NK Cells IL-12-Induced Activation of Resting Mouse an Accessory Role for B Cells In

An Accessory Role for B Cells in the IL-12-Induced Activation of Resting Mouse NK Cells

This information is current as Evette A. Haddad, Laura K. Senger and Fumio Takei of September 25, 2021. J Immunol 2009; 183:3608-3615; Prepublished online 26 August 2009; doi: 10.4049/jimmunol.0901391 http://www.jimmunol.org/content/183/6/3608 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2009/08/26/jimmunol.090139 Material 1.DC1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

An Accessory Role for B Cells in the IL-12-Induced Activation of Resting Mouse NK Cells1

Evette A. Haddad,* Laura K. Senger,* and Fumio Takei2*†

IL-12 is a potent proinflammatory cytokine. The effects of IL-12 are thought to be mediated by IFN-␥ production by NK, NKT, and T cells. In this study, we show that although IL-12 stimulates NK and NK1.1؉ T cells in bulk mouse splenocytes, it does not significantly stimulate purified NK cells, indicating that other cells are required. IL-12 stimulates T cell-deficient spleen cells and those depleted of macrophages. Unexpectedly, the depletion of dendritic cells also has little effect on the stimulation of spleen cells with IL-12. In contrast, B cell depletion almost completely inhibits IL-12-induced IFN-␥ production and B cell-deficient spleen cells are poorly stimulated with IL-12. Furthermore, purified NK cells are stimulated with IL-12 in the presence of purified B cells. Thus, B cells are necessary and also sufficient for the stimulation of purified NK cells with IL-12. Whereas spleen cells from

IL-18-deficient mice are not stimulated with IL-12, NK cells purified from IL-18-deficient mice are stimulated with IL-12 in the Downloaded from presence of wild-type (WT) B cells, and WT NK cells are not stimulated with IL-12 in the presence of IL-18-deficient B cells. Cell contact between B and NK cells is also required for IL-12-induced IFN-␥ production. Finally, B cell-deficient mice injected with IL-12 produce significantly less IFN-␥ and IL-18 in the sera than WT mice do. Thus, stimulation of NK cells with IL-12 requires B cell cooperation in vitro as well as in vivo. The Journal of Immunology, 2009, 183: 3608–3615.

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nterleukin 12 is a prototypic proinflammatory cytokine pro- IFN- production from NK cells and T cells and is known to be http://www.jimmunol.org/ duced by dendritic cells (DCs),3 monocytes/macrophages, involved in autoimmune and inflammatory diseases (20). Although I and activated B cells in the early phase of an immune re- IL-18 alone is not capable of driving IFN-␥ production, it has sponse to infection (1). The importance of IL-12 in immunity has strong synergistic effects with IL-12 and the combination of these been demonstrated by the profound susceptibility of IL-12-defi- two cytokines induces large amounts of IFN-␥ production (21). cient humans and mice to various pathogens (2–4). IL-12 is a Moreover, Fantuzzi et al. (22) have reported that both the neutral- potent inducer of IFN-␥ production, which plays a pivotal role in ization of IL-18 and caspase 1 deficiency, which mediates the the initiation of inflammation and the establishment of protective cleavage of the IL-18 precursor to active IL-18, significantly re- Th1 responses (5, 6). IL-12 has also been used as a therapeutic duce the production of IFN-␥ in mice receiving IL-12. These re- reagent against various microbial diseases (7, 8) and more recently sults suggest that IL-18 plays a role in the IL-12-induced produc- by guest on September 25, 2021 has proven to have potent antitumor/antimetastasis effects when tion of IFN-␥. Recently, it has been reported that stimulation of ex administered to experimental animals and patients (9–12). The vivo NK cells with IL-12 requires in vivo priming with IL-18 (23). anticancer effects of IL-12 are primarily mediated by IFN-␥.T To elucidate the mechanisms by which resting NK cells are cells, NK cells, and NKT cells, are thought to be the primary stimulated by IL-12, we stimulated spleen cells from normal mice producers of IFN-␥ in response to IL-12 (13, 14). Yet, resting with a suboptimal dose of IL-12 alone. After characterizing the human and mouse NK cells and T cells express none to low levels cells producing IFN-␥, we tested the effects of IL-12 on various of the IL-12R and only express appreciable levels of IL-12R after purified cell populations. We also examined mutant mice and con- activation (15, 16). Thus, the mechanisms by which resting NK ducted cell depletion experiments to determine the requirements cells are stimulated with IL-12 remain unclear. for IL-12-induced IFN-␥ production. Our results show that B cells IL-18 is another proinflammatory cytokine, initially identified as are unexpectedly required for IL-12-induced IFN-␥ production by an IFN-␥-inducing cytokine (17, 18). It is produced as an inactive NK cells. precursor, which must be cleaved to become biologically active, and is secreted by a variety of cells including epithelial cells, DCs, Materials and Methods and macrophages (19). Like IL-12, IL-18 is a strong inducer of Mice Wild-type (WT) and TCR␤/TCR␦ double knockout (KO) (TCR␤␦KO) C57BL/6 (B6) mice were purchased from The Jackson Laboratory and *Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Co- † bred pathogen free in our animal facility. IL-18KO and B cell-deficient lumbia, Canada; and Department of Pathology and Laboratory Medicine, University (BKO) (Igh-6tm1Cgn) B6 mice were also purchased from The Jackson Lab- of British Columbia Vancouver, British Columbia, Canada oratory and housed in our animal facility. Male or female 8- to 12-wk-old Received for publication May 4, 2009. Accepted for publication July 9, 2009. pathogen-free mice were used in this study. All animal use was approved The costs of publication of this article were defrayed in part by the payment of page by the animal care committee of the University of British Columbia and charges. This article must therefore be hereby marked advertisement in accordance animals were maintained and euthanized under humane conditions in ac- with 18 U.S.C. Section 1734 solely to indicate this fact. cordance with the guidelines of the Canadian Council on Animal Care. 1 This work was supported by grants from the Canadian Cancer Society. Abs, cytokines, and media 2 Address correspondence and reprint requests to Dr. Fumio Takei, Terry Fox Lab- oratory, British Columbia Cancer Research Centre, 675 West 10th Avenue, Vancou- Anti-CD16/CD32 FcR␥ (III/II) (2.4G2; American Type Culture Collec- ver, British Columbia, Canada V5Z 1L3. E-mail address: [email protected] tion) was purified from hybridoma supernatant. PE-, FITC-, allophycocya- 3 Abbreviations used in this paper: DC, dendritic cell; KO, knockout; WT, wild type. nin-, or PerCP-Cy5.5- conjugated mAbs to NK1.1, CD11c, CD3␧, CD19, B220, CD11b, F4/80, and IFN-␥ and matching isotype controls were pur- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 chased from BD Biosciences. Mouse rIL-12 was purchased from StemCell www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901391 The Journal of Immunology 3609

Technologies. Mouse rIL-18 was purchased from Biovision. RPMI 1640 Statistical analysis medium (StemCell Technologies) supplemented with 10% FBS (Life Technologies), penicillin, streptomycin (StemCell Technologies), and 5 ϫ All statistical analyses of experimental mean values were performed using Ϫ Ͻ 10 5 M 2-ME (Sigma-Aldrich) was used for cell culture. the Student paired t test. Values of p 0.05 were considered signiﬁcant.

Preparation of splenocytes and bulk cultures Results ␮ ϩ Freshly isolated spleens were ground through a 70- m nylon sieve to pre- NK1.1 cells are the primary producers of IFN-␥ among pare a single-cell suspension and washed with PBS. RBC were lysed with ammonium chloride solution and cells were washed twice. For bulk cul- IL-12-stimulated bulk spleen cells 6 tures, cells were dispensed at a density of 1 ϫ 10 cells/ml in 96-well plates When 106 cells/ml bulk splenocytes from naive B6 mice were and cultured with IL-12 (1 ng/ml) with or without IL-18 (10 ng/ml) for stimulated with 1 ng/ml IL-12 for 24 h, ϳ1.5 ng/ml IFN-␥ was 48 h. To prepare cells for sorting, splenocytes were treated with 2.4G2 mAb to block Fc receptors, washed, and then stained with appropriate Abs detected in culture supernatants, and higher concentrations of for 30 min. To exclude dead cells, propidium iodide was added to a final IL-12 (10 ng/m) did not induce more IFN-␥ (Fig. 1A). We there- concentration of 5 ␮g/ml. fore used 1 ng/ml IL-12 for all additional experiments. To identify early responders to IL-12, bulk spleen cells were stimulated for a Intracellular cytokine staining total of 12 h, stained for surface markers, and analyzed for intra- Splenocytes were cultured at a density of 4 ϫ 106/ml in 14-ml round- cellular IFN-␥ (Fig. 1B). The IFN-␥ response was limited to the bottom polypropylene tubes and treated with IL-12 (1 ng/ml) with or with- NK1.1ϩ compartment in spleen. Approximately 10% of NK out IL-18 (10 ng/ml) for 12– 48 h at 37°C. Brefeldin A was added during ϩ Ϫ ϩ ϩ thefinal6hofculture. Cells were stained with appropriate mAbs before (NK1.1 CD3 ) and 7% of NKT (NK1.1 CD3 ) cells were pos- Ϫ ϩ Downloaded from being fixed and permeabilized with a Cytofix/Cytoperm Plus Kit (BD itive for intracellular IFN-␥, while no T cells (NK1.1 CD3 )or Ϫ Ϫ ϩ Biosciences) as per the manufacturer’s instructions. Permeabilized cells other spleen cells (NK1.1 CD3 ) were IFN-␥ (Fig. 1B). Similar were then stained with allophycocyanin-conjugated anti-IFN-␥ mAb. A results were obtained with longer periods (24, 48, and 72 h) of FACSCalibur (BD Biosciences) was used for acquisition and FlowJo IL-12 stimulation (data not shown). IL-12 and IL-18, which are software (BD Biosciences) was used for analysis. known to synergize and induce high levels of IFN-␥ secretion from Cell depletion experiments NK and T cells (24), were also used to stimulate resting spleno-

cytes. The combination of IL-12 and IL-18 induced a much higher http://www.jimmunol.org/ B6 splenocytes were stained for the depletion of various cell populations as ϩ ϩ Ϫ ϩ ϩ Ϫ Ϫ percentage of NK cells to become IFN-␥ than stimulation with follows: CD11c NK1.1 for DCs, CD11b F4/80 CD11c NK1.1 for ϩ macrophages, and CD19ϩ for B cells. A FACSVantage SE (BD Bio- IL-12 alone (Fig. 1B); the effect on NK1.1 T cells was only sciences) was used to sort the DC, macrophage, or B cell populations and marginally higher than that with IL-12 alone. The percentage of the remaining population that was considered “depleted” of the population IFN-␥-producing cells among IL-12-stimulated splenocytes was of interest. Bulk splenocytes stained with mAbs but not sorted were used consistently ϳ10%. To confirm that NK1.1ϩ cells (NK cells and as a control. For add-back experiments, cell populations of interest and ϩ ␥ depleted cells were mixed at the same ratio as that in the original bulk NK1.1 T cells) were indeed the source of the IFN- detected in ϩ spleen population. For IL-12 stimulation, 2.5 ϫ 105 cells were cultured bulk spleen cultures stimulated with IL-12, NK1.1 cells were with 1 ng/ml IL-12 in 200 ␮l of medium in 96-well round-bottom plates for depleted from bulk splenocyte populations. Depletion of all 4 days. NK1.1ϩ cells from bulk spleen cells resulted in an almost com- by guest on September 25, 2021 ␥ ϩ DC isolation and coculture plete loss of IFN- secretion (Fig. 1C). Adding NK1.1 cells back to the depleted population restored the production of IFN-␥ to a ϩ WT splenocytes were stained with PE-conjugated anti-CD11c and CD11c level similar to that from undepleted cells ( p Ͼ 0.1). These results cells (DCs) were isolated using a StemCell Technologies EasySep Murine ϫ 3 show that stimulation of bulk spleen cells with IL-12 induces PE-positive selection kit. DCs (6 10 ) were cultured alone or mixed with ϩ 3 ϫ 104 NK or NK1.1ϩT cells in 96-well round-bottom tissue culture IFN-␥ production by NK and NK1.1 T cells but not T cells or plates (BD Falcon) in a total volume of 200 ␮l. IL-12 (1 ng/ml) was added other spleen cells. NK1.1ϩ cells comprise ϳ4% of splenocytes and at the beginning of culture and supernatants were harvested after 48 h. only 7–10% of them produce IFN-␥ upon stimulation with IL-12. Thus, ϳ4 ϫ 103/ml NK1.1ϩ cells among 106/ml bulk splenocyte B and NK cell cocultures cultured with IL-12 produced almost 1500 pg/ml (ϳ0.4 pg/cell) WT and IL-18KO B6 mouse splenocytes were stained with mAbs and NK IFN-␥. cells (NK1.1ϩCD3Ϫ) or B cells (CD19ϩ) and purified by two rounds of cell sorting to a purity of 95–98%. Cells were cultured in 96-well round- bottom plates at varying ratios of B:NK cells with the NK cell number Purified NK cells are not stimulated with IL-12 alone ϫ 4 ␮ fixed at 3 10 cells in a final volume of 200 l. Cultures were stimulated ϩ with IL-12 (1 ng/ml) for 48 h. For Transwell cultures, purified B and NK Although the above results showed that NK1.1 cells were the cells were cultured at 4:1 or 10:1 ratios in a final volume of 600 ␮lin source of IL-12-induced IFN-␥, purified NK cells (3 ϫ 105/ml) 24-well plates with cell-impermeable (0.4- ␮m pore size) Transwell inserts stimulated with IL-12 produced only very small amounts (60–150 ϫ 4 (BD Falcon). NK cells (9 10 cells) in the inserts and B cells in bottom ␥ A left ϩ wells were stimulated with IL-12 (1 ng/ml) for 4 days, unless otherwise pg/ml) of IFN- (Fig. 2 , ). Purified NK1.1 T cells stimulated indicated. with IL-12 alone produced a significantly higher level (500–1000 pg/ml) of IFN-␥ (Fig. 2A). Both populations produced large In vivo IL-12 injection amounts of IFN-␥ (10–40 ng/ml) when both IL-12 and IL-18 were ␥ IL-12 (1 ␮g) in 200 ␮l of PBS containing 0.1% endotoxin-free BSA (Sigma- added (Fig. 2A, right). These results suggested that IFN- pro- Aldrich) was administered i.p. into each mouse daily for 3 consecutive duced by IL-12-stimulated bulk splenocytes might derive mostly days. Control mice received three injections of PBS-BSA. One day after from NK1.1ϩ T cells while NK cells might secrete very little the final injection, blood was collected via cardiac puncture for serum IFN-␥. Alternatively, the stimulation of NK cells with IL-12 is cytokine analysis. indirect and requires other cells. Resting NK cells do not express IFN-␥ and IL-18 ELISA the IL-12R to appreciable levels (15). Whereas NKT cells express the IL-12R constitutively (16). Therefore, IL-12 might primarily Cytokines in culture supernatants or sera were measured by ELISA for IFN-␥ (eBioscience or BD Opteia, BD Biosciences) and IL-18 (MBL) stimulate NKT cells and induce production of cytokines, which in according to the manufacturers’ protocols. Values below 15–25 pg/ml cy- turn stimulates NK cells. To test these possibilities, spleen cells tokine were not detected. from TCR␤␦ double KO mice, which lack all T cells including 3610 B CELL HELP FOR NK CELL STIMULATION Downloaded from http://www.jimmunol.org/ FIGURE 2. NK1.1ϩT cells are not required for IL-12-induced IFN-␥ production by bulk splenocytes. A, NK1.1ϩT (NK1.1ϩCD3␧ϩ), NK (NK1.1ϩCD3eϪ), and T (NK1.1ϪCD3␧ϩ) cells were purified by FACS sorting from B6 mouse splenocytes. The purified cells (3 ϫ 104 in 200 ␮l of medium) were stimulated for 48 h as indicated. IFN-␥ in the culture supernatants was analyzed by ELISA. The results are the mean Ϯ SEM of four independent experiments, each with triplicate cultures. B, Bulk splenocytes from WT and TCR␤␦ KO mice were stimulated with 1 ng/ml IL-12 for 48 h, and IFN-␥ in the culture supernatants was analyzed. The results

are the mean Ϯ SEM of ﬁve independent experiments, each with triplicate by guest on September 25, 2021 cultures. , Undetectable cytokine levels.

NK1.1ϩ T cells, were tested. Bulk spleen cells from TCR␤␦ double KO mice stimulated with IL-12 produced the same amount of IFN-␥ as WT spleen cells ( p Ͼ 0.1, n ϭ 9; Fig. 2B), indicating that NK cells can be stimulated with IL-12 to produce IFN-␥ in the absence of NKT cells.

DCs and macrophages are not required for the stimulation of NK cells by IL-12 The above results suggested that the stimulation of NK cells with IL-12 requires the presence of other cells. Because the importance of cellular interaction with DCs for the stimulation of NK cells and NKT cells has been demonstrated (25–27), we ﬁrst investigated whether DCs are required. Some NK cells express CD11c (28, 29) and CD11cϩ NK cells are preferentially

FIGURE 1. IL-12 stimulates IFN-␥ production mainly from NK1.1ϩ cells in bulk spleen. A, Bulk B6 splenocytes (1 ϫ 106/ml) were cultured with varying doses of IL-12 in a 96-well round-bottom tissue culture plate experiments and the numbers indicate the percentages of positively for 48 h. Cell-free supernatants were assessed for IFN-␥ by ELISA. Results stained cells (mean Ϯ SD, n ϭ 3). C, NK1.1ϩ and NK1.1Ϫ cells were are the mean Ϯ SEM of three independent experiments, each with triplicate FACS sorted from B6 mouse splenocytes. NK1.1Ϫ cells (2 ϫ 105) were cultures. B, Bulk B6 mouse splenocytes (4 ϫ 106/ml) were cultured with used as the NK1.1ϩ-depleted population, while the NK1.1ϩ-restored or without IL-12 (1 ng/ml) alone or with IL-18 (10 ng/ml), stained with population was 4 ϫ 103 NK1.1ϩ cells combined with 1.96 ϫ 105 mAbs to NK1.1 and CD3␧, fixed, permeabilized, stained for intracellular NK1.1Ϫ cells. Stained unsorted splenocytes (2 ϫ 105) were used as IFN-␥ and analyzed by FACS. Individual populations were gated as shown bulk. Cells were stimulated as shown and cultured for 48 h. IFN-␥ in the in the dot plot and analyzed for intracellular IFN-␥. Filled histograms show culture supernatants was analyzed by ELISA and the results are the isotype-matched control Ab staining and open histograms show IFN-␥ mean Ϯ SEM of three independent experiments, each with triplicate staining. The data are representative of at least three independent cultures. , Undetectable cytokine levels. The Journal of Immunology 3611 stimulated by IL-12 (supplemental Fig. 14), we identified DCs by CD11cϩNK1.1Ϫ and divided bulk splenocytes into DCs and DC-depleted populations. DC-depleted spleen cells cultured for 4 days in the presence of IL-12 produced a slightly lower level of IFN-␥ than nondepleted controls (Fig. 3A). Purified DCs stimulated with IL-12 did not produce a detectable level of IFN-␥ and adding back DCs to the depleted cells did not significantly increase ( p Ͼ 0.1, n ϭ 9) the level of IFN-␥ production. Moreover, coculturing highly purified NK cells and DCs in the presence of IL-12 for 2 days did not induce the production of a significant amount of IFN-␥, whereas DCs enhanced the production of IFN-␥ by NK1.1ϩ T cells by 2-fold (Fig. 3B). Thus, DCs promote NK1.1ϩ T cell stimulation, but they do not seem to play a significant role in the stimulation of NK cells with IL-12. Like DCs, macrophages constitutively express the IL-12R (30). Therefore, we tested the effects of depletion of those cells on IL-12-induced IFN-␥ production by bulk splenocytes. Because some NK cells also express CD11b and CD11c, macrophages were identified by CD11bϩF4/80ϩCD11cϪ Downloaded from NK1.1Ϫ. The depletion of macrophages had no effect ( p Ͼ 0.1, n ϭ 9) on the amount of IFN-␥ being secreted in stimulated cultures (Fig. 3C).

B cells are required and sufficient for IFN-␥ induction from http://www.jimmunol.org/ IL-12-stimulated NK cells In contrast to the results from the DC and macrophage depletions, the removal of B cells (CD19ϩ) resulted in a striking loss of the IFN-␥ production by IL-12-stimulated splenocytes, while adding B cells back to the B cell-depleted splenocytes significantly restored the production of IFN-␥ (Fig. 4A). To further test the role of B cells in IL-12-mediated stimulation, splenocytes from BKO mice were stimulated with IL-12 and the amount of IFN-␥ produced was by guest on September 25, 2021 compared with WT. As expected from the B cell depletion exper- iment, IL-12-stimulated BKO splenocytes secreted significantly less IFN-␥ than WT splenocytes (Fig. 4B, left). BKO mouse spleens had slightly higher percentages of NK and NKT cells (data not shown), and BKO splenocytes produced larger amounts ( p Ͻ FIGURE 3. DCs and macrophages are not required for IL-12-induced 0.05, n ϭ 9) of IFN-␥ when stimulated with IL-12 plus IL-18 (Fig. IFN-␥ production by bulk splenocytes. A, CD11cϩNK1.1Ϫ cells (DCs) and 4B, right). Therefore, the low level of IFN-␥ production by IL- CD11cϪNK1.1ϩ/Ϫ cells were FACS sorted from B6 mouse splenocytes. Ϫ ϩ Ϫ 12-stimulated BKO splenocytes was unlikely due to defective NK CD11c NK1.1 / cells were used as the depleted population, while the ϫ 3 ϫ 5 or NKT cells in BKO spleen. To determine whether B cells are restored population was 2.5 10 DCs combined with (2.5 10 )de- sufficient for the stimulation of NK cells with IL-12, we highly pleted cells to restore the same ratio as in the original bulk population. Stained unsorted splenocytes were used as bulk. Bulk, depleted, and re- purified (95–98%) NK and B cells from TCR␤␦ KO mice and stored cells and sorted DCs (2.5 ϫ 103) were cultured with or without 1 cocultured them with IL-12 at varying ratios of B:NK cells (2:1, ng/ml IL-12 in 200 ␮l of medium for 4 days. There is no statistical dif- 4:1, and 10:1), with the NK cell number fixed at 30,000/well (Fig. ference between the bulk and depleted (p ϭ 0.05) groups or between the 4C). The amounts of IFN-␥ production increased in a dose-depen- depleted and restored (p ϭ 0.05) groups. B, NK and NK1.1ϩT cells were dent manner as the ratios of B:NK cells increased. Purified B cells purified from B6 mouse splenocytes by FACS sorting. CD11cϩ spleen without NK cells did not produce any IFN-␥ with IL-12 stimula- cells were purified with magnetic beads as in Materials and Methods and ϩ 5 tion (data not shown). Additionally, we could detect low levels used as DCs. NK and NK1.1 T cells (3 ϫ 10 ) were cultured in the ϩ ϫ 3 (37 Ϯ 8 pg/ml, n ϭ 3) of TNF-␣, another inflammatory cytokine, presence or absence of splenic CD11c cells (6 10 ) with or without IL-12 for 48 h. C, CD11bϩF4/80ϩNK1.1ϪCD11cϪ cells (macrophages) in the supernatants of these IL-12-stimulated B-NK cocultures Ϫ Ϫ ϩ Ϫ ϩ Ϫ and CD11b F4/80 NK1.1 / CD11c / cells (depleted) were FACS (data not shown). NK cells or B cells cultured alone did not secrete sorted from B6 splenocytes. The restored population was 3.75 ϫ 103 mac- ␣ any detectable TNF- upon IL-12 stimulation. We also tested rophages combined with 2.5 ϫ 105 depleted cells. All of the results show whether B cells promote IL-12-induced NK cell cytotoxicity. Our the amounts of IFN-␥ (mean Ϯ SEM) in the culture supernatants of three data show that IL-12-stimulated NK cells are poorly cytotoxic or four independent experiments, each with triplicate cultures. , Unde- (supplemental Fig. 2). There was not a statistically significant dif- tectable cytokine levels. ference between the WT and BKO splenocytes with respect to killing ability after IL-12 stimulation ( p ϭ 0.21). B cells provide endogenous IL-18 and cell contact in NK cell stimulation by IL-12 The above results showed that purified NK cells are not stimulated 4 The online version of this article contains supplemental material. with IL-12 alone and require B cells, whereas purified NK cells 3612 B CELL HELP FOR NK CELL STIMULATION Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. B cells are required for IFN-␥ from IL-12-treated bulk splenocytes and purified NK cells. A, CD19ϩ cells and CD19Ϫ cells were FACS sorted from B6 splenocytes as B and depleted populations, respectively. For the restored population, 1 ϫ 105 B cells were mixed with 1.5 ϫ 105 depleted cells. Stained unsorted bulk splenocytes (2.5 ϫ 105), B cells (1 ϫ 105), and depleted cells (1.5 ϫ 105) were cultured with 1 ng/ml IL-12 in 200 ␮l of medium for 4 days. B, Bulk splenocytes (2 ϫ 105) from B6 and BKO mice were cultured with IL-12 with or without 10 ng/ml IL-18 for 48 h. C, B (CD19ϩ) cells and NK (NK1.1ϩCD3␧Ϫ) cells were purified FIGURE 5. B cells provide IL-18 and contact-dependent NK stimula- 6 from TCR␤␦ KO mouse splenocytes by FACS sorting, mixed at B:NK tory signals during IL-12 activation. A, Bulk splenocytes (10 /ml) from B6 ratios of 2:1, 4:1, and 10:1, respectively, and stimulated with 1 ng/ml IL-12 and IL-18KO mice were cultured as in Fig. 2B for 48 h. B, B cells and NK for 48 h. The results show the amounts of IFN-␥ (mean Ϯ SEM) in the cells were purified from WT and IL-18KO mouse splenocytes by FACS 5 4 culture supernatants of three independent experiments, each with triplicate sorting as in Fig. 4C. Purified B cells (3 ϫ 10 ) and NK cells (3 ϫ 10 ) p Ͻ 0.01. were combined and stimulated with IL-12 for 48 h. C, B cells and NK cells ,ءء p Ͻ 0.05 and ,ء .cultures. , Undetectable cytokine levels purified from B6 mouse splenocytes were cultured in Transwells as de- scribed in Materials and Methods. They were cultured with IL-12 in a total can be stimulated with a combination of IL-12 and IL-18 in the volume of 600 ␮l for 96 h. Results show the amounts of IFN-␥ (mean Ϯ absence of B cells. Human tonsillar B cells constitutively express SEM) in the culture supernatants from three independent experiments, each .p Ͻ 0.05 ,ء .IL-18 mRNA (31) and mouse B cells secrete IL-18 upon Ly49D with triplicate cultures. , Undetectable levels of cytokine cross-linking (32). Moreover, a recent study has shown that the in vivo priming of NK cells with IL-18 is important for the stimulation of NK cells with IL-12 (23). To clarify the role of IL-18 and effect of IL-18 deficiency on NK cell stimulation, NK cells and B B cells, we first tested bulk splenocytes from IL-18KO mice. IL- cells were purified from WT and IL-18KO splenocytes and cocul- 18KO splenocytes stimulated with IL-12 produced significantly tured in various combinations in the presence of IL-12. WT B cells lower levels of IFN-␥ than WT splenocytes (Fig. 5A, left). This promoted both WT and IL-18KO NK cell stimulation with IL-12, reduced response to IL-12 was not because of a functional defi- whereas IL-18KO B cells did not have much effect on either WT ciency in the IL-18KO splenocytes since they produced high levels or IL-18KO NK cells (Fig. 5B). of IFN-␥ when stimulated with a combination of IL-12 and IL-18 To determine whether cell contact between B cells and NK cells (Fig. 5A, right). To determine whether B cells play a role in the is also required for the stimulation of purified NK cells with IL-12, The Journal of Immunology 3613

in the sera of vehicle-injected WT and BKO mice. Whereas the levels of serum IL-18 increased signiﬁcantly ( p Ͻ 0.05) following IL-12 injection in both WT and IL-18KO mice, they were significantly lower ( p Ͻ 0.01, n ϭ 4) in BKO mice than in WT mice (Fig. 6B).

Discussion The data presented above have revealed a novel role for B cells in the stimulation of mouse NK cells with IL-12 in vitro and in vivo. IL-12 is considered to be a potent stimulator of NK cells and stimulation of bulk spleen cells with a suboptimal dose of IL-12 alone induces IFN-␥ production by NK cells and NK1.1ϩ T cells. However, purified NK cells are not stimulated by IL-12 alone, whereas in the presence of B cells purified NK cells are stimulated by IL-12 and secrete significant amounts of IFN-␥. B cell depletion from normal spleen inhibits IL-12-induced IFN-␥ production, and spleen cells from B cell-deficient mice produce much lower levels of IFN-␥ than WT spleen cells. Moreover, purified B cells, but not

DCs, support IL-12-induced IFN-␥ production by purified NK Downloaded from cells. Therefore, B cells are necessary and also sufficient for NK cell stimulation with IL-12. Importantly, the critical role for B cells in IL-12-induced IFN-␥ production is not restricted to in vitro cultures. Injection of IL-12 into B cell-deficient mice results in significantly lower levels of IFN-␥ than those in WT mice. It has

been proposed that this difference might be due to IFN-␥ produc- http://www.jimmunol.org/ tion by B cells (33). However, our current study has shown that B cells do not secrete detectable levels of IFN-␥; it should also be noted that B cell-deficient spleen cells stimulated by IL-12 do produce some IFN-␥, albeit significantly less than WT spleen cells do. FIGURE 6. B cells are required for optimal IFN-␥ and IL-18 production This is most probably due to a B cell-independent stimulation of in vivo induced by IL-12 injection. B6 and BKO mice were i.p. injected NK1.1ϩ T cells, as purified NK1.1ϩ T cells are stimulated by with 1000 ng of IL-12 or PBS as vehicle for 3 consecutive days. Blood was IL-12 alone and produce IFN-␥. Similarly, the reduced IFN-␥ pro- ␥ collected 24 h after the last injection and serum levels of IFN- (A) and duction in B cell-deficient mice injected with IL-12 may also be

Ϯ by guest on September 25, 2021 IL-18 (B) were analyzed by ELISA. The results are mean SD of four due to NK1.1ϩ T cell stimulation. Nevertheless, spleen cells from ϩ ,ءء ;p Ͻ 0.05 ,ء .mice in each group. , Undetectable levels of cytokine TCR␤␦ double KO mice, which lack all T cells including NK1.1 .p Ͻ 0.005 ,ءءء p Ͻ 0.01; and T cell, produce no less IFN-␥ than WT spleen cells, indicating that NK cells are an important primary source of IFN-␥ upon IL-12 stimulation. they were cocultured in Transwells. When NK cells and B cells The precise mechanisms by which B cells help NK cell stimu- were separated by a cell-impermeable membrane, only a small lation with IL-12 are still unclear. As reported by Chaix et al. (23), amount of IFN-␥ was produced upon IL-12 stimulation, regardless spleen cells from IL-18-deficient mice stimulated by IL-12 pro- of the B:NK ratios. Coculturing NK and B cells in the same com- duce significantly less IFN-␥ than WT spleen cells. This has been partment, which facilitates cell-cell contact, resulted in the B cell suggested to be due to an in vivo priming of NK cells by IL-18. In dose-dependent production of significantly higher amounts ( p Ͻ our current study, a very small amount of IFN-␥ is produced by 0.005, n ϭ 9) of IFN-␥ (Fig. 5C). Therefore, B cell-NK cell phys- purified WT NK cells stimulated by IL-12 alone. Furthermore, NK ical contact appears important for the activation of NK cells by cells from IL-18KO mice can be stimulated by IL-12 in the pres- IL-12. ence of WT B cells, whereas WT NK cells are not stimulated by IL-12 in the presence of IL-18KO B cells. These results suggest B cell-deficient mice injected with IL-12 produce lower levels of that the production of IL-18 by B cells, rather than the in vivo ␥ IFN- and IL-18 than WT priming of NK cells by IL-18, is critical. However, we have been The above results showed that B cells are necessary and also suf- unable to detect IL-18 in the cultures of purified NK cells and B ficient for the in vitro production of IFN-␥ from NK cells induced cells stimulated with IL-12. It should be noted that purified NK with IL-12. To test whether B cells also play a role in an in vivo cells treated with IL-12 require as little as 10 pg/ml recombinant response to IL-12, we i.p. injected IL-12 into normal B6 and BKO mouse IL-18 to produce the level (ϳ1500 pg/ml) of IFN-␥ com- mice and compared the levels of serum IFN-␥. Our preliminary monly seen in our cultures (data not shown). It is then possible that studies showed that a daily injection of 1 ␮g/mouse for 3 consec- the level of IL-18 secreted by B cells in our cultures while suffi- utive days induced a significant level of IFN-␥ in the serum of WT cient for synergizing with IL-12 for IFN-␥ is too low to be detected mice. Under these conditions, ϳ1200 pg/ml IFN-␥ was detected in by ELISA. At the same time, our results with Transwell cultures the sera of WT mice, whereas a 4-fold lower level of IFN-␥ was indicate that physical contact between the B cells and NK cells is detected in the sera of BKO mice (Fig. 6A). No IFN-␥ was de- required. Therefore, IL-18 secretion may not be the only mecha- tected in the sera from vehicle (PBS plus 0.1% BSA)-injected WT nism by which B cells support IL-12-mediated NK cell stimula- or BKO mice. Since our in vitro results also suggested that en- tion. It is possible that for IL-18 production IL-12-stimulated B dogenous IL-18 is important for IL-12-induced IFN-␥ production, cells require contact-dependent costimulatory signals from the NK we also measured serum IL-18. Unlike IFN-␥, IL-18 was detected cells to produce IL-18. To examine this possibility, we have tested 3614 B CELL HELP FOR NK CELL STIMULATION spleen cells from CD40 KO and CD80/86 double KO mice. These Disclosures cultures produced only slightly lower levels of IFN-␥ than WT The authors have no financial conflict of interest. cells upon IL-12 stimulation (data not shown). An alternative possibility is that B cells may not only produce IL-18 but also present References it to NK cells, perhaps via the IL-18R. The IL-18R consists of two 1. Trinchieri, G. 2003. Interleukin-12 and the regulation of innate resistance and chains, the ␣-chain that binds IL-18 and the ␤-chain that does not adaptive immunity. Nat. Rev. Immunol. 3: 133–146. 2. Mansouri, D., P. Adimi, M. Mirsaeidi, N. Mansouri, S. Khalilzadeh, efficiently bind IL-18 but associates with the complex of IL-18/ M. R. Masjedi, P. Adimi, P. Tabarsi, M. Naderi, O. Filipe-Santos, et al. 2005. IL-18R␣ and transduces signals (34). To test whether B cell-de- Inherited disorders of the IL-12-IFN-␥ axis in patients with disseminated BCG ␣ infection. Eur. J. 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