Dendritic Cell-Mediated NK Cell Activation Is Controlled by Jagged2–Notch Interaction

Dendritic cell-mediated NK cell activation is controlled by Jagged2–Notch interaction

Mika Kijima*, Takeshi Yamaguchi*†, Chieko Ishifune*, Yoichi Maekawa*, Akemi Koyanagi‡, Hideo Yagita‡§, Shigeru Chiba¶, Kenji Kishihara*, Mitsuo Shimada†, and Koji Yasutomo*ʈ

Departments of *Immunology and Parasitology and †Digestive and Pediatric Surgery, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima 770-8503, Japan; ‡Division of Cell Biology, Bio-medical Research Center, and §Department of Immunology, Juntendo University School of Medicine, Tokyo 113-8421, Japan; and ¶Department of Cell Therapy and Transplantation Medicine, University of Tokyo Hospital, 7-3-1 Hongo, Tokyo 113-8421, Japan

Edited by Tak Wah Mak, University of Toronto, Toronto, ON, Canada, and approved February 29, 2008 (received for review October 18, 2007) Natural killer (NK) cells regulate various immune responses by exert- strated that Notch signaling controls both the development ing cytotoxic activity or secreting cytokines. The interaction of NK cells and activation of T cells (7–10). Furthermore, two papers with dendritic cells (DC) contributes to NK cell-mediated antitumor or reported that stimulation of hematopoietic stem cells by antimicrobial responses. However, the cellular and molecular mech- Jagged1 or Jagged2 promotes NK cell differentiation (11–12). anisms for controlling this interaction are largely unknown. Here, we However, whether Jagged controls mature NK cell activa- show an involvement of Jagged2–Notch interaction in augmenting tion or functional differentiation in vivo requires further NK cell cytotoxicity mediated by DC. Enforced expression of Jagged2 investigation. on A20 cells (Jag2-A20 cells) suppressed their growth in vivo, which In this report, we addressed whether or not one of the Notch was abrogated by depleting NK cells. Moreover, Jag2-A20 cells ex- ligands, Jagged2, controls NK cell responses in vivo. We found that erted a suppression on the growth of nonmanipulated A20 cells in stimulation of NK cells by Jagged2 enhanced the antitumor cyto- SCID mice in an NK-dependent manner. Consistently, coinoculation of lytic activity of NK cells in vivo and in vitro. In addition, Jagged2 A20 cells with DC overexpressing Jagged2 (Jag2-DC) suppressed the expressed on DC augmented NK cell function through interaction growth of A20 cells in mice. Stimulation of NK cells with Jagged2 with Notch. These results suggest that robust Notch signaling by directly enhanced their cytotoxicity, IFN-␥ production, and prolifera- enhanced Jagged2 expression may provide a useful strategy to tion. Ligation of Notch2 on NK cells enhanced their cytotoxic activity, eradicate tumor cells. Conversely, the inhibition of Jagged2–Notch and Jag2-DC or CpG-treated DC-mediated NK cell cytotoxicity was interaction might help cure certain autoimmune diseases caused by suppressed by a ␥-secretase inhibitor. These results indicate that the the overactivation of NK cells. Jagged2–Notch axis plays a crucial role in DC-mediated NK cell cytotoxicity. Furthermore, manipulation of this interaction may pro- Results vide an approach to induce potent tumor immunity or to inhibit Overexpression of Jagged2 on A20 Cells Accelerated A20 Rejection in certain autoimmune diseases caused by NK cell activation. BALB/c Mice. We first investigated the role of Jagged2 in NK cell activation by overexpressing Jagged2 on A20 cells. A20 is a cytotoxicity ͉ tumor immunity BALB/c-derived B cell lymphoma cell line that expresses high levels of MHC class II, low levels of MHC class I, Jagged1 and Delta1, and no Jagged2 or Delta4 (Fig. 1a). We transfected Jagged2 (Jag2-A20 atural killer (NK) cells are effector cells of the innate immune cells) or a control vector (c-A20 cells) into A20 cells, and Ϸ75% of system that exhibit direct cytotoxic function (1, 2). NK cell N Jag2-A20 cells expressed Jagged2 after zeocin selection (Fig. 1b). activation is determined by a delicate balance of signals delivered The expression levels of NKG2D ligands and MHC class I on by inhibitory and activating receptors (1–3). Inhibitory receptors Jag2-A20 cells were comparable with those of c-A20 cells (Fig. 1c). bind to MHC class I molecules and down-regulate NK cell cyto- To examine the effect of Jagged2 on antitumor immunity, both toxicity and cytokine production. Thus, missing or low self-MHC cell lines were inoculated s.c. in naı¨ve BALB/c mice, and tumor sizes expression activates NK cells and initiates target cell lysis (2, 3). were monitored (Fig. 1d). The growth of c-A20 cells was detectable Activating receptors are Ig-like or lectin-like molecules that initiate 19 days after inoculation, and c-A20 cells continued to grow in signaling cascades similar to those in T cells. Signaling through BALB/c mice (Fig. 1d). In contrast, Jag2-A20 cells were detectable activating receptors can overcome inhibitory signaling when the 28 days after inoculation, and the growth of tumors was greatly ligands for activating receptors are abundant (3). suppressed compared with that of c-A20 cells (Fig. 1d). NK cells Recent studies have revealed that NK cells interact with dendritic infiltrated more in Jag2-A20 than c-A20 tumors at 10 days after cells (DC) (4, 5) and that this interaction can mediate cytotoxic T inoculation [supporting information (SI) Fig. S1]. These results cell differentiation and NK cell activation/priming (6). For instance, suggest that overexpression of Jagged2 on A20 cells enhances the DC-mediated NK cell activation is crucial for antiviral immunity immune-mediated rejection of A20 cells. (5). However, the underlying molecular and cellular mechanisms to control DC-mediated NK cell activation are poorly understood. Notch is a transmembrane receptor activated by the inter- Author contributions: M.K. and T.Y. contributed equally to this work; M.S. and K.Y. action with Notch ligands. Mammals possess four Notch designed research; M.K., T.Y., Y.M., and A.K. performed research; C.I., Y.M., A.K., H.Y., and receptors and five Notch ligands. Notch ligands are catego- S.C. contributed new reagents/analytic tools; M.K., T.Y., Y.M., A.K., H.Y., and K.K. analyzed data; and K.Y. wrote the paper. rized by two families, Delta (1, 3, and 4) and Jagged (1 and 2) (7). The interaction between a ligand-receptor pair cleaves the The authors declare no conﬂict of interest. extracellular and transmembrane domains of Notch, resulting This article is a PNAS Direct Submission. ʈ in the translocation of the intracellular domain of Notch to the To whom correspondence should be addressed at: Department of Immunology and Parasitology, Institute of Health Biosciences, University of Tokushima Gradate School, nucleus. The interaction of the intracellular domain of Notch 3-18-15 Kuramoto, Tokushima 770-8503, Japan. E-mail: [email protected] with RBP-J allows the formation of transcriptional DNA– u.ac.jp. protein complexes, leading to specific gene expression. Notch This article contains supporting information online at www.pnas.org/cgi/content/full/ signaling generally controls cell fate choice, cell survival, and 0709919105/DCSupplemental. activation in many cell types. Recent studies have demon- © 2008 by The National Academy of Sciences of the USA

7010–7015 ͉ PNAS ͉ May 13, 2008 ͉ vol. 105 ͉ no. 19 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0709919105 Downloaded by guest on September 27, 2021 4 a c-A20 Jag2-A20 * ) 2 3 *

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06911131618 Fig. 1. Overexpression of Jagged2 on A20 cells accelerates A20 rejection. (a) days after tumor inoculation A20 cells were stained with biotin-conjugated anti-Jagged1, Jagged2, Delta1, Fig. 2. NK cells are responsible for the rejection of Jag2-A20 cells. (a) Jag2-A20 or Delta4, followed by PE-streptavidin or FITC-conjugated anti-MHC class I (Dd) (filled squares) or c-A20 (open diamonds) cells were injected s.c. into SCID mice or anti-MHC class II (solid line) and expression intensity was compared with (n ϭ 5), and tumor size was monitored. The tumor size was calculated as (axial) ϫ PE-streptavidin- or control Ig-stained cells (shaded histogram). (b) A20 cells (horizontal) diameter. Data are shown as the mean Ϯ SD. , statistical significance were transfected with a full-length murine Jagged2-encoding vector (Jag2- * (P Ͻ 0.05). These results are representatives of five independent experiments. (b) A20) or control vector (c-A20). The expression of Jagged2 on Jag2-A20 or c-A20 SCID mice injected with Jag2-A20 (squares) or c-A20 (diamonds) cells were treated was tested by staining cells with biotin-conjugated anti-Jagged2 antibody, with 200 ␮g of anti-asialo-GM1 (filled) or control rabbit (open) antibody (day Ϫ1, followed by streptavidin-PE (solid line). Cells stained with streptavidin-PE day 2, day 5, day 8; n ϭ 5). The tumor size was calculated as (axial) ϫ (horizontal) alone were used as a control (shaded histogram). (c) The expression of NKG2D diameter. Data are shown as the mean Ϯ SD. , statistical significance (P Ͻ 0.05) ligands or MHC class I on Jag2-A20 (thick line) or c-A20 (thin line) was tested * between anti-asialo-GM1-treated and control rabbit antibody-treated Jag2-A20- by staining cells with NKG2D-Fc followed by FITC-conjugated anti-human IgG inoculated mice or between anti-asialo-GM1-treated and control rabbit anti- or FITC-conjugated anti-Dd antibody, respectively. Cells stained with strepta- body-treated c-A20-inoculated mice. These results are representatives of four vidin-PE or isotype control alone were used as a control (shaded histogram). independent experiments. (d) Jag2-A20 (filled squares) or c-A20 cells (open diamonds) were injected s.c. into BALB/c mice, and tumor size was monitored. The tumor size was calculated as (axial) ϫ (horizontal) diameter. Data are shown as the mean Ϯ SD. *, statistical significance (P Ͻ 0.05). These results are representatives of five tumor growth. Thus, we next examined the tumor growth of independent experiments. (e)[3H]thymidine incorporation in c-A20 (open) or Jag2-A20 cells in NK-depleted SCID mice that had been treated IMMUNOLOGY Jag2-A20 (filled) cells in cultured medium over an 8-hour period was mea- with anti-asialo-GM1 mAb (Fig. 2b). In fact, this treatment specif- sured. Data are shown as the mean Ϯ SD. ically restored the in vivo growth of Jag2-A20 cells (Fig. 2b). It is of note that the depletion of NK cells augmented the tumor growth of c-A20 cells in SCID mice, indicating that NK activation occurred to To rule out the possibility that Jag2-A20 cells have acquired an a moderate degree upon inoculation of c-A20 cells, although the intrinsic slow-proliferation phenotype, we compared the prolifer- activation was not strong enough to reject A20 cells. Additionally, 3 ation of Jag2-A20 cells and c-A20 cells in vitro.[H]thymidine the c-A20 tumor size was larger than that of Jag2-A20 after NK cell incorporation was quite similar in both cell lines under three depletion (Fig. 2b), suggesting the presence of an NK cell- different cell concentration conditions (Fig. 1e). Taken together, independent aspect of the Jagged2-mediated suppression in this these results eliminated the possibility that the overexpressed context. Taken together, these results suggest that the overexpres- Jagged2 directly slowed down the proliferation of A20 cells, sug- sion of Jagged2 on A20 cells enhanced the antitumor response of gesting that the retarded growth of Jag2-A20 in mice might be a NK cells against A20 cells. consequence of augmented immune surveillance. Enhanced Cytotoxic Activity of NK Cells from BALB/c Mice Inoculated Growth of Jag2-A20 Cells Is Suppressed Even in SCID Mice, and NK Cells with Jag2-A20 Cells. To confirm that the enhanced tumor sup- Are Involved in Jagged2-Mediated Tumor Suppression. We next pression of Jag2-A20 cells reflected augmented cytotoxicity of examined whether mature T or B cells caused the reduced growth NK cells, we purified NK cells from the spleens of c-A20- or of Jag2-A20 cells by inoculating Jag2-A20 cells into T cell- and B Jag2-A20-inoculated BALB/c mice and examined them in an cell-deficient SCID mice. The c-A20 cells grew in SCID mice in vitro killing assay. The cytotoxic activity against A20 cells similarly in BALB/c mice (Fig. 2a). In contrast, the Jag2-A20 tumor was markedly elevated in NK cells harvested from Jag2-A20- size was much smaller in SCID mice compared with that of c-A20 inoculated BALB/c mice compared with those from c-A20- cells, which indicates that T and B cells are not major players in inoculated or untreated mice (Fig. 3a). Therefore, these results suppressing the growth of Jag2-A20 cells (Fig. 2a). Collectively, indicate that overexpression of Jagged2 in A20 cells enhanced these results suggest a role for NK cells in suppressing Jag2-A20 the killing activity of NK cells in vivo.

Kijima et al. PNAS ͉ May 13, 2008 ͉ vol. 105 ͉ no. 19 ͉ 7011 Downloaded by guest on September 27, 2021 a 80 (-) * c-A20 b c d Jag-A20 * 60 12 350 * * 10 300 )

3 250 8 * 40 200 (ng/ml)

% killing % 6 * γ 150 cpm 10 (x 4 IFN 100 20 2 50 N.D 0 0 Relative level of Granzyme Relative level B mRNA expression YAC1-mock YAC1-Jag2 (-) c-A20 Jag2-A20 0 100:1 50:1 25:1 E:T ratio

Fig. 3. Enhanced cytotoxic activity of NK cells from BALB/c mice inoculated with Jag2-A20 cells. (a) c-A20 or Jag2-A20 cells (2 ϫ 107 per mouse) were injected i.p. in BALB/c mice. NK cells were purified from spleens 4 days after injection and were cultured with 51Cr-labeled A20 cells for 5 h. Cytotoxic activity of NK cells from unimmunized (open circles), c-A20 injected (open diamonds) or Jag2-A20 injected (filled squares) mice against A20 cells were evaluated by measuring 51Cr release from A20 cells. These results are representatives of three independent experiments. The data are shown as the mean Ϯ SD. *, statistical significance (P Ͻ 0.05). (b) Purified NK cells were stimulated with YAC-1 cells transfected with control or Jagged2-encoding vectors for 2 days. The [3H]thymidine incorporation for the final 8 h was counted. Data are shown as the mean Ϯ SD. *, statistical significance (P Ͻ 0.05). (c) Purified NK cells were stimulated with A20 cells transfected with control or Jagged2-encoding vectors for 3 days. The concentration of IFN-␥ in the supernatant was measured by ELISA. Data are shown as the mean Ϯ SD. *, statistical significance (P Ͻ 0.05). N.D., not detected. (d) Purified NK cells were stimulated with OP9 cells transduced with control (open symbols) or Jagged2-encoding vectors (filled symbols) for 24 or 48 h. The expression of granzyme B in NK cells was measured by real-time PCR. All data were normalized against the G3PDH mRNA levels and expressed as fold increases relative to control values Ϯ SD obtained from naı¨veNK cells. Data are shown as the mean Ϯ SD of four samples. *, statistical significance (P Ͻ 0.05).

We next tested whether Jagged2 augments the proliferation, cells could subsequently eradicate bystander tumor cells that are IFN-␥ production and expression of effector molecules by NK cells. defective for Jagged2 expression. To this end, we inoculated We isolated DX5ϩ NK cells from BALB/c mice and incubated them Jag2-A20 and A20 cells or c-A20 and A20 cells at different sites with YAC-1 cells transfected with a control vector or a Jagged2- on the same SCID mouse and monitored the tumor size of the encoding vector (Fig. 3b), with c-A20 or Jag2-A20 cells (Fig. 3c), or bystander A20 cells (Fig. 4a). The growth of A20 cells in SCID with OP9 cells transfected with a control vector or a Jagged2- mice inoculated bilaterally with Jag2-A20 cells was suppressed encoding vector (Fig. 3d). The presence of Jagged2 on YAC-1, A20, compared with that of mice that received c-A20 cells (Fig. 4a). or OP9 cells apparently enhanced the proliferation, IFN-␥ secre- This effect of Jag2-A20 cells was diminished by treating SCID tion, or granzyme B transcription of NK cells, respectively (Fig. 3 mice with anti-asialo-GM1 mAb, indicating that NK cells are b, c, and d). These results demonstrate that Jagged2 enhances NK responsible for this effect (Fig. 4b). cell functions, including cytotoxicity, proliferation and IFN-␥ Transfection of Jagged2 in tumor cells is not a favorable clinical production. method to accomplish enhanced NK cell activity. Thus, we examined whether DC overexpressing Jagged2 (Jag2-DC) are able to Inoculation of Jag2-A20 Cells Enhanced the Antitumor Effect of NK augment NK cell activity. Jagged2 was transduced into bone Cells to Kill A20 Cells Transplanted at a Different Site. We then marrow-derived DC by a retroviral vector. Then Jag2-DCs or postulated that the exposure of NK cells to Jagged2-expressing control virus-transduced DC (c-DC) were coinjected with A20 cells could condition them to become activated killers, and these cells, and the tumor size was monitored. The inoculation of

8 a 6 b 5 c c-DC * c-A20 c-A20 ) * ) Jag2-DC 2 5 2 ) Jag2-A20 2 4 Jag2-A20 6 4 3 3 * 4 * 2 2 tumor size (cm tumor size (cm 2 * tumor size (cm * 1 1

0 8 13 19 23 26 28 32 0 8 13 19 23 26 28 0 9 14 17 21 days after tumor inoculation days after tumor inoculation days after tumor inoculation

Fig. 4. Inoculation of Jag2-A20 cells enables NK cells to kill A20 cells transplanted at a different site. (a) Jag2-A20 and A20 cells (closed squares) or c-A20 and A20 cells (open diamonds) were inoculated in different sites of the same SCID mouse (n ϭ 5), and the tumor size of the A20 cells was monitored. The tumor size was calculated as (axial) ϫ (horizontal) diameter. Data are shown as the mean Ϯ SD. *, statistical significance (P Ͻ 0.05). These results are representatives of three independent experiments. (b) SCID mice (n ϭ 5) injected with Jag2-A20 and A20 cells (filled squares) or c-A20 and A20 cells (open diamonds) were treated with 100 ␮g of anti-asialo-GM1 antibody (day Ϫ1, day 2, day 5, day 8; n ϭ 5), and the tumor size of the A20 cells was monitored. Tumor size was calculated as (axial) ϫ (horizontal) diameter. Data are shown as the mean Ϯ SD. *, statistical significance (P Ͻ 0.05). These results are representatives of three independent experiments. (c) A20 cells were inoculated with DC transduced with Jagged2 (filled squares) or control vector (open diamonds) in SCID mice, and the tumor size of A20 cells was monitored. The tumor size was calculated as (axial) ϫ (horizontal) diameter. Data are shown as the mean Ϯ SD. *, statistical significance (P Ͻ 0.05). These results are representatives of three independent experiments.

7012 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0709919105 Kijima et al. Downloaded by guest on September 27, 2021 pression of Delta1 and Jagged1 but not Delta4, compared with a CpG Control IgG Naïve DC activated DC controls (Fig. S2). 0.1 1.2 0.4 1.5 1.7 29.8 To test whether Jagged2 expressed on DC is able to directly activate NK cells, we examined NK cell cytotoxic activity after

Jagged2 coculture with Jag2-DC. The coculture of Jag2-DC and NK cells increased the cytotoxic activity of NK cells over that of unstimu- CD8 lated NK cells (Fig. 5c). We examined whether blocking Notch

CpG Mock- Jagged2- signaling decreases DC-mediated NK cell cytotoxicity by treating b Control IgG Naïve DC activated DC transduced DC transduced DC NK cells with a ␥-secretase inhibitor (GSI). GSI treatment inhibited 0.53 23.1 34.0 15.0 77.3 NK cell activation mediated by Jag2-DC (Fig. 5c). We next tested whether Jagged2-untransfected DC also activate NK cells in a

Jagged2 Notch signaling-dependent manner (Fig. 5d). NK cells cocultured with CpG-activated DC exhibited increased NK cell cytotoxicity, CD11c which was suppressed by GSI treatment (Fig. 5d). Furthermore, NK cells cocultured with CpG-activated DC up-regulated the Notch c d ** e * 40 40 4 target gene, HES1 (Fig. 5e). Taken together, these studies indicate * * that Notch signaling, possibly through Jagged2, is crucial for 30 30 3 DC-mediated enhancement of NK cell cytotoxicity. * * * * 2 20 20 Notch2 Signaling Is Involved in DC-Mediated NK Cell Activation. To % of killing % % of killing % 1 identify the receptor that interacts with Jagged2 in our system, we 10 10 tested the expression of Notch1, Notch2, Notch3, and Notch4 on unstimulated and IL-2-activated NK cells. We detected the expres- 0 0 0 20:1 10:1 5:1 naive CpG naive CpG Relative level of Hes1 mRNA expression level Relative E:T ratio sion of Notch1, Notch2, and Notch4 mRNA in unstimulated NK cells by RT-PCR, and we could detect the expression of Notch1–4 Fig. 5. DC-mediated NK cell activation is controlled by Notch signaling. (a) after IL-2 stimulation (Fig. 6a). We could also detect the expression The expression of Jagged2 on splenic DC from naı¨veor CpG-treated mice was of Notch1 and Notch2 in naı¨ve NK cells by flow cytometry, and evaluated by flow cytometry after staining cells with PE-conjugated anti- their expression was apparently up-regulated by IL-2 stimulation CD11c, FITC-conjugated anti-CD8␣ mAb, and biotin-conjugated anti-Jagged2 ϩ (Fig. 6b). mAb, followed by streptavidin-APC. Data shown are gated on CD11c cells. (b) We next examined whether ligation of Notch2 increases NK cell The expression of Jagged2 on bone marrow-derived untreated, CpG-treated, control vector-transduced, or Jagged2-transduced DC was evaluated by flow activity. NK cells were incubated with anti-Notch2 agonist antibody cytometry. Cells were stained with PE-conjugated anti-CD11c mAb and biotin- for 16 h, and NK cell cytotoxic activity against YAC-1 cells was conjugated anti-Jagged2 mAb, followed by streptavidin-APC. (c) NK cells from measured. The ligation of Notch2 enhanced NK cell activity, and BALB/c mice were cultured with Jag2-DC in the presence (black bars) or the treatment of NK cells by GSI inhibited the effect of the absence (gray bars) of 10 ␮M GSI. The cytotoxic ability of NK cells against anti-Notch2 antibody (Fig. 6c), indicating that Notch2 is one of the YAC-1 cells was measured by 51Cr release from YAC-1 cells. The activity of NK functional receptors for up-regulating NK cell function. cells without Jag2-DC stimulation was used as a control (white bars). Data are shown as the mean Ϯ SD. *, statistical significance (P Ͻ 0.05). (d) NK cells from Discussion ␮ BALB/c mice were cultured with unstimulated or CpG (2 M)-stimulated bone Notch signaling controls the development and activation of a variety marrow-derived DC for 16 h in the absence (white bars) or presence (black bars) of 10 ␮M GSI. NK cells were enriched and cocultured with 51Cr-labeled of immune cells (7, 8, 10, 13, 14). Recent studies have demonstrated YAC-1 cells, and 51Cr release from YAC-1 cells was measured. Data are shown that Jagged and Delta are able to promote the development or as the mean Ϯ SD. *, statistical significance (P Ͻ 0.05). (e) NK cells from BALB/c activation of NK cells, at least in in vitro culture systems (11, 12, 15). mice were cultured with unstimulated or CpG (2 ␮M)-stimulated bone mar- However, it remains unclear whether Notch signaling controls IMMUNOLOGY row-derived DC for 16 h. The mRNA expression of HES1 in purified NK cells was mature NK cell activation and functional differentiation in vivo, quantified by real-time PCR. The value obtained from Notch1 expression in NK and, if so, it should be important to know which cells affect NK cell cells cocultured with unstimulated DC was set as 1. Data are shown as the activation through the expression of Notch ligands. In this report, mean Ϯ SD. *, statistical significance (P Ͻ 0.05). we shed light on this issue by demonstrating that stimulation of NK cells with Jagged2 strengthens mature NK cell-killing activity in vivo and in vitro and that Jagged2 expressed on DC plays a crucial role Jag2-DC significantly suppressed the growth of A20 cells compared in DC-mediated NK cell activation through interaction with Notch. with c-DC (Fig. 4c). This result indicates the feasibility for the use These findings provide an insight that the modulation of Notch of DC as a vehicle to enable Jagged2-mediated augmentation of NK signaling could be a strategy to eradicate tumors or to suppress NK cell activity in vivo, and opens up the possibility for therapeutic cell-mediated diseases. tumor intervention. Recent studies have provided evidence that NK cells are activated by DC and macrophages (5, 6, 16–19), although cellular and Jagged2 on DC Directly Activates NK Cells. To explore the physio- molecular mechanisms controlling DC-mediated NK cell cytotox- logical roles of Jagged2 in terms of NK cell activation, we icity remain unclear. We have addressed this issue by examining examined whether Jagged2 is involved in DC-mediated NK cell whether Notch-signaling plays a crucial role. We observed that activation. We first analyzed Jagged2 expression on splenic DC. Jagged2 expressed on DC is able to enhance NK cell cytotoxic Splenic DC from untreated BALB/c mice express little Jagged2, activity through interaction between these cells and that blockade and CpG-administration to mice up-regulated Jagged2 expres- of Notch signaling significantly attenuated the DC-mediated NK sion on DC (Fig. 5a). Furthermore, the expression of Jagged2 on cell activation. Furthermore, stimulation of NK cells with an agonist ϩ DC was limited to the CD8␣ subset. Bone marrow-derived DC antibody recognizing Notch2 increased NK cell-killing activity. cultured in the presence of GM-CSF express Jagged2 (Fig. 5b). Therefore, these results collectively indicate that Notch signaling is CpG stimulation of bone marrow-derived DC up-regulated crucial for DC-mediated NK cell activation, possibly through Jagged2 expression, although the level was lower than that of DC Jagged2 interaction with Notch2. As for the receptor binding transduced with a Jagged2-expressing retrovirus (Fig. 5b). CpG Jagged2 on NK cells, we cannot completely rule out the involve- stimulation of bone marrow-derived DC also up-regulated ex- ment of other Notch receptors, in particular Notch1, because

Kijima et al. PNAS ͉ May 13, 2008 ͉ vol. 105 ͉ no. 19 ͉ 7013 Downloaded by guest on September 27, 2021 of Jagged2 on CpG-stimulated splenic and bone marrow-derived a * 25 DC by flow cytometry, suggesting that a posttranscriptional mech- anism might be involved in this expression. Therefore, TLR9- 20 * related infections or autoimmune responses may strengthen Notch- 15 * * mediated NK-DC interactions. In light of recent publications 10 demonstrating the involvement of IL-15 or NKG2D in DC- 5 mediated NK cell activation (5, 6), it is crucial to investigate the Relative level of level Relative Notch mRNA expression 0 Notch1 Notch2 Notch3 Notch4 unique physiological roles of Notch signaling in DC-mediated NK cell activation in comparison with those by other molecules. b Day 0 Day 2 Day 4 Several previous papers demonstrated that Jagged1, Jagged2, or 0.8 1.5 31.2 59.6 transient Delta1-mediated signaling stimulate NK cell development (11, 15), although it remains to be determined which Notch Notch1 receptors control NK cell development. On the other hand, the control IgG present study indicates that Jagged2 directly enhances the cytotoxic 4.6 57.0 71.7 activity of mature NK cells, which was inhibited by a ␥-secretase inhibitor, and that the ligation of Notch2 on NK cells augmented

Notch2 NK cell-killing activity. Because Notch 2 is able to bind with Delta1, Delta4, or Jagged1, at least in vitro (21) (data not shown), and DX5 CpG-activated DC up-regulate Delta1, Jagged1 and Jagged2, other Notch ligands might be involved in augmenting NK cell activation. c 50 ** However, we think the contribution of Delta1 or Delta4 would be less likely, at least in vivo, because we did not observe an accelerated 40 rejection of tumor cells by transfecting such ligands (data not

30 shown). The evaluation of Jagged1 in DC-NK interactions is currently underway. An intriguing emerging question from these 20

% of killing % studies is how mature NK cells choose the correct ligand(s) for their activation, because Detla1, Delta4, and Jagged1 as well as Jagged2 10 are expressed on activated DC (data not shown). One possibility is 0 that the expression pattern or timing of the Notch ligands is limited Control IgG Anti-Notch2 mAb to particular cells, thus tightly controlling the interaction of NK cells Fig. 6. Notch signaling contributes to DC-mediated NK cell activation. (a). with their correct ligands. Alternatively, the interaction of Notch The expression of Notch1, Notch2, Notch3, or Notch4 in naı¨ve(black bars) or and Notch ligands might be controlled by modifying the sugar IL-2-stimulated (white bars) NK cells (48 h) was evaluated by real-time PCR. All pattern of Notch by fringe genes. The fringe-mediated addition of data were normalized against the G3PDH mRNA levels and expressed as fold N-acetylglucosamine to o-fucose moieties on Notch could change increases relative to control values Ϯ SD obtained from Notch1 expression in the ligand sensitivity of Notch (22–24). In support of this, we have naïve NK cells. Data are shown as the mean Ϯ SD of three samples. *, statistical observed that NK cells express fringe genes (data not shown). Such significance (P Ͻ 0.05). (b) Notch1 and Notch2 expression on naı¨veNK cells or ϩ modulation of Notch and Notch ligand interaction by fringe may IL-2-stimulated DX5 NK cells (day 0, 2, and 4) were evaluated by flow contribute to the selection of the correct ligand(s) for NK cells cytometry. (c) NK cells from BALB/c mice were cultured in the presence of 1 during their development or activation. ␮g/ml anti-Notch2 or control antibody in the absence (white bars) or presence (black bars) of 10 ␮M GSI for 16 h. The cytotoxic ability of NK cells against We demonstrated here the possibility that Jagged2 overexpres- YAC-1 cells was measured by 51Cr release from YAC-1 cells. Data are shown as sion on DC helps suppress the growth of tumor cells. Because the mean Ϯ SD. *, statistical significance (P Ͻ 0.05). vaccination by DC or by tumor antigen-loaded DC has been widely explored as a method for cancer intervention, the inclusion of Jagged2 in DC-mediated vaccination modalities may prove bene- Notch1 is highly expressed on IL-2-activated NK cells. Our present ficial because this strategy leads to the activation of NK cells and experiments revealed the contribution of Notch signaling in NK tumor-specific CD8ϩ T cells. Furthermore, it might be possible to activation by using GSI, but GSI affects other signaling pathways suppress NK cell-mediated pathogenesis, which is warranted in the (20). Although our present data also revealed the up-regulation of context of some autoimmune diseases, by blocking the Jagged2 a Notch-targeted gene in NK cells by DC stimulation, it would be signaling pathway. Because Notch receptors are widely expressed important to confirm the contribution of Notch in NK cell activa- on a variety of cells, whereas expression of Jagged2 is rather limited, tion by using Notch gene-targeted mice. Additionally, we obtained the inhibition of Jagged2 may be preferable to that of other Notch evidence that Notch signaling directly controls the transcription of receptors for the purpose of suppressing NK cell activity with fewer granzyme B and perforin in cytotoxic T cells (Y.M. and K.Y., side effects. unpublished observation). We also demonstrated in this study that Materials and Methods stimulation of NK cells by Jagged2 rapidly up-regulated granzyme B mRNA. These results are in support of the notion that the direct Mice. Six- to 8-wk-old BALB/c and SCID mice were purchased from Japan SLC, and all mice were maintained under specific pathogen-free conditions in the transcriptional activation of cytolytic molecules by Notch signaling animal research center of the University of Tokushima. All animal studies were could account for the increased effector activity of NK cells. approved by the animal research committee of the University of Tokushima. Furthermore, we demonstrated that Jagged2-mediated stimulation of NK cells promotes their proliferation in vitro. Therefore, both Transfection, Cell Culture, and Virus Infection. The cDNA of murine Jagged2 was up-regulation of effector molecules and increased proliferation of cloned into the pTracer/zeo vector (Fig. S3) (Invitrogen) and the pKE004 retroviral NK cells mediated by Notch signaling would play a major role in vector (8). Retroviruses were constructed by transfecting vectors into plat-E cells strengthening antitumor effects. (25) (provided by T. Kitamura, Tokyo University, Tokyo) as reported (24). After transfection of Jagged2 into A20 or transduction of Jagged2 into YAC-1 or OP9 IL-2 stimulation of NK cells up-regulates Notch1 and Notch2 cells, stable A20, YAC-1, or OP9 cells expressing Jagged2 were maintained. In expression on NK cells. Although a previous report indicated that some experiments, A20 cells were cultured for three days and 1 ␮Ci of [3H]thy- MyD88-dependent stimulation in DC did not up-regulate Jagged2, midine (PerkinElmer Life Sciences) was added for the final 8 h. The incorporation evaluated by real-time PCR (9), we observed increased expression of [3H]thymidine was counted by a liquid scintillation counter.

7014 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0709919105 Kijima et al. Downloaded by guest on September 27, 2021 Bone marrow cells from BALB/c mice were collected and cultured in RPMI were stimulated with A20 cells transfected with murine Jagged2 (2 ϫ 105) for 3 medium 1640 supplemented with 10% FCS and culture supernatant con- days, and culture supernatants were collected. The concentration of IFN-␥ in the taining GM-CSF (provided by T. Iizuka, University of Minnesota, Minneap- supernatant was measured by ELISA according to the manufacturer’s protocol olis). The retroviral vector pKE004 encoding murine Jagged2 was trans- (eBiosciences). To test the mRNA expression of NK cells stimulated by Jagged2, 5 fected into plat-E cells. Bone marrow cells were infected with the retrovirus purified NK cells (2 ϫ 10 ) were stimulated with OP9 cells transfected with murine ϫ 5 0, 1, and 2 days after initial culture. The CD11cϩ and GFPϩ cells were sorted Jagged2 (1 10 ) for 24 or 48 h, and then NK cells were purified by anti-DX5 by the JSAN cell sorter (Bay Bioscience) 6 days after initial culture. In some antibody-coated beads. experiments, bone marrow-derived CD11cϩ cells were stimulated with 2 ␮M CpG (Hycult Biotechnology) for 24 h. Real-Time PCR. RNA was isolated from NK cells by TRIzol (Invitrogen), and cDNA was generated by using Omniscript Reverse Transcription (Qiagen) according to the manufacturer’s instructions. The mRNA expression levels were quantified by Flow Cytometry. NK cells, bone marrow-derived DC, or A20 cells were resus- using the ABI 7500 Real-Time PCR System (Applied Biosystems). cDNAs were pended in staining buffer (2% FCS, 0.05% sodium azide in PBS) at a cell density amplified with TaqMan probes using SYBR Premix Ex Taq (TaKaRa). The mRNA ϫ 6 of 2 10 per milliliter. In some experiments, spleen cells from BALB/c mice expression levels were normalized relative to G3PDH gene expression levels. Fold ␮ injected with 50 g of CpG or NK cells stimulated with mouse IL-2 (100 differences in mRNA levels were determined by using SDS System Software units/ml) (R&D Systems) for 3 days were resuspended in staining buffer. ver.1.4.0 (Applied Biosystems). Primer sequences are as follows: PE-conjugated anti-DX5 (eBiosciences), PE-conjugated anti-CD11c (eBio- Hes1 (forward: 5Ј-GGACAAACCAAAGACGGCCTCTGAGCACAG-3Ј, reverse: 5Ј- sciences), FITC-conjugated anti-CD8␣ (eBiosciences), FITC-conjugated anti-H- TGCCGGGAGCTATCTTTCTTAAGTGCATCC-3Ј), Granzyme B (forward: 5Ј- 2Dd (BD Biosciences), FITC-conjugated anti-MHC class II, mNKG2D-Fc chimera GGATATAAGGATGGTTCACC-3Ј, reverse: 5Ј-CACCTGTCCTAGAGCAATCC-3Ј), (R&D Systems), and FITC-conjugated anti-human IgG (Chemicon) were used G3PDH (forward: 5Ј-TCCACCACCCTGTTGCTGTA-3Ј, reverse: 5ЈACCACAGTCCAT- for cell staining. The biotin-conjugated anti-Jagged1 (HMJ1–29), Jagged2 GCCATCAC-3Ј), Notch1 (forward: 5Ј-TGAGACTGCCAAAGTGTTGC-3Ј, reverse: 5Ј- (HMJ2–1), Delta1 (HMD1–5), Delta4 (HMD4–3), Notch1 (HMN1–12), or Notch2 GTGGGAGACAGAGTGGGTGT-3Ј), Notch2 (forward: 5Ј-ACAAATACTGTGCAGAC- (HMN2–29) mAbs were used for cell staining together with streptavidin-PE or CACTTCAA-3Ј, reverse: 5Ј-AGCACCACGATGATCAGGGT-3Ј), Notch3 (forward: 5Ј- streptavidin-APC (eBiosciences) (SI Text). Anti-Jagged1, Jagged2, Delta1, Del- CAGGCGAAAGCGAGAACAC-3Ј, reverse: 5Ј-GGCCATGTTCTTCATTCCCA-3Ј), Ј Ј Ј ta4, Notch1, or Notch2 antibodies were established by immunizing Armenian Notch4 (forward: 5 -GAACGTGGATCCCCTCAAGT-3 , reverse: 5 -AGAGAGA- Ј hamsters with Jagged1-, Jagged2-, Delta1-, or Delta4-expressing CHO cells or GGGCAAGGACTCAT-3 ) Notch1-Fc or Notch2-Fc recombinant protein. The specificities for anti-Notch ϫ 6 ligand antibodies or Notch antibodies are shown in Figs. S4 and S5, respec- Tumor Inoculation. Mice were inoculated s.c. on the flank with 1 10 A20 or tively. The fluorescence intensity of Ϸ105 cells was examined by using a A20-Jag2 cells in PBS. The long and short diameters of the tumors were measured FACSCalibur flow cytometer (BD Biosciences). by using calipers at 2-day intervals; these two diameters were multiplied to obtain an estimate of the tumor area. Depletion of NK cells was accomplished by i.p. injections of 200 ␮g of polyclonal rabbit anti-asialo GM1 (Wako Chemicals) 2 days NK Cell Purification and Functional Analysis. Spleen cells from unimmunized before the first tumor inoculation and then at 3-day intervals. In some experi- ϫ 6 mice or mice that received 2 10 c-A20 or Jag2-A20 cells i.p. were collected, and ments, 1 ϫ 106 A20 and A20-Jag2 or c-A20 cells were injected s.c. in different sites ϩ IgG-positive cells were depleted by anti-mouse IgG beads (Qiagen). DX5 cells on the same mouse, or 1 ϫ 105 bone marrow-derived dendritic cells transduced were purified from the IgG-negative fraction by anti-DX5-coated beads (Miltenyi with Jagged2 were mixed with 1 ϫ 106 A20 cells and s.c. injected. The data are ϩ Biotec). The purity of the DX5 cells was Ϸ90%. NK cells were stimulated with 1 displayed as the mean Ϯ SD of the tumor areas for each group of animals at a ␮g/ml anti-Notch2 antibody for 16 h or cultured with bone marrow-derived given time point. control DC or Jagged2-transduced DC (1 ϫ 105) for 16 h in the absence or presence of 5 ␮g/ml (10 ␮M) ␥-secretase inhibitor (DAPT; Sigma). After 4 hours of coculture Statistical Analysis. The Mann–Whitney U test was applied to compare data from of NK cells with 51Cr-labeled targets at various effector/target ratios, superna- noninterval scales, such as tumor size. Normally distributed data from interval tants were harvested, and their radioactivity was determined. The percentage of scales were analyzed by Student’s t test with P Ͻ 0.05 considered to be statistically specific cytotoxicity (% cytotoxicity) was calculated by using the following for- significant. mula: % cytotoxicity ϭ (experimental lysis Ϫ spontaneous lysis)/(maximum lysis Ϫ spontaneous lysis) ϫ100. All cytotoxicity assays were carried out in triplicate. Data ACKNOWLEDGMENTS. We thank Dr. Kitamura and Dr. Iizuka for providing are presented as the mean Ϯ SD. Proliferation of NK cells was assessed by using reagents; Dr. Tagaya (National Cancer Institute, Bethesda, MD) for critically purified NK cells stimulated with YAC-1 cells transfected with pKE004 or pKE004 reading the manuscript; Mrs. Kinouchi for technical assistance; and Mrs. ␮ 3 Yamakawa for secretarial assistance. This work was supported by grants- encoding Jagged2 for 48 h. One Ci of [ H]thymidine (PerkinElmer Life Sciences) in-aid for Scientific Research on Priority Areas from the Ministry of Educa- was added for the final 8 hours. The incorporation of [3H]thymidine was counted tion, Culture, Sports, Science and Technology of Japan, and Knowledge 5 by a liquid scintillation counter. In some experiments, purified NK cells (2 ϫ 10 ) Cluster Initiative from the Ministry of Education, Science and Technology. IMMUNOLOGY

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