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Azusa Ujike, Kazuhiko Takeda,Akira Nakamura, Shin Ebihara, Kenichi Akiyama and Toshiyuki Takai

Published online: 20 May 2002, DOI: 10.1038/ni801

Mice deficient for paired immunoglobulin (Ig)-like receptor B (PIR-B) show defective regulation ofreceptor-mediated activation in antigen-presenting cells. Older PIR-B–/– mice had an increasednumber of peritoneal B1 cells. Splenic PIR-B–/– B2 cells were constitutively activated and proliferatedmuch more than those from wild-type mice upon B cell receptor ligation.T helper type 2 (TH2)-pronehumoral responses were augmented in PIR-B–/– mice upon immunization with T-dependent antigens,including increased interleukin 4 and decreased interferon-γ responses, as well as enhanced IgG1 andIgE production. Impaired maturation of dendritic cells (DCs), possibly due to perturbed intracellularsignaling, was responsible for the skewed responses.Thus, PIR-B is critical for B cell suppression, DCmaturation and for balancing TH1 and TH2 immune responses.

Department of Experimental Immunology and CREST Program of JST, Institute of Development, Aging and Cancer,Tohoku University, Seiryo 4-1, Sendai 980-8575, Japan.Correspondence should be addressed to T.T. (tostakai@idac.tohoku.ac.jp).

Impaired dendritic cell maturation andincreased TH2 responses in PIR-B–/– mice

Immunoglobulin (Ig)-like cell-surface receptors (IgLRs) are suggestedto play regulatory roles in cellular signaling and in immune responses1–4.IgLRs are composed of several subfamilies, including human killer cellIgLR (KIR)5,6, human leukocyte IgLR (LIR, also known as ILT andmyeloid IgLR)7–10, human and murine signal induction receptor protein(SIRP)11, murine gp49 molecules12 and paired IgLR (PIR)13,14. The genesencoding many of these receptors are located in clusters on human chro-mosome 19q13.4 or its syntenic position in mice14–16. The common fea-tures of IgLR members are that they possess ectodomains that are high-ly homologous to each other and include one or more inhibitory iso-forms that harbor immunoreceptor tyrosine–based inhibitory motifs(ITIMs) or ITIM-like sequences1. These receptor families also includenoninhibitory or activating-type isoforms, several of which are associat-ed with subunit molecules containing an immunoreceptor tyrosine-based activation motif (ITAM), such as Fc receptor γ subunit (FcRγ)17–20

or DAP12 (also known as KARAP) homodimers21–23. For example, nat-ural killer (NK) cells and a subset of T cells are negatively regulatedupon engagement of their inhibitory KIRs with major histocompatibili-ty complex (MHC) class I molecules expressed on target cells. Thus, lig-ation of inhibitory KIRs prevents these effector cells from damagingnormal self-cells, whereas activating KIRs that associate with a DAP12homodimer deliver an activation signal upon aggregation3.

PIR was first identified as a murine molecule that shows muchsequence similarity to KIR, LIR, murine gp49B and human FcR forIgA13,14. Several LIR molecules were isolated from a human spleencDNA library with a probe that corresponds to the PIR-B ectodomain16.PIR-B is encoded by a single-copy gene, whereas the PIR-A group isencoded by multiple genes, all of which are located on the mouse chro-mosome 7 proximal region14,16. The structural characteristics of humanPIR molecules are conserved between rats and mice24. PIR relativeshave also been identified in chickens25. PIR-A and PIR-B are expressed

on various hematopoietic cell lineages, including B cells, mast cells,macrophages, granulocytes and dendritic cells (DCs), but are notexpressed on T and NK cells14,16. Antibodies specific for common epi-topes of PIR-A and PIR-B showed that PIR expression depends uponthe developmental stage of the cell18. For example, PIR cell surfaceexpression on B lineage cells increases with cellular differentiation andactivation18. PIR-A requires FcRγ for its cell surface expression and forthe delivery of activation signals18–20,26. In contrast, PIR-B containsITIMs in its cytoplasmic portion and inhibits receptor-mediated activa-tion signaling in vitro upon cellular engagement with other activating-type receptors, such as the B cell antigen receptor (BCR)26–28. Untilnow, attempts to identify specific ligands for PIR have been unsuccess-ful. However, classical or nonclassical MHC class I molecules havebeen suggested to interact with PIR, based on the observation that PIRexpression was decreased in β2-microglobulin–deficient mice29. Thus,accumulating evidence suggests that PIR molecules play importantroles in regulating immune cell development and function.

To clarify the role of PIR-B in vivo, we generated and characterizedmice that were PIR-B–/–. The mutant mice have an increased number ofperitoneal B1 cells as they age, and these B1 and splenic B2 cells arehypersensitive to BCR ligation, which suggests PIR-B plays aninhibitory role in B cells under physiological conditions. DC matura-tion was also impaired in PIR-B–/– mice, which could be responsible forthe augmented T helper type 2 (TH2) responses in these mice. Thesefindings establish a role for PIR-B in regulating B cell activation andDC maturation as well as in skewing TH2 responses.

ResultsGeneration of PIR-B–/– micePIR-B–/– mice were generated by standard gene-targeting methods thatresulted in the deletion of sequences encoding the sixth ectodomain and

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juxtamembrane domains (Fig. 1a). PIR-B–/– mice had no PIR-B pro-tein, as detected by immunoblot analysis of splenic B cells (Fig. 1b,c);they grew normally and were fertile.

PIR-A and PIR-B mRNA are coexpressed in a wide variety ofimmune cells, including B cells, mast cells, DCs and macrophages14,16.We used the monoclonal antibody (mAb) 6C1—which recognizes acommon epitope on PIR-A and PIR-B18—to determine PIR expressionon splenic macrophages, splenic DCs and bone marrow–derived (BM)cultured mast cells. Flow cytometric analysis showed reduced butdetectable PIR staining in cells from PIR-B–/– mice (Fig. 1d), whichsuggested that these cells coexpress PIR-A and PIR-B on their surfaces.PIR-B dominates PIR-A in these cell types. However, splenic B cells

expressed only PIR-B on the cell surface because PIR staining wasabsent on cells from PIR-B–/– mice (Fig. 1d). PIR-A surface expressionis lost in FcRγ-deficient mice because PIR-A associates with the FcRγhomodimer, which is required for its efficient cell-surface expres-sion18–20,26. PIR expression in FcRγ–/– mice was consistent with the pre-dominant expression of PIR-B on macrophages, DCs and BM mastcells and the exclusive PIR-B expression in B cells, which suggestedthat the PIR-B deletion did not largely alter PIR-A expression in thesecells and vice versa. These results suggested that PIR-A and PIR-B sur-face expression is characteristic for each cell type—at least in its rest-ing state—and in this strain of mice, confirming published data14,16, aswell as in another murine IgLR, the gp49A-gp49B pair30.

Figure 1. Disruption of mousePirb. (a) Strategy for targeted disrup-tion.Organization of Pirb, constructionof the targeting vector and structureof the targeted genome are shown;restriction sites are indicated. TK,thymidine kinase gene; Neo, neomycinresistance gene; H, HindIII; P, PstI; RI,EcoRI; RV, EcoRV; S, SphI. (b) Southernblot analysis. Genomic DNA fromnewborn littermates of heterozygoteintercrosses was digested with EcoRI,and the resulting fragments were subjected to analysis with a PstI-SphI fragment of thegenomic DNA as a probe (shaded box in a).The positions of the 5.6-kb (wild-type) and4.8-kb (mutant) hybridizing fragments are shown for mice of the indicated PIR-B pheno-types. (c) Immunoblot analysis. Splenic B cells (2×106 cells) from wild-type or PIR-B–/– micewere subjected to immunoblot analysis with polyclonal goat anti-PIR.The position of ∼ 120-kD PIR-B is indicated by an arrow. (d) Flow cytometric analysis of cell-surface expressionof PIR on splenic macrophages, DCs and B cells and BM mast cells. Cells from wild-type(thick black line), PIR-B–/– (red line) or FcRγ–/– (blue line) mice were stained with PE-anti–mouse PIR-A and PIR-B (6C1) or control rat IgG1 mAb (dotted black line). Formacrophages and DCs, splenocytes were subjected to sorting first with anti-CD11c–con-jugated magnetic beads to obtain DCs. CD11c-negative cells were then positively selectedwith anti-CD11b–conjugated magnetic beads to obtain macrophages before flow cytomet-ric analysis. For B cells and BM mast cells, the positive cells were gated for staining withFITC-conjugated mAbs to B220 or c-Kit, respectively.

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Figure 2. Flow cytometric analysis of B celldevelopment in wild type and PIR-B–/– mice.(a) BM cells were stained with B220 and IgM andgated to show pro-, pre-, immature and mature B cells. Spl, spleen. Numbers denote the percentagesof cells within a specified gate as a fraction of totallymphocytes. (b) Splenic B220+ cells were stainedwith IgM and IgD and gated to show matureIgMloIgDhi, IgMhiIgDhi type 2 transitional and IgMhiIgDlo

type 1 transitional B cells. Numbers denote the per-centages of cells within a specified gate as a fractionof splenic B220+ cells. (c) Splenic B220+ cells werestained with CD21 and CD23 and gated to shownewly formed CD21–CD23– B cells, CD21+CD23+ follicular B cells and CD21+CD23– marginal zone B cells. Numbers denote the percentages of cells within a specified gate as afraction of splenic B220+ cells. (d) Peritoneal cells from 9-week-old mice were stained with CD5 and IgM, and gated for lymphocytes to show CD5+IgM+ B1 cells.The percentages ofcells within a specified gate as a fraction of all lymphocytes are indicated. Each panel is representative of three independent experiments. (e) Increase in peritoneal B1 cells of PIR-B–/–

mice with age. Numbers of total peritoneal cells and the B220+CD5+CD3– B1 cells were enumerated in PIR-B–/– and wild-type mice at each age (n=3). *P<0.05; NS, not significant.

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B1 cells increase with age in PIR-B–/– miceSplenic CD4+:CD8+ T cells and T:B cell population ratios wereunchanged in PIR-B–/– mice (data not shown), which suggested thatPIR-B deficiency does not influence canonical T cell development.Similar B220 and surface IgM expression was observed in BM cellsfrom PIR-B–/– and wild-type mice (Fig. 2a). PIR-B deficiency did notalter most mature B cell subsets, including mature recirculating follic-ular B cells, type 2 transitional B cells, most immature type 1 transi-tional B cells (Fig. 2b), marginal zone B cells, follicular B cells andnewly formed B cells (Fig. 2c).

CD5+IgM+ peritoneal B1 cells were significantly increased in olderPIR-B–/– mice (Fig. 2d,e). We did not observe any remarkable differ-ences in peritoneal T cells or B2 cells from PIR-B–/– and wild-typemice, even after aging (data not shown). B1 cells produce natural anti-bodies and are suggested to play a key role in autoimmunity31,32.Increased B1 cells are also observed in mice that lack CD2233,34 orCD7235, both of which harbor ITIM(s). Serum IgM concentrations,however, were not increased in PIR-B–/– mice at 6 or 32 weeks of age(data not shown). Also, antibodies to double-stranded DNA were notdetected in PIR-B–/– mice, at least up to 32 weeks of age (data notshown); this contrasted with CD22-deficient mice, in which hyper-IgMand autoantibody production is evident at that age33,34.

Hyperresponsive B cells in PIR-B–/– micePIR-B molecules in macrophages and B cells are constitutively phos-phorylated29, and PIR-B in splenocytes are constitutively associated

with the phosphatase SHP-1 and the Src kinase Lyn29. Thus, deletion ofPIR-B may induce B cell hypersensitivity upon BCR ligation, similarto that seen in CD22-deficient mice33,34. We analyzed the proliferativeresponse of splenic B2 cells upon anti–IgM F(ab′)2 stimulation andfound significantly enhanced proliferation of the PIR-B–/– B2 cells (Fig.3a). FcγRIIB is a unique inhibitory Fcγ receptor expressed on Bcells36,37. When stimulated with whole-molecule IgG specific for IgM,the enhanced PIR-B–/– B2 cell proliferation was more pronounced dueto masking of an inhibitory effect by FcγRIIB (Fig. 3b); this indicatedthat the inhibitory effects of PIR-B and FcγRIIB are additive. Thus,PIR-B–/– B cells are hypersensitive to stimulation via BCR ligation.When PIR-B–/– mice were challenged with T-independent (TI) antigens,trinitrophenol (TNP)-conjugated Ficoll (Fig. 3c) or TNP-lipopolysac-charide (LPS) (Fig. 3d), we observed significant increases in theiranti–TNP IgM responses compared to those of wild-type mice; this wasconsistent with hypersensitive B cells in PIR-B–/– mice. The enhancedresponse upon TNP-LPS challenge suggested that the PIR-B inhibitoryeffect could also be exerted independently of BCR ligation.

To investigate the mechanism for the B cell hypersensitivity, weexamined possible PIR-B and BCR interactions in vitro. However, wedid not observe any constitutive association of PIR-B with BCR byimmunoblot analysis (data not shown). PIR-B was not incorporatedinto the detergent-insoluble membrane fraction upon BCR ligation ofwild-type splenic B cells in vitro (data not shown). However, we foundenhanced tyrosine phosphorylation of cellular proteins in PIR-B–/–

B cells, even in the resting state, which indicated that PIR-B–/– B cells

Figure 3. Hypersensitive PIR-B–/–

B cells to BCR stimulation(a) Purified B220+ splenic B cells fromwild-type and PIR-B–/– mice were cul-tured in the presence of the indicatedconcentrations of anti–µ F(ab′)2, or 1µg ml–1 of anti-CD40 or 10 µg ml–1

LPS as positive controls. Data aremean±s.d. of triplicate cultures thatwere representative of three inde-pendent experiments. (b) B220+

splenic B cells were stimulated with10 µg ml–1 of anti-µ in the presenceor absence of 25 µg ml–1 of 2.4G2 toblock FcγRIIB. (c,d) Increasedhumoral responses to TI antigens inPIR-B–/– mice.Wild-type and PIR-B–/– mice, at 8 weeks of age, were immunized with 100 µg of TNP-Ficoll (c) or 50 µg of TNP-LPS (d). Relative amounts of TNP-specific IgM weredetermined by ELISA. Data are absorbance by preimmune sera and sera collected 7 days and 14 days after immunization at 450 nm. Open symbols, PIR-B–/– mice; closed symbols,wild-type mice. Each symbol denotes data from a single mouse. *P<0.05, **P<0.01.

Figure 4.Augmented tyrosine phosphorylationin PIR-B–/– B cells. (a) Before and after stimulation ofB cells with anti–µ F(ab′)2, B cell total cell lysate wassubjected to SDS-PAGE and immunoblotted for tyro-sine phosphorylation. Representative data from threeindependent experiments are shown. (b) The signalintensity of tyrosine-phosphorylated proteins in eachlane was estimated by densitometric scanning, nor-malized by signal intensities of actin and representedas a time-course after BCR stimulation. Each datapoint is mean±s.e.m. of three independent experi-ments.The signal intensity of wild-type B cells at time0 was plotted as 1.0. *P<0.05, **P<0.01. PIR-B–/– B cellswere more sensitive to stimulation in terms of tyro-sine phosphorylation than wild-type B cells. (c) Celllysate was first immunoprecipitated with anti-Lyn andimmunoblotted to visualize Lyn or phosphotyrosine.Lyn in PIR-B–/– B cells was more abundantly tyrosine-phosphorylated than that in wild-type B cells.

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were constitutively activated (Fig. 4a,b). The amount of total tyrosine-phosphorylated protein in resting B cells was estimated to be aboutthreefold higher in PIR-B–/– B cells than in wild-type cells, and it was1.5-fold higher 1 min after BCR stimulation (Fig. 4b). Five minutesafter stimulation, the total tyrosine phosphorylation profiles were sim-ilar between wild-type and PIR-B–/– B cells (Fig. 4a,b). Thus, the acti-vation time-course of PIR-B–/– B cells was different from that of wild-type cells. Consistent with these observations, tyrosine phosphorylationof Lyn was higher in PIR-B–/– B cells than wild-type cells before stim-ulation and increased after BCR stimulation (Fig. 4c). Thus, it is likelythat the PIR-B may also inhibit signaling pathways other than that for

BCR. PIR-B may down-regulate BCR signaling by interacting with anyunknown ligand. PIR-B–deficiency might generally render B cellsactive and hyperresponsive to stimulation via BCR.

TH2-prone immune response in PIR-B–/– micePIR-B–/– mice showed significantly augmented IgG1 and IgE responseswhen immunized with the T-dependent (TD) antigen TNP–keyholelimpet hemocyanin (TNP-KLH) (Fig. 5a). Lymph node cells isolated atday 11 from the immunized PIR-B–/– mice produced a much higheramount of interleukin-4 (IL-4) than those of wild-type mice, whereastheir interferon-γ (IFN-γ) production was significantly lower (Fig. 5b,

Figure 5. Enhanced TH2-type responses to TD antigen in PIR-B–/– mice. (a) Humoral responses to TNP-KLH.Wild-type (n=5) and PIR-B–/– (n=5) mice at 8 weeksof age were immunized with 10 µg of TNP-KLH with alum adjuvant and pertussis toxin. Serum was collected before immunization and then every 2 weeks after primaryimmunization. Mice were then reimmunized at week 8, bled at week 10 and the secondary response measured.Arrowheads represent the points at which mice were immu-nized (weeks 0 and 8).With the use of ELISA, the relative amounts of TNP-specific IgM, IgG1, IgG2a and IgGb or the total amount of IgE were determined. *P<0.05, **P<0.01.(b) Enhanced IL-4 and reduced IFN-γ production by lymphocytes in response to antigen in PIR-B–/– mice. Lymph node cells were collected from wild-type and PIR-B–/– mice10 days after primary immunization with TNP-KLH (upper panels) or OVA (lower panels).The cells were activated with indicated dose of the antigen.After 40 h of stimu-lation, IL-4 or IFN-γ in the culture supernatant was measured by ELISA. Proliferation was determined by [3H]thymidine uptake on day 3. Data are mean±s.d. of triplicate cul-tures and are representative of two separate experiments with similar results. *P<0.05, **P<0.01.

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Figure 6. DCs of PIR-B–/– mice show impaired maturation and reduced IL-12 production and induce a TH2-prone responsein wild-type mice by adoptive transfer. (a) DC maturation. DCs from wild-type (thick black line) or PIR-B–/– (red line) mice were incu-bated for 24 h in the presence or absence of OVA or anti-CD40. Cells were then stained for MHC class II (I-A), CD80 or CD86 and ana-lyzed by flow cytometry. Wild-type DCs showed increased surface expressions of all three molecules, indicating efficient DC maturation.However, PIR-B–/– DCs showed lower I-A expression and no up-regulation of CD80 and CD86 surface expression, indicating impaired DCmaturation. (b) Production of IL-12p70 by DCs during 24 h culture in response to OVA. Data are mean±s.d. of triplicate cultures. *P<0.05.(c) Antigen uptake by DCs from wild-type and PIR-B–/– mice. DCs were incubated with FITC-OVA at 37 °C or 4 °C for 3 h, washed, thenstained with PE–anti-CD11c. FITC-OVA uptake by DCs was monitored by flow cytometry. There was no significant difference in antigenuptake between wild-type and mutant DCs. Numbers denote the percentages of CD11C+ cells incorporating FITC-OVA. (d,e) PIR-B–/– DCsinduced TH2-skewed response after adoptive transfer into wild-type mice.After OVA-loading, BM DCs from PIR-B–/– or wild-type mice wereadoptively transferred intravenously into nonimmunized wild-type mice (n=3). One week later, the splenic CD4+ T cells produced a signifi-cantly higher amount of IL-4 than wild-type cells. **P<0.01.

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upper panels). Similar results were observed with another TD antigen,ovalbumin (OVA) (Fig. 5b, lower panels). Lymph node T cells from theimmunized PIR-B–/– mice proliferated strongly upon antigen stimula-tion, indicating that their proliferation ability was not impaired. Thus,PIR-B–/– mice showed enhanced TH2-prone antibody responses.

What is the mechanism for the TH2-exaggerated response in PIR-B–/–

mice? We isolated BM DCs from both wild-type and PIR-B–/– mice andloaded them with OVA. Flow cytometric analysis of surface markers onDCs before and after the OVA-loading showed that DCs from PIR-B–/–

mice were immature (Fig. 6a). Staining for MHC class II (I-A) waslower in DCs from PIR-B–/– mice than those from wild-type mice,whereas CD80 and CD86 staining was comparable between the mutantand wild-type DCs. After OVA-loading, wild-type DCs matured withconcomitant enhancement of MHC class II, CD80 and CD86 expres-sion, whereas little or no increase of these molecules was observed onmutant DCs (Fig. 6a). Further stimulation with anti-CD40 failed to res-cue the immature status of the PIR-B–/– DCs. In addition, production ofIL-12—a TH1-polarizing cytokine38—was greatly diminished uponOVA-loading of DCs from PIR-B–/– mice (Fig. 6b). The amount of flu-orescein isothiocyanate (FITC)-labeled OVA incorporated into DCswas comparable between the cells from wild-type and the mutant mice(Fig. 6c), indicating that antigen uptake was not defective in PIR-B–/–

DCs. Thus, maturation of DCs upon antigen uptake was impaired byPIR-B–deficiency.

Next we tested whether the TH2-prone response could be induced inwild-type mice by adoptive transfer of PIR-B–/– DCs. After OVA-load-ing, BM DCs from PIR-B–/– or wild-type mice were transferred intra-venously to nonimmunized wild-type mice. One week later, spleno-cytes or purified CD4+ splenic T cells were isolated and cultured for 48h. Splenic CD4+ T cells from mice adoptively transferred with PIR-B–/–

DCs produced large amounts of IL-4 (Fig. 6d,e). Thus, the TH2-proneimmune response observed in PIR-B–/– mice could be induced in wild-type mice by the adoptive transfer of PIR-B–/– DCs.

To test the possibility that intracellular signaling might differbetween PIR-B–/–and wild-type DCs, we stimulated BM DCs with gran-ulocyte-macrophage colony-stimulating factor (GM-CSF)—a cytokinethat induces DC development—and examined their protein tyrosinephosphorylation profiles (Fig. 7). Wild-type and mutant cells showeddistinct phosphotyrosine protein content, even in resting cells. The sig-nals of three protein species at ∼ 100 kD, ∼ 75 kD and ∼ 60 kD werestronger in the mutant DCs than those in wild-type cells (Fig. 7a). Incontrast, PIR-B–/– DCs lacked the major 120-kD phosphotyrosylatedprotein found in resting wild-type DCs, which was identified as PIR-B

by immunoblot analysis (Fig. 7a,b). Thus, PIR-B is tyrosine-phospho-rylated in resting wild-type DCs, similar to that found in resting Bcells29. We found that tyrosine phosphorylation of PIR-B was increasedupon GM-CSF stimulation (Fig. 7b), indicating that PIR-B is involvedin cytokine signaling. After GM-CSF stimulation, a 130–140-kD pro-tein was phosphorylated in both wild-type and mutant DCs. This pro-tein was identified as GM-CSF receptor common β chain (Fig. 7a,c).The time-course of tyrosine phosphorylation of the GM-CSF receptorβ in PIR-B–/– DCs was more transient than that observed in wild-typecells (Fig. 7c). These results suggested that PIR-B–deficiency main-tains an immature DC state even when sufficient antigen stimulation isadded to the cells, possibly due to altered tyrosine phosphorylation ofcellular proteins.

DiscussionWe have shown here that PIR-B participates in vivo in negatively regu-lating the peritoneal B1 cell compartment, humoral responses to TIantigens and TH2 responses to TD antigens. Our tyrosine phosphoryla-tion analysis of cellular proteins in PIR-B–/– B cells and DCs afterreceptor stimulation showed that the constitutive activation of PIR-B–/–

B cells, the hyperresponsive nature upon BCR stimulation and animpairment in DC maturation, due to altered intracellular signaling,were responsible for these phenotypes. In support of this idea, it is sug-gested that immature DCs are prone to inducing TH2-type responses39.

What is the ligand for PIR-B on B cells and DCs? Human PIR-Brelatives may provide some possibilities. Like PIR-B, LIR-1 (alsoknown as ILT2 or MIR-7)—a close relative of PIR-B in humans—isexpressed in B cells and monocytes8,40,41. Other close relatives—LIR-2, LIR-5 and LIR-8—are expressed in B cells and/or DCs9,42,43 as wellas in monocytes43. The ligands for LIR-1 and LIR-2 are MHC class Imolecules7. It is suggested that various immune cells have adopted therecognition of self–MHC class I as a common strategy to inhibit cel-lular activation. Given that classical or nonclassical MHC class I mol-ecules could interact with PIR29, then PIR may be a newly identifiedubiquitously expressed negative regulator of immune cells, such as B cells and DCs.

In B cells, PIR-B is constitutively tyrosine-phosphorylated29. InLyn-deficient mice, PIR-B tyrosine phosphorylation is greatlyreduced29, indicating the critical role played by Lyn in PIR-B tyrosinephosphorylation. PIR-B ligation on the chicken B cell line DT40 cellsinhibits the BCR-induced tyrosine phosphorylation of Igα, Igβ, Syk,Btk and PLC-γ244. Together with these published findings, our findingshere establish the pivotal role played by PIR-B in suppressing B cell

Figure 7. Perturbed signaling of PIR-B–/–

DCs. (a,b) Different phosphotyrosine profilesof wild-type and PIR-B–/– DCs before and afterGM-CSF stimulation. (a) Before and after stim-ulation of BM DCs with GM-CSF, total celllysate was subjected to SDS-PAGE andimmunoblotted for tyrosine phosphorylation.(b) Alternatively, the cell lysate was firstimmunoprecipitated with anti-PIR andimmunoblotted to visualize phosphotyrosine.The membrane was washed and then reblottedwith PIR antibodies to visualize PIR-B.Arrowsindicate the PIR-B band, which was robustlyphosphorylated, even in a resting state. (c) Celllysate was first immunoprecipitated withanti–GM-CSF receptor common β chain andimmunoblotted to visualize phosphotyrosineor GM-CSF receptor β. PIR-B–/– DCs respond-ed poorly to GM-CSF stimulation.

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activity in vivo. We did not observe any significant increase in theexpression of serum IgM nor antibodies to double-stranded DNA, atleast in on a 129/SvJ-C57BL/6 hybrid background in specificpathogen–free conditions. However, once a microbial infection occurs,it is possible that PIR-B may exert its potential role in modulation ofthe humoral response against infection, which is supported by theenhanced immune response of PIR-B–/– mice to TI antigens. This pos-sibility remains to be tested.

We showed here that in BM DCs, PIR-B was constitutively andabundantly tyrosine-phosphorylated in vitro; its phosphorylation wasfurther increased after GM-CSF stimulation. On the other hand, DCsfrom PIR-B–/– mice showed the constitutive phosphorylation of severalother cellular proteins. Possibly in relation to these observations, theinteraction between GM-CSF and its receptor on DCs induced only atransient stimulation of tyrosine phosphorylation of the GM-CSFreceptor common β chain. This perturbation of cellular signaling inPIR-B–deficiency might lead to impaired DC maturation, although wedo not know the detailed mechanism behind this defect. Because Januskinase 2 (Jak2) and signal transducers and activators of transcription 5(STAT5) are important for GM-CSF receptor–mediated signaling45,46, itis probable that the Jak and downstream molecules are constitutivelyactivated in the absence of PIR-B in DCs. Consistent with our findings,it has been reported that in IL-3–dependent BaF/3 cells, the recruitmentof SHP-1 to PIR-B was increased in response to IL-3, which suggestsa functional link between inhibitory receptor and cytokine receptor sig-naling47. IL-3, GM-CSF and IL-5 are cognate cytokines that share thecommon β chain and show functional redundancy48. The involvementof PIR-B in GM-CSF signaling that we show here may indicate that theinhibitory role of PIR-B is crucial in IL-3 and IL-5 functions. It willalso be interesting to determine whether PIR-B could be involved incellular signaling by other cytokines and their receptors, such as IL-2,IL-4 and IL-6. Development and maturation of DCs and skewed TH

responses could be critically regulated by cytokines49. Elucidation ofthe precise mechanism for this impaired maturation of DCs in PIR-Bdeficiency as well as the identification of a physiological ligand for PIRwill provide new insights into the control mechanisms for TH1 and TH2balancing by antigen-presenting cells in vivo.

MethodsGeneration of PIR-B–/– mice. Phage clones spanning the mouse gene encoding PIR-B(Pirb) were isolated from the genomic library of 129/SvJ origin (Stratagene, La Jolla, CA)with a mouse PIR-B cDNA probe13. Each end of the 5.0-kb EcoRV-EcoRI fragments—which contained 5′ upstream sequences, exons 1–7 and the 5′ part of exon 8 of murine Pirbgene—was first converted into the XhoI and SalI sites, respectively, by subcloning intoHincII and EcoRI–digested pBlueSK+ followed by digestion with XbaI and the addition ofSalI, a linker. The resulting 5.0-kb XhoI-SalI fragment was subcloned into the XhoI site ofpMC1-neo-pA– (Stratagene) to generate pMC1-PIRB-neo. A 2.0-kb XhoI-SalI fragmentspanning the 3′ part of exon 10 to sequences 5′ of exon 14 of Pirb was prepared by PCRamplification with a forward primer, 5′-TATCCTCGAGCTTCTCCGACGAAGACATCG-3′ (the XhoI site is underlined), and a backward primer, 5′-AGATCGTCGACTGTTCAGTTGTTCCCTTGAC-3′ (the SalI site is underlined), and a Pirb subclone as atemplate, followed by digestion with XhoI and SalI. The 2.0-kb fragment was ligated intothe SalI site of pMC1-PIRB-neo to generate pMC1-neo-PIRB. The resulting 8.0-kb XhoI-SalI insert was integrated into the XhoI site of pIC19R-MC1tk to give ptkPIRBneo. Thevector was linearized at a unique ClaI site within the polylinker of the plasmid.

The embryonic stem (ES) cell line RW4 (GenomeSystems, Palo Alto, CA) was main-tained on feeder layers from primary embryonic fibroblasts in a 37 °C, 5% CO2 humidifiedincubator. Transfection of ES cells with targeting vector, isolation of homologous recombi-nant clones and procedures for obtaining chimeric mice were as described50. HomologousRW4 ES cell recombinants were obtained at a frequency of 2.2%.

All the experiments were done on 6- to 64-week-old mice. Mice were housed and bredin the Animal Unit of The Institute of Development, Aging and Cancer (Tohoku University,Sendai, Japan)—an environmentally controlled and specific pathogen–free facility—according to guidelines for experimental animals defined by the facility. Animal protocolswere reviewed and approved by the IDAC Animal Studies Committee.

Immunoblot analysis. Splenic B cells (108) were positively selected by magnetic-activat-ed cell sorting (MACS) with B220 beads (Miltenyi Biotec, Bergisch Gladbach, Germany)and lysed. Proteins in the cleared supernatants of cell lysates were separated by SDS-PAGE with a 7.5% gel and transferred onto a polyvinylidene difluoride (Millipore,Bedford, MA) membrane. The membrane was incubated with goat anti–PIR-A/B (SantaCruz Biotech, Santa Cruz, CA) followed by probing with secondary antibody, biotin–don-key anti–goat Ig and horseradish peroxidase (HRP)-streptavidin (Amersham Pharmacia,Buckinghamshire, UK).

Antibodies and flow cytometry. For flow cytometric analysis, we used the followingmAbs: FITC-, phycoerythrin (PE)- or biotin-conjugated anti–mouse IgM (R6-60.2),anti–mouse IgD (11-26), anti–mouse CD3 (145-2C11), anti–mouse CD5 (Ly-1),anti–mouse B220 (RA3-6B2), anti–mouse CD21 (7G6), anti–mouse CD23 (B3B4), anti–c-kit (2B8), anti-CD11b (M1/70), anti-CD11c (HL3), anti-CD80 (16-10A1), anti-CD86(GL1), CD40 (3/23), anti–MHC class II (M5/114.15.2). All antibodies were from BDPharmingen (San Diego, CA). Texas red (TR)-conjugated streptavidin (Caltag, Burlingame,CA) was used to stain biotin-conjugated antibodies. PIR mAb, 6C118, was provided by M.D. Cooper and H. Kubagawa (University of Alabama at Birmingham). Anti–IgM F(ab′)2

was from ICN Laboratories (Costa Mesa, CA). Cell surface staining was done according tostandard techniques, and flow cytometric analysis was done with a FACSCalibur andCellQuest software (Becton Dickenson, Franklin Lakes, NJ). Dead cells were eliminatedfrom the analysis on the basis of propidium iodide–staining.

Proliferation response of B cells. Splenic B220+ cells were purified by MACS (Miltenyi).The cells were activated with soluble F(ab′)2 anti-IgM, anti-CD40 (CD40, BD Pharmingen)or LPS (Sigma, St. Louis, MO). After 48 h of stimulation, proliferation was determined by[3H]thymidine uptake.

Humoral response and T cell proliferation. Each mouse was immunized intraperitoneal-ly either with 100 µg of TNP-Ficoll, 50 µg of TNP-LPS (Sigma) or with 10 µg of TNP-KLH or TNP-OVA (Sigma) in aluminum hydroxide with pertussis toxin (List BiologicalLaboratories, Campbell, CA). Animals injected with TD antigens were boosted at day 56 toelicit the secondary response. Serum antibody titers to TNP were determined by ELISA bymeasuring absorbance at 450 nm. To determine IgE serum concentrations, we used OptEIA(Pharmingen). For T cell proliferation, lymph node cells were collected from wild-type andPIR-B–/– mice after 10 days of primary immunization with TNP-KLH or TNP-OVA. Thecells were activated with either the antigen or concanavalin A (Sigma). After 40 h of stim-ulation, the supernatant was collcted and IL-4 or IFN-γ present in the culture supernatantwas measured by ELISA (BD Pharmingen). T cell proliferation was determined by[3H]thymidine uptake on day 3.

DC maturation and antigen uptake. For BM DCs, cells from wild-type and PIR-B–/– micewere cultured in the presence of recombinant mouse GM-CSF (rGM-CSF, PeproTech,Rocky Hill, NJ), and the medium was changed at day 4. On day 6, the cells expressed MHCclass II, CD40, CD80, CD86 and CD11c and consisted of immature DCs51. To induce mat-uration, 50 µg ml–1 of OVA or 1 µg ml–1 of anti-CD40 were added to the DC culture, 24 hbefore the cells were collected. After stimulation, the culture supernatants were collectedand the amounts of mouse IL12 p70 in the supernatants were measured by ELISA (BDPharmingen); cells were then stained for CD80, CD86 and MHC class II as maturationmarkers and analyzed by flow cytometry. For antigen-uptake by DCs, the cells were incu-bated with 20 µg ml–1 of FITC-conjugated OVA (Molecular Probes, Eugene, OR) at 37 °Cor 4 °C for 3 h, washed and stained with PE–anti-CD11c. Uptake of FITC-OVA by DCs wasassessed by flow cytometry.

For adoptive transfer of antigen-loaded DCs, we first loaded cells with 20 µg ml–1 ofOVA for 3 h, then BM DCs from PIR-B–/– or wild-type mice were adoptively transferredintravenously to nonimmunized wild-type mice (106/mouse, n=3). One week later, thesplenocytes or CD4+ T cells were selected positively by MACS sorting with CD4 beads(Miltenyi) from the cells that were prepared from the mice and cultured for 48 h. The IL-4content in the supernatant was assessed by triplicate ELISA determinations with an OptEIAset (Pharmingen).

Immunoprecipitation analysis. Splenic B cells (2×107 cells) in 1 ml of PBS were incu-bated for 0, 1, 5 or 15 min at 37 °C with 2 µg of the F(ab′)2 fragment of goat anti–mouseIgM. Similarly, BM DCs (2×107 cells), which were cultured without rGM-CSF for 16 h, in1 ml of PBS were incubated for 0, 5, 15 or 30 min at 37 °C with 200 U of rGM-CSF. Cellswere solubilized in lysis buffer (2% NP-40, 20 mM Tris (pH 7.3) 300 mM NaCl and 10 mMEDTA) containing 2 mM sodium vanadate supplemented with proteinase inhibitors.Precleared lysates were sequentially incubated with rabbit anti-Lyn or rabbit anti–GM-CSFreceptor β (both from Santa Cruz) and protein A–conjugated sepharose 4B (Sigma). Totallysates from splenic B cells and BM DCs, or immunoprecipitates, were separated by SDS-PAGE gel, transferred to polyvinylidene difluoride membrane and detected by anti-phos-photyrosine (mAb 4G10 to mouse IgG2b, Upstate Biotechnology, Waltham, MA) and HRP-anti–mouse IgG2b with the electrochemiluminescence system (Amersham Pharmacia).Detected membranes were stripped off by detection antibodies with stripping buffer thatcontained 62.5 mM Tris (pH 6.8), 100 µM 2-mercaptoethanol and 2% SDS and were reblot-ted with appropriate antibodies. These were goat anti-PIR and HRP-donkey anti–goat IgG(Jackson Immunoresearch, West Grove, PA) or rabbit anti-Lyn or rabbit anti–GM-CSFreceptor β and HRP-anti–rabbit IgG (Amersham Pharmacia).

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nature immunology • volume 3 no 6 • june 2002 • http://immunol.nature.com

ARTICLES

548

AcknowledgmentsWe thank M. D. Cooper, H. Kubagawa,A. Kudo, H. Karasuyama, M. Ono and K.Takatsu forreagents and helpful advice and D. Snell for critical reading of the manuscript. Supportedby the CREST Program of Japan Science and Technology Corporation (JST) and a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan(to T. T.).

Competing interests statementThe authors declare that they have no competing financial interests.

Received 14 March 2002; accepted 3 May 2002.

1. Ravetch, J.V. & Lanier, L. L. Immune inhibitory receptors. Science 290, 84–89 (2000).2. Long, E. O. Regulation of immune responses through inhibitory receptors. Annu. Rev. Immunol. 17,

875–904 (1999).3. Cerwenka,A. & Lanier, L. L. Natural killer cells, viruses and cancer. Nature Rev. Immunol. 1, 41–49 (2001).4. Takai,T. & Ono, M.Activating and inhibitory nature of the murine paired Ig-like receptor (PIR) family.

Immunol. Rev. 181, 215–222 (2001).5. Colonna, M. & Samaridis, J. Cloning of immunoglobulin-superfamily members associated with HLA-C

and HLA-B recognition by human natural killer cells. Science 268, 405–408 (1995).6. Wagtmann, N., Rajagopalan, S.,Winter, C. C., Peruzzi, M. & Long, E. O. Killer cell inhibitory receptors

specific for HLA-C and HLA-B identified by direct binding and by functional transfer. Immunity 3,801–809 (1995).

7. Colonna, M. et al. A common inhibitory receptor for major histocompatibility complex class I mole-cules on human lymphoid and myelomonocytic cells. J. Exp. Med. 186, 1809–1818 (1997).

8. Cosman, D. et al. A novel immunoglobulin superfamily receptor for cellular and viral MHC class Imolecules. Immunity 7, 273–282 (1997).

9. Arm, J. P., Nwankwo, C. & Austen, K. F. Molecular identification of a novel family of human Ig super-family members that possess immunoreceptor tyrosine-based inhibition motifs and homology to themouse gp49B1 inhibitory receptor. J. Immunol. 159, 2342–2349 (1997).

10. Meyaard, L. et al. LAIR-1, a novel inhibitory receptor expressed on human mononuclear leukocytes.Immunity 7, 283–290 (1997).

11. Kharitonenkov,A. et al. A family of proteins that inhibit signalling through tyrosine kinase receptors.Nature 386, 181–186 (1997).

12. Arm, J. P. et al. Molecular cloning of gp49, a cell-surface antigen that is preferentially expressed bymouse mast cell progenitors and is a new member of the immunoglobulin superfamily. J. Biol. Chem.266, 15966–15973 (1991).

13. Hayami, K. et al. Molecular cloning of a novel murine cell-surface glycoprotein homologous to killercell inhibitory receptors. J. Biol. Chem. 272, 7320–7327 (1997).

14. Kubagawa, H., Burrows, P. D. & Cooper, M. D.A novel pair of immunoglobulin-like receptorsexpressed by B cells and myeloid cells. Proc. Natl. Acad. Sci. USA 94, 5261–5266 (1997).

15. Wende, H., Colonna, M., Ziegler,A. & Volz,A. Organization of the leukocyte receptor cluster (LRC)on human chromosome 19q13.4. Mammal. Genome 10, 154–160 (1999).

16. Yamashita,Y. et al. Genomic structures and chromosomal location of p91, a novel murine regulatoryreceptor family. J. Biochem. 123, 358–368 (1998).

17. Nakajima, H., Samaridis, J.,Angman, L. & Colonna, M. Human myeloid cells express an activating ILTreceptor (ILT1) that associates with Fc receptor γ-chain. J Immunol. 162, 5–8 (1999).

18. Kubagawa, H. et al. Biochemical nature and cellular distribution of the paired immunoglobulin-likereceptors, PIR-A and PIR-B. J. Exp. Med. 189, 309–317 (1999).

19. Maeda,A., Kurosaki, M. & Kurosaki,T. Paired immunoglobulin-like receptor (PIR)-A is involved in acti-vating mast cells through its association with Fc receptor γ chain. J. Exp. Med. 188, 991–995 (1998).

20. Ono, M.,Yuasa,T., Ra, C. & Takai,T. Stimulatory function of paired immunoglobulin-like receptor-A inmast cell line by associating with subunits common to Fc receptors. J. Biol. Chem. 274,30288–30296 (1999).

21. Lanier, L. L., Corliss, B. C.,Wu, J., Leong, C. & Phillps, J. H. Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 391, 703–707 (1998).

22. Olcese, L. et al. Killer-cell activatory receptors for MHC class I molecules are included in a multi-meric complex expressed by human killer cells. J. Immunol. 158, 5083–5086 (1997).

23. Tomasello, E. et al. Association of signal-regulatory proteins β with KARAP/DAP-12. Eur. J. Immunol.30, 2147–2156 (2000).

24. Dennis, G. Jr, Stephan, R. P., Kubagawa, H. & Cooper, M. D. Characterization of paired Ig-like recep-tors in rats. J. Immunol. 163, 6371–6377 (1999).

25. Dennis, G. Jr, Kubagawa, H. & Cooper, M. D. Paired Ig-like receptor homologs in birds and mammalsshare a common ancestor with mammalian Fc receptors. Proc. Natl. Acad. Sci. USA 94, 13245–13250(2000).

26. Yamashita,Y., Ono, M. & Takai,T. Inhibitory and stimulatory functions of paired Ig-like receptor (PIR)family in RBL-2H3 cells. J. Immunol. 161, 4042–4047 (1998).

27. Bléry, M. et al. The paired Ig-like receptor PIR-B is an inhibitory receptor that recruits the protein-tyrosine phosphatase SHP-1. Proc. Natl. Acad. Sci. USA 95, 2446–2451 (1998).

28. Maeda,A., Kurosaki, M., Ono, M.,Takai,T. & Kurosaki,T. Requirement of SH2-containing protein tyro-sine phosphatases SHP-1 and SHP-2 for paired immunoglobulin-like receptor B (PIR-B)-mediatedinhibitory signal. J. Exp. Med. 187, 1355–1360 (1998).

29. Ho, L. H., Uehara,T., Chen, C.-C., Kubagawa, H. & Cooper, M. D. Constitutive tyrosine phosphoryla-tion of the inhibitory paired Ig-like receptor PIR-B. Proc. Natl. Acad. Sci. USA 96, 15086–15090 (1999).

30. Rojo, S. et al. Natural killer cells and mast cells from gp49B null mutant mice are functional. Mol. Cell.Biol. 20, 7178–7182 (2000).

31. Hardy, R. R. & Hayakawa, K. B cell development pathways. Annu. Rev. Immunol. 19, 595–621 (2001).32. Murakami, M. et al. Antigen-induced apoptotic death of Ly-1 B cells responsible for autoimmune dis-

ease in transgenic mice. Nature 357, 77–80 (1992).33. O’Keefe,T. L.,Williams, G.T., Davies, S. L. & Neuberger, M. S. Hyperresponsive B cells in CD22-defi-

cient mice. Science 274, 798–801 (1996).34. Otipoby, K. L. et al. CD22 regulates thymus-independent responses and the lifespan of B cells. Nature

384, 634–637 (1996).35. Pan, C., Baumgarth, N. & Parnes, J. R. CD72-deficient mice reveal nonredundant roles of CD72 in B

cell development and activation. Immunity 11, 495–506 (1999).36. Daëron, M. et al. The same tyrosine-based inhibition motif, in the intracytoplasmic domain of FcγRIIB,

regulates negatively BCR-,TCR-, and FcR-dependent cell activation. Immunity 3, 635–646 (1995).37. Takai,T., Ono, M., Hikida, M., Ohmori, H. & Ravetch, J.V.Augmented humoral and anaphylactic

responses in FcγRII-deficient mice. Nature 379, 346–349 (1996).38. Magram, J. et al. IL-12-deficient mice are defective in IFNγ production and type 1 cytokine respons-

es. Immunity 4, 471–481 (1996).39. MacDonald,A. S., Straw,A. D., Bauman, B. & Pearce, E. J. CD8– Dendritic cell activation status plays an

integral role in influencing TH2 response development. J. Immunol. 167, 1982–1988 (2001).40. Samaridis, J. & Colonna, M. Cloning of novel immunoglobulin superfamily receptors expressed on

human myeloid and lymphoid cells: structural evidence for new stimulatory and inhibitory pathways.Eur. J. Immunol. 27, 660–665 (1997).

41. Wagtmann, N., Rojo, S., Eichler, E., Mohrenweiser, H. & Long, E. O.A new human gene complexencoding the killer cell inhibitory receptors and related monocyte/macrophage receptors. Curr. Biol.7, 615–618 (1997).

42. Cella, M. et al. A novel inhibitory receptor (ILT3) expressed on monocytes, macrophages, and den-dritic cells involved in antigen processing. J. Exp. Med. 185, 1743–1751 (1997).

43. Borges, L., Hsu, M. L., Fanger, N., Kubin, M. & Cosman, D.A family of human lymphoid and myeloid Ig-like receptors, some of which bind to MHC class I molecules. J. Immunol. 159, 5192–5196 (1997).

44. Maeda,A. et al. Paired immunoglobulin-like receptor-B (PIR-B) inhibits BCR-induced activation of Sykand Btk by SHP-1. Oncogene 18, 2291–2297 (1999).

45. Quelle, F.W. et al. JAK2 associates with the βc chain of the receptor for granulocyte-macrophagecolony-stimulating factor, and its activation requires the membrane-proximal region. Mol. Cell. Biol.14, 4335–4341 (1994).

46. Mul,A. L.,Wakao, H., O’Farrell,A. M., Harada, N. & Miyajima,A. Interleukin-3, granulocyte-macrophagecolony stimulating factor and interleukin-5 transduce signals through two STAT5 homologs. EMBO J.14, 1166–1175 (1995).

47. Wheadon, H., Paling, N. R. D. & Welham M. J. Molecular interactions of SHP1 and SHP2 in IL-3-sig-nalling. Cell. Signal. 14, 219–229 (2002).

48. Miyajima,A., Kitamura,T., Harada, N.,Yokota,T. & Arai, K. Cytokine receptors and signal transduction.Annu. Rev. Immunol. 10, 295–331 (1992).

49. Shortman, K. & Liu,Y.-J. Mouse and human dendritic cell subtypes. Nature Rev. Immunol. 2,151–161 (2002).

50. Takai,T., Li, M., Sylvestre, D., Clynes, R. & Ravetch, J.V. FcR γ chain deletion results in pleiotrophiceffector cell defects. Cell 76, 519–529 (1994).

51. Inaba, K. et al. Generation of large numbers of dendritic cells from mouse bone marrow culturessupplemented with granulocyte-macrophage colony-stimulating factor. J. Exp. Med. 176,1693–1702 (1992).

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