Induction of IL-13 Expression in NK Cells: Role of CD244 and SLAM-Associated

This information is current as Ning Gao, Pamela Schwartzberg, Julie A. Wilder, Bruce R. of September 26, 2021. Blazar and Dorothy Yuan J Immunol 2006; 176:2758-2764; ; doi: 10.4049/jimmunol.176.5.2758 http://www.jimmunol.org/content/176/5/2758 Downloaded from

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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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

B Cell Induction of IL-13 Expression in NK Cells: Role of CD244 and SLAM-Associated Protein

Ning Gao,* Pamela Schwartzberg,† Julie A. Wilder,‡ Bruce R. Blazar,§ and Dorothy Yuan1*

NK cells are an important component of the innate immune system that can also interact with B cells in a mutually productive manner. We have previously shown that activated B cells can induce NK cells to up-regulate their secretion of IFN-␥. In this study, we show that B cells, and, particularly, marginal zone B cells, can, in addition, induce NK cells via direct cell-cell interactions to express mRNA encoding the Th2 cytokine IL-13. The induction of NK cell IL-13 mRNA expression requires the ligation of the CD244 receptor by the CD48 ligand on B cells via signaling pathways that depend upon expression of the X-linked lymphopro- liferative disease product, SH2D1A/DSHP/SAP (SLAM-associated protein, or SAP) in NK cells. Thus, the positive signals attributed to the B cell activation of CD244 on murine NK cells appears to be more similar to the activity of CD244 on human

cells. The induction of IL-13 mRNA by B cells may account for the effect of NK cells on the generation of Th2-type responses in Downloaded from the presence of some adjuvants. The Journal of Immunology, 2006, 176: 2758–2764.

he presence of clonally distributed receptors on B lym- induce NK cells to express IL-13 as well and might therefore ex- phocytes confer on them two important functions. They plain why NK cells can sometimes exert Th2-like effects. T can process and present Ags to T cells and, in response to Parameters of induction of IL-13 mRNA expression by NK cells appropriate stimulation, they can be induced to secrete Abs that are can be measured quite precisely because even after in vitro IL-2 http://www.jimmunol.org/ important in the control of pathological insult. In addition to these propagation NK cells do not produce measurable amounts of IL-13 Ag-specific functions, we showed previously that in vivo-preacti- mRNA. We found that, whereas highly purified resting B cells can vated B cells can up-regulate IFN-␥ production by NK cells (1) via induce NK cells to synthesize IL-13 mRNA, marginal zone (MZ) direct cell-cell interactions. Whereas it is clear that secretion of B cells are much more effective than follicular B cells. By the use IFN-␥ by NK cells plays an important role in Th1 responses, we of Abs specific for possible ligand/receptors as well as mice with have found that under some conditions of immunization, NK cells targeted disruptions of encoding cell surface molecules, we can also increase the IgG1 response (2) that is usually associated found that activation of NK cells by B cells requires the interaction with Th2 cytokines. Although murine NK cells have not been of CD48 and its counterreceptor, CD244 (2B4), expressed on NK shown to secrete IL-4 (3, 4), a closely related Th2 cytokine, IL-13, cells. CD244 is a member of the Ig superfamily (14), which in- by guest on September 26, 2021 can be produced by NK cells (5, 6). IL-13 shares many properties cludes other membrane-associated , such as CD150 (sig- with IL-4, but also has many distinct functions. For example, IL-13 naling activation molecule (SLAM)2), CD2, and is more important than IL-4 in the development of airway hyper- CD48. These proteins are expressed to varying degrees in subsets reactivity and mucus secretion as well as control of some parasites of immune cells and may function as ligands or receptors. Murine (7–9). The production of a Th2 cytokine by NK cells provides CD244 has been shown to mediate both activating and inhibitory another source of cytokines for the alternative pathway of macro- signals. The results reported herein show, for the first time, that phage activation (10). The induction of production of IL-13 ligation of the CD244 receptor on NK cells by CD48 expressed on may be mediated by other cytokines as well as cell surface ligands B cells results only in activation. As for human NK cells (15, 16) such as CD30L (11). NK cells can be also induced to produce this activation pathway can be shown to be mediated via the IL-13 by cytokines such as IL-2 and IL-18 (5, 12, 13), but whether SLAM-associated protein (SAP). The induction by B cells of the they can be stimulated by surface ligands expressed by other cell production of Th2 as well as Th1 cytokines by NK cells provides types is not known. Since we have shown that B cells can up- further evidence for a B cell regulatory role for NK cells in vivo. regulate NK cell IFN-␥ production, we tested whether they can Materials and Methods Cell preparations and culture *Department of Molecular Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390; †National Research Institute, National For B cell preparations, T were depleted from splenocytes of Institutes of Health, Bethesda, MD 20892; ‡Lovelace Respiratory Research Institute, Ͻ ␥Ϫ/Ϫ § BALB/c-Ifg tm1 (IFN- ) (17), or C57BL/6 (both from The Jackson Albuquerque, NM 87108; and Department of Pediatrics, Division of Blood and Mar- Laboratory), or D0.11 mice (Ref. 18; provided by Dr. M. Siegelman, Uni- row Transplantation, and Cancer Center, University of Minnesota Hospital and Can- cer Center, Minneapolis, MN 55455 versity of Texas Southwestern Medical Center, Dallas, TX) mice, and frac- tionated by Percoll gradient centrifugation as previously described (19). Received for publication July 28, 2005. Accepted for publication December 28, 2005. The high-density fraction (20) was further purified by binding to CD43 The costs of publication of this article were defrayed in part by the payment of page MicroBeads (Miltenyi Biotec) to deplete remaining non-B cells. B cells charges. This article must therefore be hereby marked advertisement in accordance were found routinely to be Ͼ95% positive for the CD19 marker. For in with 18 U.S.C. Section 1734 solely to indicate this fact. vivo modulation of CD48, IFN-␥Ϫ/Ϫ mice were injected two times with 1 Address correspondence and reprint requests to Dr. Dorothy Yuan, Department of 300 ␮g/animal of an ammonium sulfate cut of anti-CD48 (HM48-1) 2 days Molecular Pathology, University of Texas Southwestern Medical Center, Dallas, TX apart (21). B cells were isolated 1 day after the last injection. NK cells were 75390. E-mail address: [email protected] purified from BALB/c IFN-␥Ϫ/Ϫ, C57BL/6, or D0.11 mice or from Ϫ/Ϫ Ϫ/Ϫ 2 Abbreviations used in this paper: SLAM, signaling lymphocyte activation molecule; CD2 (22), CD244 (23), and C.B-17 SCID mice (all bred and pro- SAP, SLAM-associated protein; QM, quasi-monoclonal; MZ, marginal zone; SH2, vided by Dr. M. Bennett, University of Texas Southwestern Medical Cen- Ϫ Ϫ Src homology 2. ter). The generation of SAP / mice has been described (24). Spleen cells

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 2759 from the mice were first passed over a nylon wool column to remove FACS analysis and cell sorting adherent cells, then depleted of T cells by complement-mediated lysis. NK cells were then isolated by positive selection using anti-DX5 Abs and mag- B cells isolated from QM mice were labeled with fluorescein-conju- netic beads (Miltenyi Biotec). Purified cells were cultured in 1000 U/ml gated -anti-IgM, PE-conjugated CD23, and biotinylated CD21 followed by IL-2 as described previously (25). NK cells from D0.11 mice did not ex- allophycocyanin-conjugated streptavidin. The cells were sorted on a FACS hibit properties different from BALB/c mice. When necessary, residual T DIVA (BD Biosciences). For FACS analysis, the FACScan flow cytometer cells if detected by FACS in the propagated NK cells were depleted with (BD Biosciences) was used. biotinylated anti-CD3⑀ and streptavidin-conjugated magnetic beads (Milte- nyi Biotec). Quasi-monoclonal (QM) mice originally generated by Cas- Results calco et al. (26) were provided by Dr. R. Noelle (Dartmouth Medical Cen- Parameters of B cell induction of IL-13 mRNA synthesis by NK ter, Hanover, NH). Each cell type was either cultured alone or together cells (1 ϫ 106/ml total cell concentration) in the presence of 100 U/ml IL-2 with or without other additives in 24- or 48-well Falcon tissue culture plates We found that IL-13 mRNA is synthesized at levels below the (BD Biosciences) or in tissue culture plates containing Transwell inserts. sensitivity of detection by RT-PCR in numerous preparations of For some experiments, B cells were irradiated at 2 ϫ 106/ml at 1200 cGy 137Cs gamma-irradiation (Gamma Cell 40; Atomic Energy). propagated NK cells from different strains. Upon coculture with resting B cells, however, significant amounts of IL-13 mRNA can Abs and reagents be detected. In the experiments depicted in Fig. 1, highly purified, Hamster anti-CD40L (CD154; Ref. 27) Abs were provided by Dr. R. No- resting B cells were cultured with IL-2 propagated NK cells for 2 elle (Dartmouth Medical School). Ammonium sulfate cut preparations of days at varying ratios of B:NK cells. Subsequently, the cocultured hamster anti-CD48 (HM48-1) were prepared as described previously (21). cells were assessed by RT-PCR analysis. The use of labeled prim- ␥ ␥ Rat anti-CD28 (28), anti-Fc R, (2.4G2 (29)), anti-IFN- , R4-6A2, (30), ers allowed us to determine, semiquantitatively, the level of IL-13 Downloaded from anti-IgM, and anti-CD48 (1G10) Abs (originally obtained from Dr. J. mRNA induced in the cultures. To adjust for possible differences Bromberg, Mount Sinai School of Medicine, New York, NY), hamster anti-TCR␣␤ (31), mouse anti-NK1.1 (PK136; Ref. 32), and anti-2B4 in cell as well as RNA recovery from the cocultures, relative ex- (CD244; Ref. 33) were purified from hybridoma culture supernatants using pression of IL-13 mRNA was determined as a function of the level Gamma Bind (Pharmacia Fine Chemicals). Conjugated hamster anti-CD48 of Ly49 mRNA in each culture for all experiments. Note that al- (HM48-1), mouse anti-DX5, rat anti-CD3⑀, allotype-specific mouse anti-␮, though Ly49 can be expressed by B1 B cells (35) we have never and rat anti-CD19 were purchased from BD Biosciences. Conjugated rat anti-CD23 and anti-CD21 were provided by Dr. T. Waldschmidt (Univer- http://www.jimmunol.org/ sity of Iowa, Iowa City, IA). rIFN-␥ was purchased from BioSource In- ternational. rIL-12 was a generous gift from Dr. N. Street, (University of Texas Southwestern Medical Center). rIL-18 was purchased from R&D Systems.

Semiquantitative RT-PCR analysis RNA was prepared using the TRIzol reagent (Invitrogen Life Technolo- gies), and RT-PCR was performed as previously described (34). Primers for assessment of ␮M mRNA abundance have been described elsewhere (25). Sequences of primers for IL-13 mRNA were: forward, 5Ј-AGTTC by guest on September 26, 2021 TACAGCTCCCTGGTTCTC; reverse, 5Ј-GGATGGTCTCTCCTCATT AGAAGG, which yields a 452-bp product. Sequences of primers for IFN-␥ mRNA were: forward, 5Ј-GTGGCATAGATGTGGAAGA; reverse, 5Ј- AGCTGGTGGACCACTCGGAT, which yields a 303-bp product. Ly49 primers were consensus sequences (forward, 5Ј-TCCCAAGATGAGT GAGC; reverse, 5ЈTTAGATGGGCCATTGTCAATC) which yields a 650- to 657-bp product from most Ly49 family members including both BALB/c and C57BL/6 strains. All primers were ascertained to span intronic regions. Amplified products for each set of primers were authen- ticated by size and restriction enzyme analysis. To quantify RT-PCR products, at least one of each primer pair was 3Ј end-labeled with [␥-32P]ATP and used to spike reaction mixtures. Amplified products were quantified using the ImageQuant software package (Molecular Dynamics). For all primer pairs, titration curves were performed to ascertain that the cycle number used fell within the linear range as cDNA concentrations were increased.

ELISA analysis of IL-13 protein expression ELISA plates (Greiner BioOne) were coated with anti-IL-13 (mAb 413, 2 ␮g/ml; R&D Systems) in 0.1 ml of PBS and incubated overnight at room temperature. Plates were then washed with PBS containing 0.1% Tween 20 (pH 7.4) and blocked with 1% BSA/5% sucrose in PBS for2hatroom temperature. After thorough washing, samples and standards (IL-13, 413 ML; R&D Systems) were added and the plates were incubated overnight at FIGURE 1. Parameters of B cell induction of NK cell IL-13 mRNA 4°C. Detection proceeded the following day by adding biotinylated anti- expression. 5 ϫ 105 IL-2 propagated NK cells from D0.11 TCR-transgenic, IL-13 (BAF413, 0.2 mg/ml; R&D Systems) in 0.1% BSA/PBS and incu- IFN-␥Ϫ/Ϫ, or SCID mice were incubated with syngeneic, resting B lym- bating for2hatroom temperature. Following thorough washing, strepta- phocytes at the ratios indicated for 48 h in medium containing 100 U/ml vidin-HRP was added (0.625 mg/ml diluted in 0.1% BSA/PBS) and plates IL-2. RNA extracted from the cocultured cells was subjected to RT-PCR were incubated for 30 min longer at room temperature. After washing, analyses using ␥-32P-labeled primers. A, ImageQuant visualization of RT- tetramethylbenzidine substrate (Sigma-Aldrich) was added to the plates PCR products obtained from representative cocultures. B, Semiquantitative until the maximum standard on the plate developed to an OD450 of at least 1.0. Plates were read on Bio-Rad ELISA Microplate Reader model 550 determination of the relative intensity of IL-13 mRNA normalized to the intensity of Ly49 mRNA amplified from each of the cocultures for one of OD450 after2NH2SO4 was added to the wells to stop the reaction. IL-13 levels in the samples were quantified by comparison to the standard curve at least two replicate experiments. C, An aliquot of B cells was first irra- generated using rIL-13 (DeltaSoft). Detection limits were routinely be- diated before they were cocultured with NK cells from D0.11 mice for 48 h tween 0.016 and 0.062 ng/ml IL-13. and subjected to RT-PCR analysis. 2760 B CELL INDUCTION OF NK CELL DIFFERENTIATION detected the mRNA in our B cell preparations (see Fig. 1C). Fig. 1B shows that the number of B cells required for induction is relatively high. Reproducible induction was only observed with greater than 1:1 ratios of B:NK cells. FACS analysis confirmed that the NK cell cultures used in these experiments do not contain detectable numbers of T or NKT cells, whereas Ͼ97% of the cells express DX5 (data not shown). To further ascertain that the in- duction does not require NKT cells, we propagated NK cells from SCID mice and found that they can be also induced to produce IL-13 mRNA (Fig. 1). Similar results were obtained from NK cells propagated from IFN-␥Ϫ/Ϫ NK cells (Fig. 1B). Therefore, the in- ␥ ␥ duction does not require IFN- . Moreover, the absence of IFN- in FIGURE 2. Surface determinants required for induction of IL-13 the cultures indicates that the induction is unlikely to be mediated mRNA. A, IL-2-propagated NK cells from IFN-␥Ϫ/Ϫ mice were cultured by small numbers of contaminating adherent cells that can be stim- with resting B cells at the ratios indicated. Alternatively, NK cells were ulated by NK-derived IFN-␥ to produce cytokines that can stim- placed in the bottom chamber of a Transwell culture chamber and B cells ulate IL-13 mRNA synthesis. were placed in the upper chamber at B:NK ratio of 2:1. After 48 h cells The expression of IL-13 mRNA is reflected in the amount of were harvested from the bottom chamber and analyzed by RT-PCR. B, NK IL-13 protein secreted into the medium which is quite low (range, and B cells were cultured for 48 h with or without the Abs as indicated. The

60–200 pg/ml) and rather variable. However, this range does not relative IL-13 mRNA level in each culture was determined by RT-PCR Downloaded from differ from those previously reported for IL-13 secreted by NK analysis and normalized to the level of Ly49 mRNA. The percent change resulting from the addition of Abs was calculated by dividing the relative cells (12, 36). As a result of the low levels of protein expression, IL-13 mRNA levels in cocultures containing additives by the relative levels we monitored the expression of IL-13 by mRNA expression in all obtained from control cultures with no additives. Expt. 1 and Expt. 2 in- subsequent experiments. dicate the results of two independent experiments. In Expt. 2 some of the Since B cells have not been reported to produce IL-13 mRNA, conditions were not done. it is unlikely that the IL-13 mRNA is derived from B cells in the http://www.jimmunol.org/ cocultures. Furthermore, we have not been able to induce IL-13 mRNA expression in B cells stimulated with various B cell mito- gens including LPS, anti-IgM, or anti-CD40. However, to confirm Since the CD48 receptor is expressed by both B and NK cells, that B cells are not responsible for the IL-13 mRNA expression in it is not clear which cell type is affected. To test whether expres- cocultures, we irradiated them before culturing the cells with NK sion of CD48 on B cells is necessary for the induction, we mod- cells. The dramatic decrease in the expression of mRNA for mem- ulated the CD48 receptor in vivo by the injection of anti-CD48 brane IgM (micromolar) confirmed that the transcriptional activity mAbs (21). We have shown in previous experiments that virtually of the B cells was severely compromised by the irradiation. De-

all CD48 can be removed from B cells by this treatment without by guest on September 26, 2021 spite this disruption, the IL-13 mRNA can be induced in the co- detectable effects on other B cell surface determinants (38). As cultures of these cells with NK cells (Fig. 1C), indicating that expression of this mRNA is not dependent on the ability of B cells shown in Fig. 3A, incubation of modulated B cells with IL-2-prop- to synthesize mRNA. agated NK cells reduced the induction of IL-13 to a significant extent. Therefore, the expression of CD48 on B cells is critical. Surface receptors involved in the induction of IL-13 mRNA Some residual CD48 remaining after in vivo modulation could be production responsible for the low level of induction obtained from the CD48- Since irradiated B cells are effective inducers of IL-13 mRNA modulated cells when the cells were cocultured at higher B:NK synthesis by NK cells, the induction is unlikely to be mediated by ratios. However, it is also possible that less effective ligands other cytokines produced by B cells. To determine whether cell contact than CD48 is responsible for the induction. is necessary for the induction, B cells were separated from NK Two different receptors, CD2 and CD244, that can recognize cells by a semipermeable membrane. Fig. 2A shows that upon CD48 are expressed on NK cells. To determine whether either of separation the induction of IL-13 mRNA was completely abro- these is required for the induction of IL-13 mRNA, we prepared gated. Therefore, contact between the two cell types is a necessary IL-2-propagated NK cells from mice with targeted disruptions of condition for induction but we cannot eliminate the possibility that either the CD2 or the CD244 gene. Fig. 3B shows that NK cells the induction requires, in addition, another cytokine(s) produced that do not express CD2 were not compromised in their ability to by NK cells. be stimulated by B cells. In contrast, absence of CD244 on NK As an initial attempt to identify the surface ligands involved in cells severely diminished their response. The inability of the interaction between NK and B cells, we tested the ability of CD244Ϫ/Ϫ NK cells to be stimulated by B cells is not due to an various Abs to inhibit the induction. RT-PCR analyses (Fig. 2B) intrinsic defect in their ability to express IL-13 mRNA because show that the induction of IL-13 mRNA was not affected by the ample message was induced when the cells were stimulated by inclusion of two hamster Abs against CD28 and CD40L. These immobilized anti-NK1.1 or anti-Fc␥R Abs (Fig. 3C). As expected receptors, which have counterreceptors on B cells, were previously NK cells from CD244Ϫ/Ϫ mice did not respond to anti-CD244. shown to be expressed on NK cells (28, 37). Neither was any effect found for anti-TCR ␣␤ mAb included as a control for nonspecific Surprisingly, however, in three of three experiments, they also did effects since these propagated NK cells contain few T cells or for not express IL-13 mRNA in response to anti-CD28 but can, how- ␥ anti-IgM or anti-Fc␥R mAbs. However, induction was inhibited by ever, express IFN- mRNA (Fig. 3D). In contrast, in the absence as much as 70% by two different anti-CD48 mAbs. This inhibition of CD2, the response to CD28, while variable, was not compro- is independent of possible effects of ligation of the Fc␥RIII recep- mised. Thus, despite the ability of both CD2 and CD244 to bind to tor on NK cells, since the addition of anti-Fc␥R Abs along with the CD48, in terms of IL-13 mRNA induction, it appears that CD244 anti-CD48 Abs had no effect. on NK cells is the major receptor. The Journal of Immunology 2761

FIGURE 4. Induction of NK-cell IL-13 mRNA by a subpopulation of B cells. A, Spleen cells from QM mice were depleted of T cells as described in Materials and Methods and stained with fluoroscein-conjugated anti-␮a, Downloaded from PE-CD23, and biotinylated CD21 followed by allophycocyanin-streptavi- din. The IgMϩ cells were gated and sorted based on CD23 and CD21 staining. The CD23high, CD21low, and follicular B cells (FO) were sorted from the CD23low, CD21high MZ cells by FACS. Each of the sorted pop- ulations was cultured with IL-2-propagated NK cells from D011 mice at the indicated ratios for 48 h and subsequently evaluated by RT-PCR anal- http://www.jimmunol.org/ ysis (B).

FIGURE 3. Interaction between CD48 on B cells and CD244 on NK cells is required for the induction of IL-13 mRNA expression. A, IFN-␥Ϫ/Ϫ mice were treated with two consecutive injections (300 ␮g/animal) of ei- to induce IL-13 (see Fig. 2). In contrast to the induction of IL-13 ther rat anti-CD48 on days Ϫ4 and Ϫ2 or with control rat Ig before the mRNA expression, we failed to note any difference in the ability of splenocytes were isolated from each animal on day 0. Resting B cells the two B cell subpopulations to up-regulate IFN-␥ mRNA ex- prepared from the mice were cultured with NK cells propagated from IFN- pression (data not shown). ␥Ϫ/Ϫ mice as described in Fig. 1. Results from the average of two exper- by guest on September 26, 2021 iments are shown in combination with the SEM. B, IL-2-propagated NK Induction of NK-cell IL-13 mRNA expression requires SAP cells from C57BL/6, CD2Ϫ/Ϫ, or CD244Ϫ/Ϫ mice were cultured with rest- ing B cells from IFN-␥Ϫ/Ϫ mice for 48 h. C, The same NK cells were The XLP gene product, SH2D1A/DSHP/SAP (SAP), is a 128- stimulated by culturing in wells precoated with purified Abs, as indicated residue protein that consists almost entirely of a single Src homol- for 48 h. RNA extracted from each culture was then analyzed by semi- ogy 2 (SH2) protein interaction domain. Because the SH2 domain quantitative RT-PCR to determine the relative level of IL-13 mRNA as a of SAP binds a conserved tyrosine-containing motif found in the function of Ly49 mRNA in the same cultures. Where indicated, error bars intracellular domains of CD2 family members, including CD244, indicate the SEM of three independent experiments. resulting in the recruitment of tyrosine kinases, it is possible that the CD244-CD48 interaction with B cells is mediated via SAP. In two independent experiments, we found that NK cells propagated MZ B cells preferentially induce NK-cell IL-13 mRNA from SAP-deficient mice (24) were severely compromised in their production response to B cells (Fig. 5A). The cells were not impaired in their The relatively large number of B cells required for the induction of ability to express IL-13 or IFN-␥ in response to other stimuli pro- IL-13 mRNA production by NK cells suggests that only a sub- vided in the form of immobilized Abs (Fig. 5B). The absence of a population of B cells are efficient inducers. Since activated NK response to immobilized anti-CD244 confirms that induction of cells have been shown to be recruited to the MZ of splenic follicles IL-13 mRNA expression via this receptor requires SAP but induc- (39), we tested whether MZ B cells preferentially induce NK cells. tion of IFN-␥ mRNA appears to be also compromised. The de- MZ B cells constitute Ͻ10% of the splenic B cells in most species creased response is not due to differential expression of CD244 on Ϫ Ϫ Ϫ Ϫ of mice. However, they are enriched in some transgenic mice (40). SAP / NK cells (41). In contrast to CD244 / NK cells, they We used splenocytes from the QM mouse (26) which contains an can also respond to anti-CD28. This response is unlikely to be unusually high proportion of MZ B cells. These cells were sepa- mediated via activation of the Fc␥RIIIR because it was not affected rated from follicular B cells by FACS, based on high CD21 and by the addition of soluble anti-Fc␥R which should block this low CD23 expression (Fig. 4B). The isolated cells were cultured interaction. with IL-2-propagated NK cells at two different B:NK ratios. ␥ Clearly, the MZ B cells were much more effective inducers al- Expression of NK-cell IL-13 and IFN- expression in response though the level of expression of CD48 on these cells does not to cytokines differ from that of follicular B cells (data not shown). MZ B cells To further ascertain that the inability of CD244Ϫ/Ϫ or SAPϪ/Ϫ do express, however, higher levels of ␮M RNA corresponding to mice to respond to B cells was not attributed to defective cytokine the higher expression of surface IgM in this population. However, responses, NK cells from the various strains used in this study this increased expression is not likely to be the critical factor be- were stimulated with IL-12, IL-18, or a combination of the two cause inhibition with anti-IgM did not reduce the ability of B cells cytokines. Fig. 6 shows that NK cells from all of the strains could 2762 B CELL INDUCTION OF NK CELL DIFFERENTIATION

FIGURE 6. Defective expression of CD244 or SAP does not affect the response of the NK cells to stimulation by IL-12 and IL-18. IL-2-propa- gated NK cells from each of the strains as indicated were cultured with either IL-12 (10 ng/ml) or IL-18 (10 ng/ml) alone or with a combination of the two cytokines for 48 h. The relative level of IL-13 or IFN-␥ mRNA as a function of Ly49 mRNA in each culture was determined by RT-PCR analysis and quantified by ImageQuant analysis.

absence of expression in similar cultures (13). One possible dif-

ference is that Hoshino et al. (5) isolated NK cells after repeated Downloaded from injection of animals with IL-2 in vivo. This type of priming may

Ϫ/Ϫ result in NK cells that differ from those that have been propagated FIGURE 5. SAP NK cells are compromised in their response to B in vitro. Similarly, although IL-13 mRNA was detected in 24-h cells. A, NK cells propagated from either B6.SAPϪ/Ϫ or intact control mice cultures of purified NK cells after addition of only IL-2, these cells were cultured with syngeneic B cells (representative of two experiments) in the ratios as indicated. B, NK cells from B6.SAPϪ/Ϫ mice were cultured were previously cultured with IL-12 and IL-18 for 3 days before with various immobilized Abs as indicated for 48 h. Error bars when shown sorting (12) and may therefore be more activated than those cul- http://www.jimmunol.org/ represents the SD for three experiments. RNA extracted from each culture tured with only IL-2. was analyzed by RT-PCR analysis and quantified by ImageQuant analysis. We have shown that the stimulation of NK cell IL-13 mRNA expression by B cells requires the expression of CD244 on NK cells because NK cells from mice deficient in expression of this express both IL-13 as well as IFN-␥ mRNA in response to a com- receptor cannot synthesize IL-13 mRNA in response to B cells. A bination of IL-12 and IL-18. Therefore, stimulation by B lympho- number of studies have shown that human CD244 is an activating cytes is unlikely to require cytokines. These results also confirm receptor in that stimulation by its ligand, CD48, results in activa- previous reports that IL-12 is a much better stimulator of IFN-␥ tion of cytotoxicity as well as IFN-␥ secretion (16, 46). Whether than IL-13 mRNA expression and that the combination of IL-12 CD244 exerts inhibitory or activating signals in murine NK cells by guest on September 26, 2021 and IL-18 may exert more of a synergistic effect on IL-13 than for is, however, not clear. Earlier data indicated an activating role (14) IFN-␥ mRNA expression. but more recent studies have indicated inhibitory (23) as well as activating functions (47–49). Insofar as induction of cytokine ex- Discussion pression, we have not detected any inhibitory activity of CD244. NK cells have been usually associated with Th1 responses because We have not observed, for example, increased expression of IFN-␥ of their ability to secrete IFN-␥. Although human NK cells can mRNA in NK cells from CD244Ϫ/Ϫ mice in response to activation secrete IL-5 (42, 43), a Th2 cytokine, there is conflicting data by B cells (data not shown). Expression of CD48, the ligand for regarding whether murine NK cells can synthesize this cytokine (5, CD244, on tumor cells has been shown to down-regulate the pro- 12, 44). In contrast, both human and murine NK cells can produce duction of this cytokine (23), suggesting that CD244 can impart an IL-13 (5, 13). IL-13 was initially described as a protein preferen- inhibitory signal for cytokine secretion. However, inasmuch as we tially produced by activated mouse Th2 cells (reviewed in Ref. 45) have not been able to examine the production of IL-13 by indi- and shares many biological functions with IL-4. vidual NK cells, we cannot rule out altered expression in subpopu- Our studies show that highly purified B cells can induce IL-2- lations of cells. In contrast, the finding that the inhibitory activity propagated NK cells to produce IL-13 mRNA. This induction is of CD244 is not mediated via SAP (23, 41) is consistent with our independent of IFN-␥ production by NK cells (Fig. 1) or cytokine finding that induction by B cells as an indicator of positive sig- production by B cells and requires contact between the two cell naling does require the participation of SAP (Fig. 5C). Although types (Fig. 2). Although NKT cells can produce Th2 cytokines,we both CD2 and CD244 can recognize CD48, disruption of CD2 on have shown that the IL-13 mRNA in this case is not produced by NK cells had no effect on their ability to respond to B cells, pos- these cells (Fig. 1). The expression of CD244 on NK cells is ab- sibly because CD2 has a lower affinity for CD48 than CD244. solutely required for this positive interaction (Fig. 3). Furthermore, More importantly, CD2 receptors do not contain the conserved the SLAM-associated protein, SAP, is required for the transmis- immunoreceptor tyrosine-based switch motifs (TxYxxV/I) (50) in sion of the signal from CD244 (Fig. 5). The expression of CD48, their cytoplasmic tails that enable them to bind to SAP. In this the ligand for CD244 on B cells is also important for the activation regard, the mouse B-NK cell interaction is more similar to human of IL-13 mRNA expression. However, since MZ B cells are more cells in that the signal transduced by CD244 requires the mediation effective stimulators than follicular B cells (Fig. 4), both of which of SAP, and patients with defects in SAP cannot respond via their express CD48, other costimulatory molecules may be involved. CD244 receptor (16, 51, 52). In contrast, their response to CD2 In contrast to earlier reports indicating up-regulation of the tran- ligation is not affected (16). scripts after only4hofinvitro stimulation of NK cells with 100 Interestingly, in two different SAP-deficient mouse strains, Th2 U of IL-2 (5), we do not detect IL-13 mRNA in otherwise non- responses are more affected than Th1 responses (24, 53, 54). Thus, stimulated IL-2-propagated cultures. Others have also reported the the effect of CD244 on T cells in response to TCR stimulation (47, The Journal of Immunology 2763

54) may be more similar to B cell stimulation of NK cytokine ing in down-regulation of the proinflammatory cytokines usually expression in that CD244 performs a costimulatory role. In this produced by (reviewed in Ref. 10) and possibly al- regard, it is worthy to note that despite a significant decrease in the tering the nature of the cytokine circuit. It should be noted that level of stimulation by CD48-deficient B cells, residual induction although NKT cells have been frequently associated with Th2 re- remained. Furthermore, although immobilized anti-CD28 can in- sponses (61, 62), depletion of only NK cells can affect IgE (63) as duce both wild-type and CD2Ϫ/Ϫ NK cells to express IL-13 well as IgG1 responses under specific conditions of immunization mRNA, NK cells from CD244Ϫ/Ϫ were not responsive. Thus, it is (2). In addition, although IL-13 has generally been associated with possible that induction of IL-13 mRNA expression by B cells re- allergic inflammation (45), the cytokine may also play a role in quires, in addition, the simultaneous ligation of another receptor other diseases as well (64). It remains to be shown whether the B such as CD28, and CD244 could function in NK cells in a similar cell induction of IL-13 expression by NK cells plays a significant manner as in T cells (54), by augmenting the activity of other role in these responses. For example, the cytokine produced under receptors. Therefore, when CD48 is blocked on B cells, the other these conditions can amplify a cytokine burst involving mast cells molecules, such as CD80 or CD86, can still stimulate NK expres- and eosinophils in the same manner that NK production of IFN-␥ sion of IL-13 mRNA, albeit at a much reduced level. The role of amplifies the cytokine loop initiated by macrophages. Thus, with alternative ligands that can stimulate NK cells in conjunction with the involvement of B cells, the interplay between these cytokine CD48 may also explain our finding that MZ B cells are better circuits mediated by the innate system can significantly affect the stimulators than follicular B cells despite the fact that they express outcome of the adaptive immune response. similar levels of CD48. MZ B cells express higher levels of CD80 and CD86 (56) as well as other ligands such as LFA-1 (57). These Acknowledgments Downloaded from coreceptors all have counterreceptors on NK cells that could signal We thank Dr. M. Wabl for permission to use the QM mouse and Dr. R. via collaboration with CD244. The differential induction by MZ vs Noelle for providing the mice. We thank Dr. Michael Bennett and Tom follicular B cells also suggest that not all cells that express CD48 Waldschmidt for providing mice and reagents, respectively. The hybrid- are effective inducers of IL-13 mRNA expression by NK cells. In oma HM48-1was provided by Dr. Hideo Yagita. contrast, another possibility that has not been addressed is that

follicular B cells may express costimulators that result in the gen- Disclosures http://www.jimmunol.org/ eration of an inhibitory signal via CD244. The authors have no financial conflict of interest. It is important to clarify that we have documented two types of NK-B cell interactions. Other than the stimulatory effect of B cells References on NK cells documented herein, NK cells can also activate partial 1. Michael, A., J. J. Hackett, M. Bennett, V. Kumar, and D. Yuan. 1989. Regulation ␥ B cell differentiation as evidenced by the induction of germline of B lymphocytes by natural killer cells: role of IFN- . J. Immunol. 142: 1095–1101. transcripts (25). This effect, which also requires direct cell contact, 2. Yuan, D., R. Bibi, and T. Dang. 2004. The role of adjuvant on the regulatory is principally mediated by the CD48 receptor on B cells respond- effects of NK cells on B cell responses as revealed by a new model of NK cell deficiency. Int. Immunol. 16: 707–716. ing to CD2 expressed by NK cells (38). This interaction does not, 3. Stetson, D. B., M. Mohrs, R. L. Reinhardt, J. L. Baron, Z. E. Wang, L. Gapin, however, differentiate between MZ or follicular B cells, both of M. Kronenberg, and R. M. Locksley. 2003. Constitutive cytokine mRNAs mark by guest on September 26, 2021 which can readily respond to NK induction. Therefore, it is un- natural killer (NK) and NK T cells poised for rapid effector function. J. Exp. Med. 198: 1069–1076. likely to be mediated by IL-13 production by NK cells. 4. Katsumoto, T., M. Kimura, M. Yamashita, H. Hosokawa, K. Hashimoto, Examination of the expression of IL-13 and IFN-␥ mRNA for A. Hasegawa, M. Omori, T. Miyamoto, M. Taniguchi, and T. Nakayama. 2004. two of the major cytokines produced by murine NK cells confirms STAT6-dependent differentiation and production of IL-5 and IL-13 in murine NK2 cells. J. Immunol. 173: 4967–4975. previous findings that IL-12 preferentially induces IFN-␥ but is a 5. Hoshino, T., R. T. Winkler-Pickett, A. T. Mason, J. R. Ortaldo, and H. A. Young. poor inducer of IL-13 mRNA. But the combination of the two 1999. IL-13 production by NK cells: IL-13-producing NK and T cells are present in vivo in the absence of IFN-␥. J. Immunol. 162: 51–59. cytokines can induce high levels of both cytokines (13). Previous 6. Loza, M. J., and B. Perussia. 2001. Final steps of maturation: reports have indicated that polarization of T cell responses by cy- a model for type 1-type 2 differentiation? Nat. Immunol. 2: 917–924. tokines is not affected in SAP-deficient mice (24). Although we 7. McKenzie, G. J., A. Bancroft, R. K. Grencis, and A. N. McKenzie. 1998. A distinct role for interleukin-13 in Th2-cell-mediated immune responses. Curr. found that NK cells from these mice respond poorly to either IL-12 Biol. 8: 339–342. or IL-18, significant levels of both IL-13 and IFN-␥ expression can 8. Barner, M., M. Mohrs, F. Brombacher, and M. Kopf. 1998. Differences between be induced by the combination of the two cytokines (Fig. 6). IL-4R␣-deficient and IL-4-deficient mice reveal a role for IL-13 in the regulation of Th2 responses. Curr. Biol. 8: 669–672. Therefore, the response of NK cells to these cytokines is indepen- 9. Urban, J. F., Jr., N. Noben-Trauth, D. D. Donaldson, K. B. Madden, S. C. Morris, dent of the SAP-mediated phosphorylation. M. Collins, and F. D. Finkelman. 1998. IL-13, IL-4R␣, and Stat6 are required for We have not been able to induce freshly isolated NK cells to the expulsion of the gastrointestinal nematode parasite Nippostrongylus brasil- iensis. Immunity 8: 255–264. produce IL-13 mRNA. Upon activation by poly(I:C) in vivo, how- 10. Gordon, S. 2003. Alternative activation of macrophages. Nat. Rev. Immunol. 3: ever, NK cells can be rapidly recruited to the MZ of splenic fol- 23–35. 11. Harlin, H., E. Podack, M. Boothby, and M. L. Alegre. 2002. TCR-independent licles (39) where they may encounter B cells residing in this re- CD30 signaling selectively induces IL-13 production via a TNF receptor-asso- gion. Whereas we have shown that these MZ B cells can induce ciated factor/p38 mitogen-activated protein kinase-dependent mechanism. J. Im- NK-cell IL-13 secretion, how the cytokine affects B cell responses munol. 169: 2451–2459. 12. Lauwerys, B., N. Garot, J.-G. Renault, and F. Houssiau. 2000. Cytokine produc- is unclear. The IL-13 receptors on B cells may need to be up- tion and killer activity NK/T-NK cells derived with IL-2, IL-15, or the combi- regulated before they can respond to IL-13 (58). IL-13 can enhance nation of IL-12 and IL-18. J. Immunol. 165: 1847–1853. germinal center B cell differentiation in the presence of anti-CD40 13. Chakir, H., A. M. Lemay, and J. R. Webb. 2001. Cytokine expression by murine DX5ϩ cells in response to IL-12, IL-18, or the combination of IL-12 and IL-18. (59, 60) but the effect on MZ B cells has not been examined. We Cell. Immunol. 212: 71–81. have previously documented a partial dependence on NK cells of 14. Mathew, P. A., B. A. Garni-Wagner, K. Land, A. Takashima, E. Stoneman, M. Bennett, and V. Kumar. 1993. Cloning and characterization of the 2B4 gene the IgG1 response to TI-2 Ags given in the presence of the adju- encoding a molecule associated with non-MHC-restricted killing mediated by vant RIBI (2). It is possible, therefore, that the induction of NK activated natural killer cells and T cells. J. Immunol. 151: 5338–5347. secretion of this cytokine under some conditions of immunization 15. Tangye, S. G., S. Lazetic, E. Woollatt, G. R. Sutherland, L. L. Lanier, and J. H. Phillips. 1999. Cutting edge: human 2B4, an activating NK cell receptor, is responsible for the participation of NK cells in a TI-2 response. recruits the protein tyrosine phosphatase SHP-2 and the adaptor signaling protein IL-13 can activate macrophages via the alternate pathway, result- SAP. J. Immunol. 162: 6981–6985. 2764 B CELL INDUCTION OF NK CELL DIFFERENTIATION

16. Tangye, S. G., J. H. Phillips, L. L. Lanier, and K. E. Nichols. 2000. Functional 42. Warren, H. S., B. F. Kinnear, J. H. Phillips, and L. L. Lanier. 1995. Production requirement for SAP in 2B4-mediated activation of human natural killer cells as of IL-5 by human NK cells and regulation of IL-5 secretion by IL-4, IL-10, and revealed by the X-linked lymphoproliferative syndrome. J. Immunol. 165: IL-12. J. Immunol. 154: 5144–5152. 2932–2936. 43. Loza, M. J., L. Zamai, L. Azzoni, E. Rosati, and B. Perussia. 2002. Expression 17. Dalton, D. K., S. Pitts-Meek, S. Keshav, I. S. Figari, A. Bradley, and of type 1 (interferon ␥) and type 2 (interleukin-13, interleukin-5) cytokines at T. A. Stewart. 1993. Multiple defects of immune cell function in mice with distinct stages of natural killer cell differentiation from progenitor cells. Blood 99: disrupted interferon-␥ genes. Science 259: 1739–1742. 1273–1281. 18. Murphy, K. M., A. B. Heimberger, and D. Y. Loh. 1990. Induction by of 44. Samson, S. I., O. Richard, M. Tavian, T. Ranson, C. A. Vosshenrich, F. Colucci, ϩ ϩ intrathymic apoptosis of CD4 CD8 TCRlo in vivo. Science 250: J. Buer, F. Grosveld, I. Godin, and J. P. Di Santo. 2003. GATA-3 promotes 1720–1723. maturation, IFN-␥ production, and liver-specific homing of NK cells. Immunity 19. Layton, J., P. Krammer, T. Hamaoka, J. Uhr, and E. Vitetta. 1985. Small and 19: 701–711. large B cell subsets respond differently to T cell-derived B cell growth and dif- 45. Hershey, G. K. 2003. IL-13 receptors and signaling pathways: an evolving web. ferentiation factors. J. Mol. Cell. Immunol. 2: 155–167. J. Allergy Clin. Immunol. 111: 677–690. 20. Weiss, E., A. Michel, and D. Yuan. 1989. Role of transcriptional termination in 46. Johnson, L. A., R. H. Goldfarb, and P. A. Mathew. 2000. Regulation of IFN-␥ ␮ the regulation of mRNA expression in B lymphocytes. J. Immunol. 142: production following 2B4 activation in human NK cells. In Vivo 14: 625–629. 1046–1052. 47. Lee, K. M., S. Bhawan, T. Majima, H. Wei, M. I. Nishimura, H. Yagita, and 21. Blazar, B. R., P. A. Taylor, A. Panoskaltsis-Mortari, H. Yagita, J. S. Bromberg, V. Kumar. 2003. Cutting edge: the NK cell receptor 2B4 augments antigen- and D. A. Vallera. 1998. A critical role for CD48 antigen in regulating alloen- specific T cell cytotoxicity through CD48 ligation on neighboring T cells. J. Im- graftment and lymphohematopoietic recovery after bone marrow transplantation. munol. 170: 4881–4885. Blood 92: 4453–4463. 48. Assarsson, E., T. Kambayashi, J. D. Schatzle, S. O. Cramer, A. von Bonin, 22. Teh, S. J., N. Killeen, A. Tarakhovsky, D. R. Littman, and H. S. Teh. 1997. CD2 Ϫ ϩ P. E. Jensen, H. G. Ljunggren, and B. J. Chambers. 2004. NK cells stimulate regulates the positive selection and function of antigen-specific CD4 CD8 T proliferation of T and NK cells through 2B4/CD48 interactions. J. Immunol. 173: cells. Blood 89: 1308–1318. 174–180. 23. Lee, K. M., M. E. McNerney, S. E. Stepp, P. A. Mathew, J. D. Schatzle, M. Bennett, and V. Kumar. 2004. 2B4 acts as a non-major histocompatibility 49. Bloch-Queyrat, C., M. C. Fondaneche, R. Chen, L. Yin, F. Relouzat, A. Veillette, complex binding inhibitory receptor on mouse natural killer cells. J. Exp. Med. A. Fischer, and S. Latour. 2005. Regulation of natural cytotoxicity by the adaptor Downloaded from 199: 1245–1254. SAP and the Src-related kinase Fyn. J. Exp. Med. 202: 181–192. 24. Czar, M. J., E. N. Kersh, L. A. Mijares, G. Lanier, J. Lewis, G. Yap, A. Chen, 50. Sidorenko, S. P., and E. A. Clark. 2003. The dual-function CD150 receptor sub- A. Sher, C. S. Duckett, R. Ahmed, and P. L. Schwartzberg. 2001. Altered lym- family: the viral attraction. Nat. Immunol. 4: 19–24. phocyte responses and cytokine production in mice deficient in the X-linked 51. Benoit, L., X. Wang, H. F. Pabst, J. Dutz, and R. Tan. 2000. Defective NK cell lymphoproliferative disease gene SH2D1A/DSHP/SAP. Proc. Natl. Acad. Sci. activation in X-linked lymphoproliferative disease. J. Immunol. 165: 3549–3553. USA 98: 7449–7454. 52. Nakajima, H., M. Cella, A. Bouchon, H. L. Grierson, J. Lewis, C. S. Duckett, 25. Gao, N., T. Dang, and D. Yuan. 2001. IFN-␥-dependent and -independent initi- J. I. Cohen, and M. Colonna. 2000. Patients with X-linked lymphoproliferative

ation of switch recombination by NK cells. J. Immunol. 167: 2011–2018. disease have a defect in 2B4 receptor-mediated NK cell cytotoxicity. Eur. J. Im- http://www.jimmunol.org/ 26. Cascalho, M., A. Ma, S. Lee, L. Masat, and M. Wabl. 1996. A quasi-monoclonal munol. 30: 3309–3318. mouse. Science 272: 1649–1652. 53. Wu, C., K. B. Nguyen, G. C. Pien, N. Wang, C. Gullo, D. Howie, M. R. Sosa, 27. Noelle, R., M. Roy, D. Shepherd, I. Stamenkovic, J. Ledbetter, and A. Arufoo. M. J. Edwards, P. Borrow, A. R. Satoskar, et al. 2001. SAP controls T cell 1992. A 39-kDa protein on activated helper T cells binds CD40 and transduces responses to virus and terminal differentiation of TH2 cells. Nat. Immunol. 2: the signal for cognate activation of B cells. Proc. Natl. Acad. Sci. USA 89: 410–414. 6550–6554. 54. Cannons, J. L., L. J. Yu, B. Hill, L. A. Mijares, D. Dombroski, K. E. Nichols, 28. Nandi, D., J. A. Gross, and J. P. Allison. 1994. CD28-mediated costimulation is A. Antonellis, G. A. Koretzky, K. Gardner, and P. L. Schwartzberg. 2004. SAP ␶ ␬ necessary for optimal proliferation of murine NK cells. J. Immunol. 152: regulates TH2 differentiation and PKC- -mediated activation of NF- B1. Immu- 3361–3369. nity 21: 693–706. 29. Unkeless, J. 1979. Characterization of a monoclonal antibody directed against 55. Morra, M., R. A. Barrington, A. C. Abadia-Molina, S. Okamoto, A. Julien, mouse and lymphocyte Fc receptors. J. Exp. Med. 150: 580–596. C. Gullo, A. Kalsy, M. J. Edwards, G. Chen, R. Spolski, et al. 2005. Defective B

30. Spitalny, G., and E. Havell. 1984. Monoclonal antibody to murine ␥ interferon cell responses in the absence of SH2D1A. Proc. Natl. Acad. Sci. USA 102: by guest on September 26, 2021 inhibits lymphokine-induced antiviral and macrophage tumoricidal activities. 4819–4823. J. Exp. Med. 159: 1560–1565. 56. Oliver, A., F. Martin, and J. Kearney. 1999. IgMhighCD21high lymphocytes en- 31. Kubo, R. T., W. Born, J. W. Kappler, P. Marrack, and M. Pigeon. 1989. Char- riched in the splenic marginal zone generate effector cells more rapidly than the ␣␤ acterization of a monoclonal antibody which detects all murine T cell recep- bulk of follicular B cells. J. Immunol. 162: 7198–7207. tors. J. Immunol. 142: 2736–2742. 57. Lu, T. T., and J. G. Cyster. 2002. Integrin-mediated long-term B cell retention in 32. Koo, G., and J. Peppard. 1984. Establishment of monoclonal anti-NK-1.1 anti- the splenic marginal zone. Science 297: 409–412. body. Hybridoma 3: 301–303. 58. Andrews, R., L. Rosa, M. Daines, and G. Khurana Hershey. 2001. Reconstitution 33. Garni-Wagner, B. A., A. Purohit, P. A. Mathew, M. Bennett, and V. Kumar. of a functional human type II IL-4/IL-13 receptor in mouse B cells: demonstra- 1993. A novel function-associated molecule related to non-MHC-restricted cy- tion of species specificity. J. Immunol. 166: 1716–1722. totoxicity mediated by activated natural killer cells and T cells. J. Immunol. 151: 59. Poudrier, J., P. Graber, S. Herren, D. Gretener, G. Elson, C. Berney, 60–70. J. F. Gauchat, and M. H. Kosco-Vilbois. 1999. A soluble form of IL-13 receptor 34. Abuodeh, R., H. Wei, and D. Yuan. 1998. Effect of upstream RNA processing on ␣1 promotes IgG2a and IgG2b production by murine germinal center B cells. selection of ␮ S versus ␮M poly(A) sites. Nucleic Acids Res. 26: 5417–5424. J. Immunol. 163: 1153–1161. 35. Ochi, H., and T. Watanabe. 2000. Negative regulation of B cell receptor-mediated signaling in B-1 cells through CD5 and Ly49 co-receptors via Lyn kinase activ- 60. Poudrier, J., P. Graber, S. Herren, C. Berney, D. Gretener, M. H. Kosco-Vilbois, ␣ ity. Int. Immunol. 12: 1417–1423. and J. F. Gauchat. 2000. A novel monoclonal antibody, C41, reveals IL-13R 1 36. Hoshino, T., R. H. Wiltrout, and H. A. Young. 1999. IL-18 is a potent coinducer expression by murine germinal center B cells and follicular dendritic cells. Eur. of IL-13 in NK and T cells: a new potential role for IL-18 in modulating the J. Immunol. 30: 3157–3164. immune response. J. Immunol. 162: 5070–5077. 61. Jahng, A. W., I. Maricic, B. Pedersen, N. Burdin, O. Naidenko, M. Kronenberg, 37. Carbone, E., G. Ruggiero, G. Terrazzano, C. Palomba, C. Manzo, S. Fontana, Y. Koezuka, and V. Kumar. 2001. Activation of natural killer T cells potentiates H. Spits, K. Karre, and S. Zappacosta. 1997. A new mechanism of NK cell or prevents experimental autoimmune encephalomyelitis. J. Exp. Med. 194: cytotoxicity activation: the CD40-CD40 ligand interaction. J. Exp. Med. 185: 1789–1799. 2053–2060. 62. Yoshimoto, T., B. Min, T. Sugimoto, N. Hayashi, Y. Ishikawa, Y. Sasaki, 38. Gao, N., T. Dang, W. A. Dunnick, J. T. Collins, B. R. Blazar, and D. Yuan. 2005. H. Hata, K. Takeda, K. Okumura, L. Van Kaer, et al. 2003. Nonredundant roles ϩ Receptors and counterreceptors involved in NK-B cell interactions. J. Immunol. for CD1d-restricted natural killer T cells and conventional CD4 T cells in the 174: 4113–4119. induction of immunoglobulin E antibodies in response to interleukin 18 treatment 39. Salazar-Mather, T. P., R. Ishikawa, and C. A. Biron. 1996. NK cell trafficking and of mice. J. Exp. Med. 197: 997–1005. cytokine expression in splenic compartments after IFN induction and viral in- 63. Korsgren, M., C. G. A. Persson, F. Sundler, T. Bjerke, T. Hansson, fection. J. Immunol. 157: 3054–3064. B. J. Chambers, S. Hong, L.Van Kaer, H. G. Ljunggren, and O. Korsgren. 1999. 40. Martin, F., and J. F. Kearney. 2002. Marginal-zone B cells. Nat. Rev. Immunol. Natural killer cells determine development of allergen-induced eosinophilic air- 2: 323–335. way inflammation in mice. J. Exp. Med. 189: 553–562. 41. Mooney, J. M., J. Klem, C. Wulfing, L. A. Mijares, P. L. Schwartzberg, 64. Young, D. A., L. D. Lowe, S. S. Booth, M. J. Whitters, L. Nicholson, M. Bennett, and J. D. Schatzle. 2004. The murine NK receptor 2B4 (CD244) V. K. Kuchroo, and M. Collins. 2000. IL-4, IL-10, IL-13, and TGF-␤ from an exhibits inhibitory function independent of signaling lymphocytic activation mol- altered peptide ligand-specific Th2 cell clone down-regulate adoptive transfer of ecule-associated protein expression. J. Immunol. 173: 3953–3961. experimental autoimmune encephalomyelitis. J. Immunol. 164: 3563–3572.