTLV-1-transformed human and rat T cell lines, possibly due to trans-activation by Tax protein encoded by the pX region of HTLV-1 (10, 21, 36). HTLV-1 is not only the etiological agent of adult T cell leukemia but also causes HTLV-1-associated diseases,

Downloaded from such as myelopathy, uveitis, arthropathy, and Sjo¨gren’s syndrome (37). It has been shown that the HTLV-1 pX transgenic mice de- veloped inﬂammatory arthropathy, neuroﬁbronatosis, and exoci- nopathy (38–41). The anti-mouse OX40L mAb we established in

cells from C57BL/6 mice (1 ϫ 105/well) were cocultured with the irradi- FIGURE 5. Contributions of CD80/86, CD70, and OX40L to costimu- ated preactivated B cells (5 ϫ 104/well) in the presence of anti-CD3 mAb lation of T cells by anti-IgM plus anti-CD40-stimulated B cells. A, Cell and 2 ␮g/ml each of the indicated mAbs (total concentration of mAbs was surface expression of costimulatory molecules on preactivated B cells. Pre- adjusted to 8 ␮g/ml with rat IgG) for 48 h. The proliferative response was activated B cells were prepared by stimulating splenic B cells from assessed by pulsing the cultures with 0.5 ␮Ci/well [3H]thymidine for the C57BL/6 mice with anti-IgM Ab plus anti-CD40 mAb for 72 h. Cells were last 9 h. C, Inhibitory effects on IL-2 production. Cell-free supernatants stained with biotinylated mAb against the indicated molecules (shaded his- were collected from the cultures in B at 48 h, and IL-2 content was mea- tograms) or control IgG (blank histograms) followed by PE-labeled strepta- sured by ELISA. The data in B and C are expressed as the mean Ϯ SD of vidin. B, Inhibitory effects of anti-CD80/86, anti-OX40L, and/or anti-CD70 triplicate wells. Similar results were obtained from three independent ex- .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .mAb on T cell proliferation costimulated by activated B cells. Splenic T- periments 7064 T CELL COSTIMULATION BY ACTIVATED B CELLS

the present study will be useful to address the pathological function of OX40L in these mouse models. It has been reported that OX40L, as estimated by OX40-Ig binding, was expressed on mouse splenic B cells upon stimulation with anti-IgD dextran or CD40 ligand (CD40L)-expressing L cells, and the highest expression levels were obtained when stimulated by both (18). In contrast, we could not detect significant levels of OX40L expression on anti-IgM Ab- or anti-CD40 mAb-stimulated B cells (Fig. 4, A and B). This discrepancy may be due to higher cross-linking of surface Ig by anti-IgD dextran than by anti-IgM Ab or a signaling difference between surface IgD and IgM and higher cross-linking of CD40 by CD40L-expressing L cells than anti-CD40 mAb. In our present results, however, a high expression of OX40L was obtained by the stimulation with anti-IgM Ab plus anti-CD40 mAb, consistent with the previous observation that the highest OX40L expression was obtained after stimulation by anti- IgD dextran and CD40L. Therefore, it is likely that OX40L is preferentially expressed on surface Ig-stimulated, Ag-specific B cells upon interaction with CD40L-expressing Th cells, especially when surface Ig cross-linking by Ags and CD40 cross-linking by CD40L are limiting. Stu¨ber et al. also demonstrated that cross- linking of OX40L on activated B cells by OX40-Ig resulted in an enhanced proliferation and an increase in Ig secretion in vitro (18), and that administration of anti-OX40 Ab profoundly inhibited the anti-hapten IgG response in vivo and the development of periar- teriolar lymphoid sheath-associated B cell foci probably by interrupting the interaction of OX40 on activated T cells with OX40L on activated B cells (23), suggesting a critical role for the OX40- OX40L interaction in differentiation of activated B cells into Ig- producing plasma cells. In this respect, our novel finding of con- comitant expression of OX40 and OX40L on activated B cells is intriguing, since it suggests that the OX40/OX40L-mediated proliferation and differentiation could be driven by B-B cell interactions in the B cell foci. Further studies are now under way to

by guest on October 1, 2021. Copyright 1999 Pageant Media Ltd. characterize the function of OX40 expressed on activated B cells. It has been shown that ligation of OX40 by anti-OX40 mAbs or OX40L provided a costimulatory signal for T cell proliferation (10–12). We also observed a potent costimulatory activity of mouse OX40L for proliferation of anti-CD3-stimulated naive T cells (Fig. 2). We then examined the costimulatory function of OX40L expressed on activated B cells. The anti-IgM plus anti- CD40-stimulated B cells expressed multiple costimulatory molecules, including CD80, CD86, CD70, and OX40L (Fig. 5A). Anti- http://classic.jimmunol.org CD80/86 mAbs only partially inhibited the costimulatory activity of activated B cells, and the CD80/86-independent part was mostly inhibited by the combination of anti-OX40L and anti-CD70 mAbs (Fig. 5B), indicating substantial contributions of OX40L and CD70. The residual part may be mediated by other costimulatory molecules, such as CD44H (42) and CD54 (43), which have also

Downloaded from been implicated in the costimulatory activity of CD40L-activated B cells. Although it is possible that the anti-OX40L and anti-CD70 mAbs might act in an inhibitory manner by negatively signaling T FIGURE 6. Costimulation of CD28Ϫ T cells by OX40L and CD70 on cells, this is unlikely, since neither mAb inhibited anti-CD3- and activated B cells. A, Cell surface expression of costimulatory molecules on anti-CD28-stimulated T cell proliferation (data not shown). The preactivated B cells from CD28Ϫ/Ϫ mice. Preactivated B cells were pre- additional experiment with CD28Ϫ T cells from CD28-deﬁcient Ϫ Ϫ pared by stimulating splenic B cells from CD28 / mice with anti-IgM Ab mice (Fig. 6) clearly indicated that OX40L and CD70 expressed on plus anti-CD40 mAb for 72 h. Cells were stained with biotinylated mAb activated B cells can costimulate T cells in a CD28-independent against the indicated molecules (shaded histograms) or with control IgG (blank histograms) followed by PE-labeled streptavidin. B, Inhibitory effects of anti-OX40L and/or anti-CD70 mAb on proliferation of CD28Ϫ T cells costimulated by activated B cells. Splenic T cells from CD28Ϫ/Ϫ mice the cultures with 0.5 ␮Ci/well [3H]thymidine for the last 9 h. C, Inhibitory (1 ϫ 105/well) were cocultured with the irradiated preactivated B cells effects on IL-2 production. Cell-free supernatants were collected from the (5 ϫ 104/well) in the presence of anti-CD3 mAb and 2 ␮g/ml each of the cultures in B at 48 h, and IL-2 content was measured by ELISA. Data in B indicated mAbs (the total concentration of mAbs was adjusted to 4 ␮g/ml and C are expressed as the mean Ϯ SD of triplicate wells. Similar results were .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .with rat IgG) for 48 h. The proliferative response was assessed by pulsing obtained from three independent experiments The Journal of Immunology 7065

ϩ manner. It has been reported that CD4 T cells from CD28-defi- 9. Oshima, H., H. Nakano, C. Nohara, T. Kobata, A. Nakajima, N. A. Jenkins, cient mice can express CD40L upon anti-CD3 stimulation (44), D. J. Gilbert, N. G. Copeland, T. Muto, H. Yagita, et al. 1998. Characterization of murine CD70 by molecular cloning and mAb. Int. Immunol. 10:517. which is a prerequisite for the expression of OX40L and CD70 on 10. Akiba, H., M. Atsuta, H. Yagita, and K. Okumura. 1998. Identification of rat B cells. We also showed that OX40 can be expressed on CD28Ϫ OX40 ligand by molecular cloning. Biochem. Biophys. Res. Commun. 251:131. T cells upon anti-CD3 stimulation (Fig. 3). Taken together, these 11. Baum, P. R., R. B. Gayle III, F. Ramsdell, S. Srinivasan, R. A. Sorensen, M. L. Watson, M. F. Seldin, E. Baker, G. R. Sutherland, K. N. Clifford, et al. results suggest that OX40L and CD70 may mostly account for the 1994. Molecular characterization of murine and human OX40/OX40 ligand sys- CD28-independent pathway of T cell costimulation during Ag- tems: identification of a human OX40 ligand as the HTLV-1-regulated protein specific T-B cognate interactions. It has been shown that CD28- gp34. EMBO J. 13:3992. 12. Godfrey, W. R., F. F. Fagnoni, M. A. Harara, D. Buck, and E. G. Engleman. deficient mice can be primed with soluble Ags in vivo so as to 1994. Identification of a human OX-40 ligand, a costimulator of CD4ϩ T cells generate Ag-specific T cell proliferative responses in vitro (6, 7). with homology to tumor necrosis factor. J. Exp. Med. 180:757. CD40/CD40L have been implicated as the predominant mediator 13. Paterson, D. J., W. A. Jefferies, J. R. Green, M. R. Brandon, P. Corthesy, M. Puklavec, and A. F. Williams. 1987. Antigens of activated rat T lymphocytes of the CD28-independent pathway of T cell costimulation (45– including a molecule of 50,000 Mr detected only on CD4 positive T blasts. Mol. 47). Although some reports suggested transmission of a costimu- Immunol. 24:1281. latory signal via CD40L into T cells (48, 49), it is generally ac- 14. Mallett, S., S. Fossum, and A. N. Barclay. 1990. Characterization of the MRC OX40 antigen of activated CD4 positive T lymphocytes–a molecule related to cepted that CD40L acts indirectly by up-regulating the expression nerve growth factor receptor. EMBO J. 9:1063. of costimulatory molecules on APC. Our present results suggest 15. Calderhead, D. M., J. E. Buhlmann, A. J. van den Eertwegh, E. Claassen, that OX40L and CD70 can at least partly account for the CD40/ R. J. Noelle, and H. P. Fell. 1993. Cloning of mouse Ox40: a T cell activation marker that may mediate T-B cell interactions. J. Immunol. 151:5261. CD40L-dependent pathway of T cell costimulation. We are now 16. Latza, U., H. Durkop, S. Schnittger, J. Ringeling, F. Eitelbach, M. Hummel, addressing this possibility by administrating anti-OX40L and/or C. Fonatsch, and H. Stein. 1994. The human OX40 homolog: cDNA structure, anti-CD70 mAb into CD28-deficient or CD40L-deficient mice im- expression and chromosomal assignment of the ACT35 antigen. Eur. J. Immunol. 24:677. munized with various Ags. 17. Durkop, H., U. Latza, P. Himmelreich, and H. Stein. 1995. Expression of the Our present study revealed substantial contributions of OX40L human OX40 (hOX40) antigen in normal and neoplastic tissues. Br. J. Haematol. and CD70 to CD28-independent T cell costimulation. CD28Ϫ T 91:927. 18. Stuber, E., M. Neurath, D. Calderhead, H. P. Fell, and W. Strober. 1995. Cross- cells are not unique to CD28-deficient mice. CD28 is expressed on linking of OX40 ligand, a member of the TNF/NGF cytokine family, induces most CD4ϩ T cells, but on only half of CD8ϩ T cells (50, 51). It proliferation and differentiation in murine splenic B cells. Immunity 2:507. has been suggested that CD8ϩ CD28Ϫ T cells might represent 19. Ohshima, Y., Y. Tanaka, H. Tozawa, Y. Takahashi, C. Maliszewski, and G. Delespesse. 1997. Expression and function of OX40 ligand on human den- suppressor T cells in humans (52, 53). It has also been reported dritic cells. J. Immunol. 159:3838. ϩ Ϫ that CD4 CD28 T cells accumulated in PBL from SLE patients, 20. Imura, A., T. Hori, K. Imada, T. Ishikawa, Y. Tanaka, M. Maeda, S. Imamura, which might be relevant to autoantibody production in these pa- and T. Uchiyama. 1996. The human OX40/gp34 system directly mediates adhe- sion of activated T cells to vascular endothelial cells. J. Exp. Med. 183:2185. tients (51). At present, costimulatory molecules mediating activa- 21. Miura, S., K. Ohtani, N. Numata, M. Niki, K. Ohbo, Y. Ina, T. Gojobori, Ϫ tion of these CD28 T cells have not been well characterized. Our Y. Tanaka, H. Tozawa, M. Nakamura, et al. 1991. Molecular cloning and char- present results suggest that OX40L and CD70 may be responsible acterization of a novel glycoprotein, gp34, that is specifically induced by the Ϫ human T-cell leukemia virus type I transactivator p40tax. Mol. Cell. Biol. 11: for the costimulation of these CD28 T cells. 1313. In the present study we demonstrated the T cell costimulatory 22. Ohshima, Y., L. P. Yang, T. Uchiyama, Y. Tanaka, P. Baum, M. Sergerie, P. Hermann, and G. Delespesse. 1998. OX40 costimulation enhances interleu- by guest on October 1, 2021. Copyright 1999 Pageant Media Ltd. function of OX40L expressed on activated B cells. It has been kin-4 (IL-4) expression at priming and promotes the differentiation of naive hu- reported that OX40L was also expressed on human DC and endo- man CD4ϩ T cells into high IL-4-producing effectors. Blood 92:3338. thelial cells. We observed that murine DC and endothelial cell 23. Stuber, E., and W. Strober. 1996. 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