Cells CD27-Mediated Activation of Murine NK

CD27-Mediated Activation of Murine NK Cells1

Kazuyoshi Takeda,2*† Hideo Oshima,*‡ Yoshihiro Hayakawa,§ Hisaya Akiba,*† Machiko Atsuta,*† Tetsuji Kobata,*† Kimio Kobayashi,¶ Mamoru Ito,¶ Hideo Yagita,*† and Ko Okumura*†

CD27, a member of the TNF receptor superfamily, has been implicated in T cell activation, T cell development, and T cell- dependent Ab production by B cells. In the present study we examined the expression and function of CD27 on murine NK cells. Murine NK cells constitutively expressed CD27 on their surface. Stimulation with immobilized anti-CD27 mAb or murine CD27 ligand (CD70) transfectans solely could induce proliferation and IFN-␥ production of freshly isolated NK cells and enhanced the proliferation and IFN-␥ production of anti-NK1.1-sutimulated NK cells. Although NK cell cytotoxicity was not triggered by anti-CD27 mAb or against CD70 transfectants, prestimulation via CD27 enhanced the cytotoxic activity of NK cells in an IFN- ␥-dependent manner. These results suggest that CD27-mediated activation may be involved in the NK cell-mediated innate immunity against virus-infected or transformed cells expressing CD70. The Journal of Immunology, 2000, 164: 1741–1745.

t has been well established that T cells require a costimula- Abs and reagents tory signal in addition to an Ag-specific TCR-mediated signal Purified anti-CD16/32 mAb (2.4 G2), FITC-conjugated or no azide/low I for full activation (1, 2). Such a costimulatory signal can be endotoxin anti-mouse NK1.1 mAb (PK 136), biotin-conjugated or no provided by CD28 or some members of the TNF receptor super- azide/low endotoxin anti-mouse CD27 mAb (LG.3A10), peridinin chloro- family, including CD27, CD30, OX40 (CD134), and 4-1 BB phyl protein (PerCP)3-conjugated anti-mouse CD3⑀ mAb (145-2C11), bi- (CD137), on the T cell surface (3–6). otin-conjugated anti-mouse CD30 mAb (mCD30.1), biotin-conjugated anti- mouse CD40 mAb (3/23), biotin-conjugated anti-mouse 4-1 BB mAb With respect to NK cells, it has been recently revealed that many (1AH2), no azide/low endotoxin anti-mouse CD70 mAb (FR-70), isotype activating or inhibitory NK cell receptors (KIR, NKR-P1 family, control for hamster IgG (G235-2356), rat IgG1(R3-34), rat IgG2a (R35-95) and CD94-NKG2, etc.) and other molecules (CD2, CD16, etc.) and rat IgG2b(R95-1), and PE-conjugated streptavidin were purchased regulate NK cell activation (7). It has been also reported that CD28 from PharMingen (San Diego, CA). The hybridoma-producing anti-mouse OX-40 mAb (OX-86) was obtained from European Cell Culture Collection was also involved in NK cell activation (7–11). However, there has (Wilshire, U.K.), and the hybridomas producing anti-mouse IFN-␥-neu- been little information about the expression and function of TNF tralizing mAb (R4-6A2), anti-mouse MHC class II mAb (M5/114), anti- receptor superfamily members on NK cells. mouse B220 mAb (RA3-3A1), anti-mouse CD8 mAb (3.155), and anti- In the present study we examined the expression and function of mouse CD4 mAb (RL172.4) were obtained from American Type Culture by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd. some TNF receptor superfamily members on murine NK cells. We Collection (Manassas, VA). The mAbs were prepared and biotinylated in our laboratory followings described standard method (13). Human rIL-2 found that murine NK cells constitutively express CD27, and the was provided by Shionogi Pharmaceutical (Osaka, Japan). Recombinant stimulation with anti-CD27 mAb or CD27 ligand (CD70) en- murine IL-12 (4.9 ϫ 106 U/mg) was provided by Genetic Institute hances NK cell proliferation and IFN-␥ production. The physio- (Andover, MA). logical relevance of the findings is discussed. Cell preparation Splenic mononuclear cells were prepared by pressing spleens through a Materials and Methods stainless steel mesh followed by treatment with RBC lysis solution as pre- Mice viously described (14). NK cells were purified as previously described (8).

http://classic.jimmunol.org In brief, B and T cells were depleted from splenocytes by passage through Male C57BL/6 (B6) mice, 6 wk of age, were purchased from Clear Japan Ϫ Ϫ a nylon wool column (Wako, Osaka, Japan) and then by treatment with a (Tokyo, Japan). B6 RAG-2 / mice were bred in Central Institute for mixture of hybridoma supernatants (anti-MHC class II, anti-B220, anti-CD4, Experimental Animals (Kawasaki, Japan) and used at 6–8 wk of age (12). and anti-CD8) and low toxic rabbit complement (Cedarlane, Hornby, Canada). All mice were maintained under specific pathogen-free conditions. After Percoll (Pharmacia Biotech) gradient centrifugation, the purity of NK (NK1.1ϩ CD3Ϫ) cells was Ͼ95% as determined by flow cytometry. *Department of Immunology, Juntendo University School of Medicine, Bunkyo-ku, Cell lines Tokyo, Japan; †CREST (Core Research for Evolutional Science and Technology) of Downloaded from Japan Science and Technology Corp., Chiyoda-ku, Tokyo, Japan; ‡Department of NK-susceptible YAC-1 target cells and NK-resistant P815 target cells were § First Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan; Department maintained in RPMI 1640 medium supplemented with 10% heat-inacti- of Pathogenic Biochemistry, Research Institute of Wakan-yaku, Toyama Medical and vated FCS, 2 mM L-glutamine, and 25 mM NaHCO in humidified 5% CO Pharmaceutical University, Toyama, Japan; and ¶Central Institute for Experimental 3 2 Animals, Nogawa, Miyamae, Kawasaki, Japan at 37°C. P815 cells stably transfected with mouse CD70cDNA (P815- CD70) were prepared as previously described (15). Received for publication October 22, 1999. Accepted for publication December 1, 1999. Flow cytometry The costs of publication of this article were defrayed in part by the payment of page Expression of respective molecules on NK cells was analyzed by three- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. color flow cytometry as described previously (14, 16). To avoid the non- specific binding of Abs to Fc␥R on NK cells, the cells were preincubated 1 This work was supported by a Grant-in-Aid for Scientific Research from the Min- with anti-mouse CD16/32 (2.4G2) mAb before staining, which diminished istry of Education, Science, and Culture, Japan. 2 Address correspondence and reprint requests to Dr. Kazuyoshi Takeda, Department of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bukyou-ku, 3 Abbreviations used in this paper: PerCP, peridinin chlorophyl protein; TRAF, TNF Tokyo 113-8421, Japan. receptor-associated factor.

FIGURE 1. Constitutive expression of CD27 on murine NK cells. Freshly isolated NK cells were prepared from C57BL/6 splenocytes by depletion of B and T cells, and then stimulated with 500 U/ml IL-2 for 7 days. After Fc receptor blocking, fresh NK and IL-2-stimulated NK cells were stained with FITC-conjugated anti-NK1.1 mAb, PerCP-conjugated anti-CD3 mAb, and biotin-conjugated mAb against the indicated TNF receptor family molecules, followed by PE-conjugated streptavidin. Expression of respective molecules on NK1.1ϩ CD3Ϫ cells was analyzed by ﬂow cytometry. Solid lines indicate the staining with respective mAb, and broken lines indicate the staining with isotype-matched control IgG.

the binding of isotype controls to NK cells. Then the cells were incubated streptavidin. Expression of the respective TNF receptor family with a saturating amount of biotinylated mAbs. After washing with PBS molecule was analyzed on gated NK1.1ϩ CD3Ϫ cells. While twice, the cells were incubated with FITC-conjugated anti-mouse NK1.1 CD30, CD40, OX40, and CD134 were not detectable on either mAb, PerCP-conjugated anti-CD3⑀ mAb, and PE-conjugated streptavidin. After washing with PBS twice, the stained cells were analyzed on a FAC- fresh or IL-2-activated NK cells, CD27 was constitutively ex- Scan (Becton Dickinson, San Jose, CA), and data were processed using the pressed on both fresh and IL-2-activated NK cells (Fig. 1). As CellQuest program (Becton Dickinson). recently reported (18), CD137 was expressed on IL-2-activated NK cells, but not on fresh NK cells. These results indicated that Cell culture CD27 is unique among the TNF receptor family molecules tested Ϫ Ϫ Spleen cells from B6 RAG-2 / mice were incubated in a tissue culture in that it is constitutively expressed on freshly isolated NK cells. grade polystyrene dish at 37°C for1htodeplete adherent cells. Harvested ϩ Ϫ cells were routinely Ͼ95% NK1.1 CD3 as estimated by ﬂow cytometry. Proliferation of NK cells upon cross-linking of CD27 Puriﬁed NK cells were cultured in RPMI 1640 medium supplemented with

10% heat-inactivated FCS, 2 mM L-glutamine, and 25 mM NaHCO3 in Since CD27 has been reported to provide a costimulatory signal ϫ 5 humidified 5% CO2 at 37°C as previously reported (17). NK cell (3 10 ) for T cell proliferation (15, 19–24), the effect of CD27 cross- ␮ were stimulated with immobilized anti-NK1.1 mAb (10 g/ml) and/or anti- linking by immobilized anti-CD27 mAb on the proliferation of NK mouse CD27 mAb (10 ␮g/ml) in the presence or the absence of IL-2 (25 U/ml) and/or IL-12 (5 U/ml) on a 96-well flat-bottom culture plate (Costar, cells was first examined. To exclude the possible contribution of

by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd. Cambridge, MA). Abs in PBS were immobilized by overnight incubation at 4°C. In the coculture experiments, purified NK cells (3 ϫ 105/well) were cocultured with irradiated (15,000 rad) P815 or P815-CD70 cells (3 ϫ 104/well) in 96-well round-bottomed culture plate in the presence of 25 U/ml IL-2. In some experiments, coculture was performed in the presence of anti-NK1.1 mAb (10 ␮g/ml) and/or anti-CD70 mAb (5 ␮g/ml). The cultures were incubated for 2 days and pulsed with 0.5 ␮Ci/well of [3H]thymidine (Amersham, Aylesbury, U.K.) for the last 16 h. Incorpo- rated radioactivity was measured in a Microbeta counter (Microbeta Plus, Wallac, Turku, Finland). Cell-free culture supernatants were harvested after incubation for 3 days and subjected to cytokine assay. http://classic.jimmunol.org ELISA IFN-␥ levels in the culture supernatants were evaluated by using a mouse IFN-␥-specific ELISA kit (OptEIA) purchased from PharMingen accord- ing to the manufacturer’s instruction. Cytotoxicity assay Downloaded from Cytotoxic activity was assessed by a standard 51Cr release assay (14). Briefly, 51Cr-labeled target cells (5 ϫ 103) were added to serial dilutions of effector cells. For assessing redirected lysis, anti-NK1.1 mAb or anti-CD27 mAb were added at 10 ␮g/ml. After 4-h incubation, supernatants were harvested and counted with a gamma counter. Specific cytotoxicity was calculated as previously described (14). Results Constitutive expression of CD27 on murine NK cells FIGURE 2. Proliferation of NK cells upon CD27 and/or NK1.1 cross- linking. Freshly isolated RAG-2Ϫ/Ϫ splenic NK cells were stimulated for 2 To examine the expression of TNF receptor family molecules on days with immobilized anti-CD27 mAb (10 ␮g/ml) and/or immobilized NK cells, freshly isolated splenic NK cells from B6 mice and anti-NK1.1 mAb (10 ␮g/ml) in the absence (A) or the presence (B) of 25 IL-2-activated NK cells were subjected to three-color staining with U/ml IL-2. Proliferative response was assessed by pulsing the culture with FITC-conjugated anti-NK1.1 mAb, Per-CP-conjugated anti-CD3⑀ 0.5 ␮Ci/well of [3H]TdR for the last 16 h. Data are expressed as the mAb, and biotin-conjugated mAb against CD27, CD30, CD40, mean Ϯ SD of triplicate cultures. Data shown are representative of three .p Ͻ 0.05 ,ء .OX40(CD134), or 4-1BB(CD137) followed by PE-conjugated independent experiments with similar results The Journal of Immunology 1743

FIGURE 4. Induction of proliferation and IFN-␥ production of NK cells by CD27/CD70 interaction. Freshly isolated RAG-2Ϫ/Ϫ splenic NK cells were cocultured with irradiated P815 or CD70-transfected P815 cells in the presence or the absence of anti-NK1.1 mAb (10 ␮g/ml) and/or anti-CD70 mAb (5 ␮g/ml) with 25 U/ml IL-2. After 2 days, the proliferative response was assessed by pulsing the culture with 0.5 ␮Ci/well of [3H]thymidine (3H-TdR) for the last 16 h (A). Cell-free culture supernatants were collected FIGURE 3. IFN-␥ production by NK cells upon CD27 and/or NK1.1 after 3 days, and IFN-␥ was measured by ELISA (B). Data are expressed cross-linking. Freshly isolated RAG-2Ϫ/Ϫ splenic NK cells were stimulated as the mean Ϯ SD of triplicate cultures. Data shown are representative of .p Ͻ 0.05 ,ء .with immobilized anti-CD27 mAb (10 ␮g/ml) and/or immobilized anti- three independent experiments with similar results NK1.1 mAb (10 ␮g/ml) in the presence of 25 U/ml IL-2 without (A)or with (B) 5 U/ml IL-12. Cell-free culture supernatants were collected from the cultures after 3 days, and IFN-␥ was measured by ELISA. Data are CD27 ligand (CD70) stimulates NK cells to proliferate and to expressed as the mean Ϯ SD of triplicate cultures. Data shown are repre- produce IFN-␥ Ͻ ء sentative of three independent experiments with similar results. , p 0.05. To exclude the possible contribution of Fc receptors on NK cells to the NK cell activation by immobilized mAb, we determined whether the natural ligand for CD27 (CD70) could induce the proliferation and IFN-␥ production of NK cells. Puriﬁed NK cells Ϫ Ϫ contaminated T cells, NK cells were puriﬁed from B6 RAG-2 / Ϫ/Ϫ

by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd. from RAG-2 mice were cocultured with irradiated P815 or Ϫ/Ϫ splenocytes, which lack T cells and NKT cell (12). RAG-2 NK CD70-transfected P815 (P815-CD70) cells in the presence of IL-2. cells expressed CD27 at the same level as the B6 NK cells shown When NK cells were cocultured with P815-CD70 cells, substantial in Fig. 1 (data not shown). The cross-linking of CD27 induced a levels of proliferation and IFN-␥ production were observed, which low, but signiﬁcant, level of proliferation, which was comparable were abrogated by anti-CD70 mAb (Fig. 4). Moreover, CD70 en- to that induced by NKR-P1 cross-linking by immobilized anti- hanced the proliferation and IFN-␥ production induced by anti-NK1.1 NK1.1 mAb (Fig. 2A). Co-cross-linking of CD-27 and NKR-P1 mAb, which were again abrogated by anti-CD70 mAb. These results showed an additive effect. In the presence of a submitogenic con- indicated that CD27 on NK cells can induce proliferation and IFN-␥ centration of IL-2, anti-CD27 mAb also induced a substantial level production of NK cells upon interaction with its ligand, CD70.

http://classic.jimmunol.org of proliferation, albeit to a lesser extent than anti-NK1.1 mAb, and enhanced the proliferation induced by anti-NK1.1 mAb (Fig. 2B). CD27 cannot trigger NK cell cytotoxicity These results suggested that CD27 expressed on freshly isolated It has been reported that cross-linking of NK1.1 could trigger NK NK cells is functional in inducing NK cell proliferation. cell cytotoxicity (25–27). We then examined whether CD27 could trigger NK cell cytotoxicity by using anti-CD27 mAb-redirected cytotoxic assay against FcR-bearing P815 target cells or by using IFN-␥ production by NK cells upon cross-linking of CD27

Downloaded from CD70-transfected P815 cells as the target cells. Freshly isolated We next examined the IFN-␥ induction by NK cells upon CD27 NK cells lysed the classical NK target YAC-1 cells efﬁciently (data cross-linking with anti-CD27 mAb. When NK cells were incu- not shown), but not P815 cells. Although anti-NK1.1 mAb efﬁ- bated with immobilized anti-CD27 mAb in the presence of 25 ciently directed NK cell cytotoxicity against P815, anti-CD27 U/ml IL-2 for 3 days, a substantial level of IFN-␥ was detected in mAb did not (Fig. 5). Moreover, P815-CD70 cells did not show the culture supernatant, which was comparable to that induced by increased susceptibility to NK cells in the presence or the absence anti-NK1.1 mAb (Fig. 3A). The combination of anti-CD27 and of anti-NK1.1 mAb (Fig. 5). These results indicated that CD27 anti-NK1.1 mAbs showed an additive effect. In the presence of a cannot trigger cytotoxic activity to NK cells, unlike NK1.1. suboptimal concentration of IL-12, anti-CD27 mAb induced a con- ␥ siderable level of IFN-␥ production, which was again comparable IFN- -mediated enhancement of NK cell cytotoxicity by to that induced by anti-NK1.1 mAb, and enhanced the IFN-␥ pro- prestimulation via CD27 duction induced by anti-NK1.1 mAb (Fig. 3B). These results in- Since IFN-␥ has been known to enhance the NK cell cytotoxicity dicated that CD27 cross-linking by anti-CD27 mAb can induce (28, 29), it would be possible that NK cell cytotoxicity could be IFN-␥ production by NK cells as potently as NKR-P1 cross-link- augmented by prestimulation via CD27, which induce IFN-␥ pro- ing by anti-NK1.1 mAb. duction by NK cells as shown in Fig. 3. To address this possibility, 1744 NK CELL ACTIVATION VIA CD27

FIGURE 5. CD27 does not trigger NK cell cytotoxicity. NK cells were freshly isolated from RAG-2Ϫ/Ϫ spleen, and cytotoxicity was tested against 51Cr-labeled P815 or CD70-transfected P815 cells in the presence or the absence of anti-NK1.1 mAb (10 ␮g/ml) or anti-CD27 mAb (10 ␮g/ml) at the indicated E:T cell ratios. Data are expressed as the mean Ϯ SD of triplicate wells and are representative of three independent experiments .p Ͻ 0.05 compared with P815 alone ,ء .with similar results

puriﬁed NK cells were stimulated with immobilized anti-CD27 FIGURE 6. IFN-␥-mediated enhancement of NK cell cytotoxicity by Ϫ Ϫ mAb for 3 days in the presence of a suboptimal dose of IL-2, and prestimulation via CD27. Freshly isolated RAG-2 / splenic NK cells ␮ then cytotoxic activity was tested against YAC-1 and P815 target were stimulated for 3 days with immobilized anti-CD27 mAb (10 g/ml) ␮ cells. As shown in Fig. 6, the prestimulation with anti-CD27 mAb and/or immobilized anti-NK1.1 mAb (10 g/ml) in the presence of 25 U/ml IL-2. Neutralizing anti-IFN-␥ mAb (10 ␮g/ml) was added to some signiﬁcantly augmented the cytotoxic activities against both target wells. After 3 days, NK cells were harvested, and cytotoxicity against cells to levels comparable to those induced by anti-NK1.1 pre- YAC-1 (A) and P815 (B) was tested at the indicated E:T cell ratios. Data stimulation and further enhanced the anti-NK1.1-induced cytotox- are expressed as the mean Ϯ SD of triplicate cultures. Data shown are p Ͻ ,ء .icity. The enhancement by anti-CD27 prestimulation was com- representative of three independent experiments with similar results by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd. pletely abrogated by neutralizing anti-IFN-␥ mAb. These results 0.05 compared with IL-2 alone. indicated that CD27-mediated stimulation can augment NK cell cytotoxicity indirectly by inducing IFN-␥ production. NK cells (30). In the present study we showed that the great ma- Discussion jority of murine NK cells express CD27 before and after IL-2 In this study we demonstrated that murine NK cells express CD27 stimulation. Expression of CD27 on freshly isolated NK cells was constitutively and that stimulation of CD27 with immobilized anti- unique among the TNF receptor family molecules tested, while CD27 mAb or its ligand, CD70, induces proliferation and IFN-␥ CD137 was expressed on IL-2-activated NK cells as previously

http://classic.jimmunol.org production of freshly isolated NK cells as potently as stimulation reported (18). Moreover, we demonstrated that CD27 on murine of the NK1.1 (NKR-P1) molecule, which has been implicated in NK cells is functional in inducing proliferation and IFN-␥ produc- murine NK cell activation in previous studies (7, 17, 25–27). Al- tion, while such functions have not been explored in previous stud- though CD27 stimulation does not directly trigger cytolytic func- ies with human NK cells. Our present observation of the failure to tion of NK cells, unlike NKR-P1, it can augment NK cell cyto- trigger cytolytic function of murine NK cells by anti-CD27 mAb toxicity indirectly by inducing autocrine IFN-␥ production. These or against CD70 transfectants is consistent with previous observa-

Downloaded from results suggest the involvement of CD27 in the regulation of NK tions, which reported that human NK cells did not show enhanced cell expansion and functions. cytotoxicity against CD70 transfectants in a 4-h 51Cr release assay Expression and function of CD27 on T cells have been well (31). Furthermore, we clariﬁed that the enhanced cytotoxic activity of characterized in both human and murine systems (5, 6, 15, 19–24). murine NK cells after prestimulation with immobilized anti-CD27 CD27 is expressed on the majority of human peripheral blood T mAb is mediated by autocrine IFN-␥, suggesting that the increased cells, and its engagement by anti-CD27 mAb or CD70 induced NK cell activity after coculture with CD70 transfectans in the human proliferation of naive T cells in the presence of a suboptimal dose system might be also mediated by IFN-␥ (31). These results imply of PMA, PHA, or anti-CD3 mAb (5, 6, 19–24). Also in the murine that CD27 plays a similar role on murine and human NK cells. system, CD27 is expressed on the vast majority of mature T cells, The signaling mechanism by which CD27 activates NK cells and its engagement by anti-CD27 mAb or murine CD70 costimu- remains to be determined in the future study. It has been shown lated proliferation of Con A- or anti-CD3-stimulated T cells (15, that cross-linking of CD27 induced tyrosine phosphorylation of 20). In contrast, the expression and function of CD27 on NK cells cytoplasmic proteins, including ZAP-70, in human T cells, might have not been well characterized. In the human system, it has been enhance the TCR/CD3-mediated signaling (22). It remains to be reported that CD27 was expressed at low levels by ϳ30–40% of determined whether CD27 stimulation may also induce protein peripheral blood NK cells and was up-regulated on IL-2-activated tyrosine phosphorylation of NK cells. In this respect, it may be The Journal of Immunology 1745

interesting to compare the signaling via CD27 with that via the 12. Shinkai, Y., G. Rathbun, K. P. Lam, E. M. Oltz, V. Stewart, M. Mendelsohn, NKR-P1 molecule, which not only induces IFN-␥ production but J. Charron, M. Datta, F. Young, A. M. Stall, et al. 1992. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell also triggers NK cell lytic function. It has been shown that 68:855. NKR-P1 is associated with the FcR ␥-chain, which activates 13. Kaneko, Y., S. Hirose, M. Abe, H. Yagita, K. Okumura, and T. Shirai. 1996. 2ϩ CD40-mediated stimulation of B1 and B2 cells: implication in autoantibody pro- ZAP-70 and/or Syk and induces an increase in intracellular Ca duction in murine lupus. Eur. J. Immunol. 26:3061. that is requisite for triggering NK cell lytic function (32–34). In 14. Takeda, K., S. Seki, K. Ogasawara, R. Anzai, W. Hashimoto, K. Sugiura, ϩ ϩ contrast, it has been shown that CD27 ligation affected calcium M. Takahashi, M. Satoh, and K. Kumagai. 1996. Liver NK1.1 CD4 ␣␤ T cells activated by IL-12 as a major effector in inhibition of experimental tumor me- mobilization in NK cells only in the induction mediated by CD2 tastasis. J. Immunol. 156:3366. (30), which might be responsible for the failure to trigger the cy- 15. Oshima, H., H. Nakano, C. Nohara, T. Kobata, A. Nakajima, N. A. Jenkins, tolytic function of NK cells. In addition to the tyrosine phosphor- 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. ylation events, we recently revealed that CD27 recruits TNF re- 16. Akiba, H., H. Oshima, K. Takeda, M. Atsuta, H. Nakano, A. Nakajima, ceptor-associated factor-2 (TRAF2) and TRAF5, which activate C. Nohara, H. Yagita, and K. Okumura. 1999. CD28-independent costimulation NF-␬B and c-Jun N-terminal kinase and have been implicated in of T cells by OX40 ligand and CD70 on activated B cells. J. Immunol. 162:7058. 17. Arase, H., N. Arase, and T. Saito. 1996. Interferon ␥ production by natural killer cellular proliferation and cytokine production (35). An important (NK) cells and NK1.1ϩ T cells upon NKR-P1 cross-linking. J. Exp. Med. 183: role for TRAF-mediated signaling in T cells has been substantiated 2391. 18. Melero, I., J. V. Johnston, W. W. Shufford, R. S. Mittler, and L. Chen. 1998. by the impaired proliferation of TRAF5-deficient T cell in re- NK1.1 cells express 4-1BB (CDw137) costimulatory molecule and are required sponse to CD27-mediated costimulation (36). Further studies are for tumor immunity elicited by anti-4-1BB monoclonal antibodies. Cell. Immu- underway to characterize the signaling pathway responsible for the nol. 190:167. 19. Hintzen, R. Q., R. de John, S. M. Lens, and R. A. van Lier. 1994. CD27: marker CD27-mediated activation of NK cells. and mediator of T-cell activation? Immunol. Today 15:307. It has been established that NK cells play important roles in the 20. Gravestein, L. A., J. D.Nieland, A. M. Kruisbeek, and J. Brost. 1995. Novel mAb innate immunity against infection and tumor development (29, 37, reveal potent co-stimulatory activity of murine CD27. Int. Immunol. 7:551. 21. Agematsu, K., T. Kobata, K. Sugita, G. J. Freeman, M. P. Beckmann, 38). NK cells preferentially lyse virus-infected cells and trans- S. F. Schlossman, and C. Morimoto. 1994. Role of CD27 in T cell immune formed cells by their cytotoxic function. IFN-␥ produced by NK response: analysis by recombinant soluble CD27. J. Immunol. 153:1421. 22. Kobata, T., K. Agematsu, J. Kameoka, S. F. Schlossman, and C. Morimoto. 1994. cells not only exerts anti-viral and proinflammatory effects, but ϩ CD27 is a signal-transducing molecule involved in CD45RA naive T cell co- also induces Th1-and CTL-mediated acquired immunity. In the stimulation. J. Immunol. 153:5422. present study we showed that CD27-mediated stimulation by anti- 23. Hintzen, R. Q., S. M. Lens, K. Lammers, H. Kuiper, M. P. Beckmann, and ␥ R. A. van Lier. 1995. Engagement of CD27 with its ligand CD70 provides a CD27 mAb or CD70 enhanced IFN- production and the cytotoxic second signal for T cell activation. J. Immunol. 154:2612. activity of NK cells. CD70 was originally characterized as a 24. Brown, G. R., K. Meek, Y. Nishioka, and D. L. Thiele. 1995. CD27-CD27 ligand/ marker of Reed-Sternberg cells in Hodgkin’s disease and non- CD70 interactions enhance alloantigen-induced proliferation and cytolytic activity in CD8ϩ T lymphocytes. J. Immunol. 154:3686. Hodgkin’s lymphomas (39). It has also been found that CD70 is 25. Karlhofer, F. M., and W. M. Yokoyama. 1991. 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