CD22 Is Both a Positive and Negative Regulator of B Lymphocyte Antigen Receptor Signal Transduction: Altered Signaling in CD22-Deficient Mice

CORE Metadata, citation and similar papers at core.ac.uk

Provided by Elsevier - Publisher Connector

Immunity, Vol. 5, 551–562, December, 1996, Copyright 1996 by Cell Press CD22 Is Both a Positive and Negative Regulator of B Lymphocyte Antigen Receptor Signal Transduction: Altered Signaling in CD22-Deficient Mice

Shinichi Sato, Ann S. Miller, Makoto Inaoki, sialic acid–bearing ligands expressed by lymphocytes, Cheryl B. Bock, Paul J. Jansen, Mimi L. K. Tang, neutrophils, monocytes, and erythrocytes, as well as and Thomas F. Tedder* nonhematopoietic cells (Aruffo et al., 1992; Engel et al., Department of Immunology 1993; Law et al., 1995; Powell et al., 1993; Sgroi et al., Duke University Medical Center 1993; Stamenkovic et al., 1991). In vitro, CD22 ligation Durham, North Carolina 27710 regulates B cell function (reviewed by Tedder et al., 1997), and CD22 binding to T cells provides a costimulatory signal (Sgroi et al., 1995; Tuscano et al., 1996a). Summary Thus, CD22 may also regulate B cell migration or cell– cell interactions. B cell activation following antigen receptor cross-link- A positive signaling role for CD22 in B cell function ing can be augmented in vitro by ligation of cell surface is suggested by studies where the addition of CD22 CD22, which associates with the SHP1 protein tyrosine monoclonal antibodies (MAbs) to human B cell cultures phosphatase. The targeted deletion of CD22 in mice stimulates proliferation directly or provides potent co- demonstrated that CD22 differentially regulates anti- stimulation with anti-immunoglobulin antibodies, cyto- gen receptor signaling in resting and antigen-stimu- kines, or CD40 MAbs (Pezzutto et al., 1987; Tuscano et lated B lymphocytes. B cells from CD22-deficient mice al., 1996a). Treatment of B cells with a solid-phase CD22 exhibited the cell surface phenotype and augmented MAb prior to anti-immunoglobulin stimulation enhances intracellular calcium responses characteristic of chron- proliferation and reduces the concentration of anti- ically stimulated B cells, as occurs in SHP1-defective immunoglobulin needed for an equivalent response mice. Thus, CD22 negatively regulates antigen recep- without pretreatment (Doody et al., 1995). Since CD22 tor signaling in the absence of antigen. However, acti- MAb binding also enhances the increase in intracellular calcium flux ([Ca2ϩ]) observed following surface immu- vation of CD22-deficient B lymphocytes by prolonged i noglobulin cross-linking, CD22 must contribute to the IgM cross-linking resulted in modest B cell prolifera- earliest signaling cascades that occur during B cell acti- tion, demonstrating that CD22 positively regulates an- vation (Pezzutto et al., 1988). Tyrosine phosphorylation tigen receptor signaling in the presence of antigen. of CD22 also occurs rapidly following CD22 or antigen receptor ligation (Leprince et al., 1993; Schulte et al., Introduction 1992; Tuscano et al., 1996b), suggesting that these two receptors are closely linked. In fact, a small percentage The balance among humoral immune responses, toler- (0.2%–2%) of surface immunoglobulin may physically ance induction, and the development of autoimmunity associate with CD22 (Leprince et al., 1993; Peaker and is regulated by signaling thresholds established through Neuberger, 1993). Consistent with a costimulatory role B lymphocyte antigen receptors (Goodnow, 1996). for CD22 in augmenting B cell proliferation, a number of These responses are further controlled by an array of effector molecules bind tyrosine-phosphorylated CD22 cell surface and cytoplasmic molecules that regulate via their src homology 2 (SH2) domains: p53/p56lyn, antigen receptor signaling during B lymphocyte devel- p72syk, phosphatidylinositol (PI) 3-kinase, and phospholi- opment and activation. Cell surface CD22 can regulate pase C-␥1 (PLC-␥1) (Law et al., 1996; Leprince et al., signal transduction through B lymphocyte antigen re- 1993; Tuscano et al., 1996b). amino acid cytoplasmic domain of CD22 140ف ceptors in vitro, but its in vivo function remains unde- The fined (reviewed by Law et al., 1994; Tedder et al., 1997). includes six tyrosines that are targets for rapid phos- CD22 is absent from the surface of pre-B cells and newly phorylation following surface immunoglobulin or CD22 emerging immunoglobulin Mϩ (IgMϩ) B cells, is present ligation (Schulte et al., 1992; Wilson et al., 1991). Two at a low density on immature (B220loIgMhi) B cells, and regions of the CD22 cytoplasmic domain are homolo- is fully expressed by mature (B220hiIgDϩ) B cells of the gous to the tyrosine-based activation motif (TAM) (re- bone marrow (Erickson et al., 1996; Symington et al., viewed by Cambier, 1995; Weiss and Littman, 1994). 1982). In the periphery, CD22 is expressed at mature TAMs are found in multiple signaling receptors of immu- levels on all B cell subsets (Erickson et al., 1996). CD22 nological importance, where they serve as platforms for expression increases on mitogen-stimulated B cells, but the docking of p72syk/ZAP-70 kinases and may serve as is lost during plasma cell differentiation. CD22 may platforms for src family tyrosine kinases. The cyto- thereby provide regulatory signals during multiple plasmic domain of mouse CD22 also contains three re- stages of B cell development. gions homologous to the tyrosine-based inhibition motif CD22 is a member of the “sialoadhesin” subclass of (TIM), which provides docking sites for SH2 domains of the immunoglobulin superfamily, whose members func- the SHP1 (previously termed SH-PTP1, PTP1C, SHP, or tion as mammalian lectins (Engel et al., 1993; Kelm et HCP) protein tyrosine phosphatase (Matthews et al., al., 1994; Powell et al., 1993; Sgroi et al., 1993). Although 1992; Plutzky et al., 1992; Shen et al., 1991). TIMs are the actual biological significance of CD22-mediated ad- found in transmembrane proteins involved in terminat- hesion remains unknown, the two amino-terminal immu- ing signal transduction through other receptors, includ- noglobulin domains of CD22 mediate interactions (Engel ing Fc␥RIIB and the inhibitory receptors p58 and p70 of et al., 1995a; Law et al., 1995) with structurally diverse natural killer cells (Burshtyn et al., 1996; Fry et al., 1996). Immunity 552

A negative signaling role for CD22 in B cell function has Table 1). However, there was a reduction in the number been suggested since tyrosine-phosphorylated CD22 of IgMϩB220hi (36%, p < 0.01) and HSAloB220hi (55%, p < associates with SHP1 following surface immunoglobulin 0.001) mature B cells in the bone marrow of CD22Ϫ/Ϫ cross-linking (Campbell and Klinman, 1995; Doody et mice (Figure 2A; Table 1). The number of IgMϩB220ϩ al., 1995; Lankester et al., 1995; Law et al., 1996). While mature B cells in blood was also reduced (66%, p < CD22 is unique in containing both TAMs and TIMs, fur- 0.001) in CD22Ϫ/Ϫ mice (Figure 2B; Table 1). Therefore, ther experiments examining the interactions of CD22 the number of mature B cells that recirculate through with the surface immunoglobulin complex and the role the blood and bone marrow was reduced in CD22Ϫ/Ϫ of CD22-associated SHP1 in regulating antigen receptor mice. signaling are needed to clarify the role of CD22 in regu- Overall, the numbers of spleen IgMϩB220ϩ B cells lating B cell function. found in CD22Ϫ/Ϫ mice and wild-type littermates were In the current study, CD22-deficient mice were gener- similar, although differences in the marginal zone sub- ated to elucidate the function of CD22 in vivo. The population were apparent (Figures 2C and 3A; Table 1). phenotype of the CD22-deficient mice demonstrates The number of marginal zone B cells (IgMhiCD21hi) was p < 0.04) in CD22Ϫ/Ϫ littermates) %50ف that CD22 expression differentially regulates signaling decreased by through the antigen receptor complex in resting and (Figure 3A; Table 1). This was consistent with the number lower %40ف activated B cells. of IgMhiIgDlo or IgMhiCD23Ϫ B cells being (p < 0.03) than in wild-type littermates, since these cells include marginal zone B cells, immature B cells, and Results B-1 B cells (Figure 3A; Table 1). Immunohistochemical analysis of B cell subpopulations within the spleen dem- Generation of CD22-Deficient Mice onstrated that the marginal zone appeared to be slightly CD22-deficient mice were generated by mutation of the wider, with a lower cell density than in CD22Ϫ/Ϫ mice, CD22 gene in the HM1 embryonic stem (ES) cell line although the overall architecture and organization of using homologous recombination (Selfridge et al., 1992). the spleen was similar between CD22Ϫ/Ϫ and wild-type Exons encoding a 5Ј portion of untranslated sequence, littermates (data not shown). The decrease in marginal the mRNA translation start site and leader peptide, and zone B cells may relate to the low number of circulating a portion of the first immunoglobulin domain were re- B cells, since the marginal zone in normal mice contains placed with a neomycin resistance marker that termi- a high proportion of B cells that have recently emigrated nates translation (Figures 1A–1D). Following transforma- from the circulation. The number of CD5ϩB220lo B-1a tion, two ES cell clones had the predicted mutation as cells was normal in CD22Ϫ/Ϫ mice (Table 1). The number determined by Southern blot analysis of restriction of follicular or mantle zone B cells, defined as either endonuclease–digested genomic DNA. Chimeric mice IgMloIgDhi or IgMloCD23ϩ, was slightly increased in CD22- generated from one ES cell clone transmitted the muta- deficient mice (Figure 3A; Table 1). The decrease in tion to their progeny. Mice heterozygous for the targeted circulating B cells and spleen marginal zone B cells allele were mated, and CD22ϩ/Ϫ and CD22Ϫ/Ϫ mice were correlated with higher numbers of B cells (43%, p < 0.01) obtained at the expected Mendelian frequency (Fig- within peripheral lymph nodes of CD22Ϫ/Ϫ mice (Table 1). ure 1E). CD22Ϫ/Ϫ mice were deficient in cell surface There was a 57% increase in the number of CD22 expression as determined by immunofluores- CD5ϩB220lo B-1a cells within the peritoneum, with a cence staining with the CY34 MAb specific for immuno- compensatory decrease in the number of CD5ϪB220hi globulin domains 1 and 2 (Law et al., 1995; van der B-2 cells (Figure 2D; Table 1). Changes in the CD5ϪMac- Merwe et al., 1996) and the NIM-R6 MAb specific for 1lo B-1b population of cells were similar to those ob- membrane-proximal immunoglobulin domains (Law et served for the B-1a population (data not shown). While al., 1995) (Figure 1F). Lymphocytes from CD22ϩ/Ϫ mice these results suggest a general increase in the number reacted with the CY34 MAb with a mean fluorescence of B-1 lineage cells in the peritoneum, these differences intensity that was 59 Ϯ 4% (n ϭ 20) of wild-type lit- were not statistically significant and the number of termates (Figure 1F). Immunohistochemical staining of IgMϩB220ϩ B cells found within the peritoneal cavity of frozen sections of spleens from CD22Ϫ/Ϫ mice with the CD22Ϫ/Ϫ mice was similar to that of wild-type littermates CY34 and NIM-R6 antibodies did not reveal detectable (Table 1). secreted, cell surface, or cytoplasmic CD22 protein The effect of deleting CD22 was dose dependent, (data not shown). Therefore, the CD22Ϫ/Ϫ mice bear a since the changes described above were also observed genuine null mutation. CD22-deficient mice thrived and in hemizygous mice, although the changes were always reproduced as well as their wild-type littermates and did more subtle (Table 1). In all cases, however, there were not present any obvious anatomical or morphological no obvious differences in the size (light scatter proper- abnormalities during the first 4 months of life. ties) of CD22Ϫ/Ϫ B lymphocytes isolated from bone marrow, blood, lymph nodes, or spleen when compared B Cell Development in CD22-Deficient Mice with B lymphocytes from wild-type littermates (data not CD22Ϫ/Ϫ mice had normal frequencies of CD43ϩB220lo shown). There were no detectable changes in the devel- pro-B cells and IgMϪB220lo pro/pre-B cells in the bone opment or number of CD3ϩ, CD4ϩ, or CD8ϩ T lympho- marrow (Figure 2A; Table 1). The frequencies of IgMϩ cytes or myelomonocytic cells in any of the above tis- B220lo B cells were increased in CD22ϩ/Ϫ (26%, p < 0.03) sues or thymus (data not shown). These data therefore and CD22Ϫ/Ϫ mice (42%, p < 0.02), suggesting a small suggest that CD22 plays a modest functional role in the increase in the number of immature B cells (Figure 2A; development and tissue localization of B lymphocytes. CD22 Regulates B Lymphocyte Signaling 553

Figure 1. Targeted Disruption of the CD22 Gene (A) Intron–exon organization of the CD22 gene containing 15 exons based on the results of this study and those of Law et al. (1993). Closed squares indicate untranslated sequence (UT) and open squares indicate translated regions. Domains are indicated: leader (L), immunoglobulin-like domains (Ig), transmembrane domain (TM), and cytoplasmic (Cyto) domains. (B) Restriction map of the targeted region of the CD22 gene. The vertical arrow indicates a HincII restriction site that did not cut efficiently, thereby generating two restriction fragments. (C) Structure of the CD22 targeting vector showing the location of the thymidine kinase (tk) and neomycin-resistance (neor ) genes in the 5Ј to 3Ј orientation. (D) Structure and restriction map of the predicted targeted allele. Gene targeting in ES cells by homologous recombination would result in acquisition of new EcoRI and HincII sites and loss of a BstEII site, as indicated. (E) Southern blot analysis of tail DNA from CD22ϩ/ϩ, CD22ϩ/Ϫ, and CD22Ϫ/Ϫ littermates digested with the listed enzymes and hybridized with the 5Ј probe indicated in (B) and (D). (F) Immunofluorescence analysis of CD22 expression by splenocytes from CD22ϩ/ϩ, CD22ϩ/Ϫ, and CD22Ϫ/Ϫ littermates with flow cytometry analysis (solid lines). Dotted histograms represent immu- nofluorescent staining with unreactive, isotype-matched control MAbs. Secondary antibodies were FITC-conjugated goat anti–

mouse IgG1 antibodies for the CY34 MAb and phycoerythrin-conjugated goat anti–rat IgG antibodies for the NIM-R6 MAb.

CD22 Regulates IgM Expression Levels in the bone marrow, blood, spleen, and lymph nodes B cells from CD22Ϫ/Ϫ mice expressed a cell surface was decreased substantially in CD22Ϫ/Ϫ mice (Figure 2; phenotype that was reminiscent of weakly stimulated B Table 2). The lowest levels of IgM were observed on cells. Cell surface levels of IgM on mature B220hi B cells bone marrow and circulating B cells from CD22Ϫ/Ϫ mice, Immunity 554

Figure 3. B Lymphocyte Subpopulations Present in the Spleen of CD22-Deficient Mice (A) Splenocytes were isolated from wild-type, CD22ϩ/Ϫ, and CD22Ϫ/Ϫ littermates and examined by two-color immunofluorescence stain- Figure 2. Lymphocytes Present in Lymphoid Tissues of CD22-Defi- ing with flow cytometry analysis as described in Figure 2. cient Mice (B) Flow cytometry analysis of MHC class II antigen (I-A) and CD44 / Single cell suspensions of leukocytes were isolated from wild-type, expression on spleen B cells from wild-type (broken lines), CD22ϩ Ϫ Ϫ/Ϫ CD22ϩ/Ϫ, and CD22Ϫ/Ϫ littermates and examined by two-color immu- (thin unbroken line), and CD22 (thick lines) littermates. All results nofluorescence staining with flow cytometry analysis. We analyzed are representative of those obtained with seven 2-month-old mice 10,000 cells with the forward and side light scatter properties of of each genotype. lymphocytes for each sample. Values represent the percentage of the total gated lymphoid cell population that falls into the indicated Ϫ/Ϫ quadrants. Populations of cells lacking surface antigen expression Thus, B cells from CD22 mice characteristically ex- were determined using unreactive isotype-matched MAbs as con- press an IgMloIgDhiCD21/CD35lo-med phenotype with ele- trols. Horizontal broken lines in some histograms are provided for vated levels of class II, CD23, and CD44 reminiscent of reference. These results are representative of those obtained with weakly stimulated B cells. This distinct phenotype is six or seven 2-month-old mice of each genotype. equivalent to that of anergic B cells that have been chronically exposed to soluble autoantigens in vivo (Cooke et al., 1994; Cyster and Goodnow, 1995; Good- .(p < 0.001) less IgM than wild-type B now et al., 1988) %40ف which had cells (Table 2). B cells from CD22ϩ/Ϫ mice expressed 2؉ intermediate surface IgM levels (Table 2). By contrast, Augmented [Ca ]i Response B220loIgMϩ immature B cells in the bone marrow of in CD22-Deficient B Cells CD22Ϫ/Ϫ mice had slightly increased levels of IgM (Fig- The loss of CD22 dramatically altered early B cell signal- ure 2A; Table 2). Cell surface IgD levels on mature B ing responses since splenic B cells from CD22-deficient 2ϩ cells from the bone marrow, blood, and lymph nodes of mice demonstrated more acute increases in [Ca ]i fol- CD22Ϫ/Ϫ mice were also lower, although to a lesser ex- lowing surface IgM cross-linking than B cells from wild- 2ϩ tent than IgM (Table 2). The majority of spleen B cells type littermates (Figure 4A). Differences in [Ca ]i levels from CD22Ϫ/Ϫ mice expressed low to medium levels of were largest when suboptimal concentrations (10 ␮g/ 2ϩ CD21/CD35, indicating that they were mature B cells ml) of anti-IgM antibodies were used, but [Ca ]i levels (data not shown). B cells from CD22-deficient mice ex- in CD22-deficient B cells were also significantly in- pressed levels of major histocompatibility complex creased at every timepoint after 170 s (p < 0.05 in three (MHC) class II antigens (I-A) and CD44 that were signifi- experiments) when spleen B cells were stimulated by cantly higher than wild-type B cells (Figure 3B; Table 2). optimal concentrations (40 ␮g/ml) of anti-IgM antibod- CD23 levels were also higher on B cells from CD22Ϫ/Ϫ ies. B cells from CD22ϩ/Ϫ mice showed intermediate mice (Figure 3A; Table 2). However, CD18 expression responses. Similar results were obtained when CD19 levels and markers of cell activation such as CD86 (B7-2) was cross-linked with optimal concentrations (80 ␮g/ml) Ϫ/Ϫ 2ϩ were not increased in CD22 mice (data not shown). of MAb (Figure 4B), with significantly increased [Ca ]i CD22 Regulates B Lymphocyte Signaling 555

Table 1. Frequency and Numbers of B Lymphocytes in CD22-Deficient Mice

Percentage of Lymphocytes Number of Cells (ϫ 106)a Tissue Phenotype Wild Type CD22ϩ/Ϫ CD22Ϫ/Ϫ Wild Type CD22ϩ/Ϫ CD22Ϫ/Ϫ

Bone marrow CD43ϩB220lo 9 Ϯ 310Ϯ511Ϯ3 B220loIgMϪ 46 Ϯ 739Ϯ541Ϯ12 B220loIgMϩ 19 Ϯ 524Ϯ4* 27 Ϯ 7* B220hiIgMϩ 14 Ϯ 416Ϯ49Ϯ3** B220hiHSAlo 22 Ϯ 625Ϯ610Ϯ4** Blood B220ϩIgMϩ 51 Ϯ 545Ϯ929Ϯ5** 6.7 Ϯ 0.8 6.4 Ϯ 1.4 2.3 Ϯ 1.1** Spleen B220ϩIgMϩ 48 Ϯ 450Ϯ554Ϯ4** 44 Ϯ 10 47 Ϯ 14 43 Ϯ 16 IgMloIgDhi 21 Ϯ 524Ϯ630Ϯ5** 19 Ϯ 623Ϯ924Ϯ9 IgMhiIgDlo 8.0 Ϯ 3.0 7.3 Ϯ 2.1 5.5 Ϯ 0.8* 7.8 Ϯ 4.6 6.8 Ϯ 2.6 4.4 Ϯ 2.1* IgMhiCD21hi 3.0 Ϯ 1.2 2.7 Ϯ 1.5 1.9 Ϯ 0.8* 2.9 Ϯ 1.7 2.5 Ϯ 1.6 1.5 Ϯ 0.9* B220loCD5ϩ 2.0 Ϯ 0.5 2.5 Ϯ0.8 2.6 Ϯ 0.5 2.2 Ϯ 1.0 2.8 Ϯ 2.0 2.6 Ϯ 2.4 Lymph nodes B220ϩIgMϩ 14 Ϯ 317Ϯ420Ϯ5** 0.7 Ϯ 0.2 1.0 Ϯ 0.6 1.0 Ϯ 0.3** Peritoneum B220ϩIgMϩ 59 Ϯ 13 67 Ϯ 10 61 Ϯ 12 1.4 Ϯ 0.7 1.8 Ϯ 1.0 1.6 Ϯ 0.7 B220loCD5ϩ 31 Ϯ 542Ϯ16 49 Ϯ 14** 0.7 Ϯ 0.3 1.1 Ϯ 0.5 1.1 Ϯ 0.7 B220loIgDlo 53 Ϯ 867Ϯ8* 64 Ϯ 9* B220hiCD5Ϫ 37 Ϯ 336Ϯ10 25 Ϯ 7** 0.9 Ϯ 0.4 1.0 Ϯ 0.6 0.6 Ϯ 0.3 B220hiIgDhi 16 Ϯ 213Ϯ712Ϯ6

Values represent mean (Ϯ SD) results obtained from six or seven different 2-month-old mice. Numbers represent the percentage of lymphocytes (based on side and forward light scatter properties) expressing the indicated cell surface markers as determined in Figures 2 and 3 using two-color immunofluorescence staining. a B cell numbers were calculated based on the total number of cells harvested from the indicated tissues. For blood, the values indicate the number of cells per milliliter. Asterisk, the percentage or number of cells was significantly different than in wild-type littermates, p Ͻ 0.05; double asterisk, p Ͻ 0.01, as determined using the paired Student’s t test.

levels at timepoints between 190 and 330 s (p < 0.05 in other causes. By contrast, the proliferation of purified three experiments). However, these differences were B cells from CD22Ϫ/Ϫ mice in response to LPS, CD38, not observed when suboptimal concentrations (40 ␮g/ and CD40 was similar to that of wild-type littermates ml) of anti-CD19 MAb was used. It is unlikely that the over a range of mitogen concentrations (Figure 5). Prolif- small changes in B lymphocyte subpopulations noted eration in response to CD40 ligation plus IL-4 was higher 2ϩ Ϫ/Ϫ earlier could result in such dramatic changes in [Ca ]i in CD22 mice at all CD40 concentrations (112 Ϯ 77% responses. Therefore, the exaggerated calcium re- at 1 ␮g/ml, p < 0.01 in four experiments). Proliferation sponse to surface IgM cross-linking in CD22-deficient at low concentrations of anti-CD38 MAb (plus IL-4) was B cells is likely to result directly from alterations in the also higher (134 Ϯ 59% at 0.1 ␮g/ml, p < 0.002 in four activation or maturation states of these cells or their signaling capacity. Table 2. IgM and IgD Expression Levels in CD22-Deficient Mice

Expression Relative to B Cell Proliferation in CD22-Deficient Mice Wild-Type Mice The effect of CD22 loss on B cell responses to IgM, (percent Ϯ SD) interleukin-4 (IL-4), lipopolysaccharide (LPS), CD38, and ϩ/Ϫ Ϫ/Ϫ CD40 was examined, since each of these mitogenic Antigen Source of B Cells CD22 CD22 signals activates B cells through divergent signaling IgM Bone marrow B220lo 105 Ϯ 26 111 Ϯ 10** cascades. The proliferative response of purified B cells B220hi 89 Ϯ 19 62 Ϯ 12** ϩ from CD22-deficient mice to cross-linking of surface Blood B220 66 Ϯ 12* 57 Ϯ 30* Spleen B220ϩ 97 Ϯ 22 78 Ϯ 16** IgM was significantly lower (45 Ϯ 15% at 20 ␮g/ml, p < Lymph node B220ϩ 96 Ϯ 20 64 Ϯ 14** 0.0005 in four experiments) over a range of antibody IgD Bone marrow IgMlo 95 Ϯ 15 73 Ϯ 5** concentrations (Figure 5). Similar results were obtained Blood IgMϩ 104 Ϯ 783Ϯ9* when the B cells were activated using suboptimal con- Spleen IgMlo 103 Ϯ 12 100 Ϯ 8 centrations of anti-IgM antibodies (5 ␮g/ml) with in- Lymph node IgMϩ 101 Ϯ 18 90 Ϯ 14* lo creasing concentrations of IL-4 added to the cultures CD23 Spleen IgM 138 Ϯ 54* 149 Ϯ 75* Class II Spleen IgMϩ 109 Ϯ 19 146 Ϯ 22** (36 Ϯ 13% at 50 U/ml, p < 0.0007 in four experiments) CD44 Spleen IgMϩ 117 Ϯ 23 139 Ϯ 26** (Figure 5). The decrease in proliferation on day 3 did not result from changes in the kinetics of the maximal Relative cellsurface receptor densities were determined by comparing the channel numbers of the mean linear fluorescence intensity proliferative response (data not shown). Proliferation of between B cells from wild-type and CD22ϩ/Ϫ or CD22Ϫ/Ϫ mice. Val- Ϫ/Ϫ CD22 and wild-type B cells in response to antigen ues represent the mean percentage of wild-type expression levels receptor cross-linking with intact rabbit anti–mouse IgM obtained from five to seven mice of each genotype. All samples antibodies paralleled that obtained with F(abЈ)2 antibod- were stained in parallel and analyzed sequentially by flow cytometry ies (data not shown). This suggests that negative signal- with identical instrument settings as shown in Figures 2 and 3. ing through Fc receptors is not affected by the loss of Asterisk, differences between test and wild-type mice were significant, p Ͻ 0.05; double asterisk, p Ͻ 0.01. CD22 and that the decrease in proliferation results from Immunity 556

Figure 4. Signal Transduction through Sur- face IgM and CD19 in B Cells from CD22- Deficient Mice Splenocytes from wild-type, CD22ϩ/Ϫ, and CD22Ϫ/Ϫ mice were isolated, loaded with indo-1, and stained with FITC-labeled anti- B220 MAb. B cells were examined for relative 2ϩ [Ca ]i levels by flow cytometry after gating on the B220ϩ population of cells. Suboptimal or optimal concentrations of antibodies reactive with IgM (A) or CD19 (B) were added to the cell mixtures at the indicated times 2ϩ (arrows). An increase in [Ca ]i over time is shown as an increase in the ratio of indo-1 fluorescence. Values represent the ratios of fluorescence intensity of cell populations after MAb treatment relative to the fluorescence intensity of untreated cells. These results are representative of those obtained from three littermate pairs of mice.

experiments) in CD22-deficient B cells (Figure 5). Similar 1990). However, following overnight activation with anti- results were obtained in all cases with unfractionated IgM antibodies, B cells from CD22Ϫ/Ϫ mice generated 2ϩ splenocytes (data not shown). Proliferation of B cells acute increases in [Ca ]i levels following surface IgM from CD22ϩ/Ϫ mice varied considerably among mice, but cross-linking, while B cells from wild-type mice did not 2ϩ ranged between that of wild-type and CD22-deficient B generate detectable changes in [Ca ]i levels (Figure 6). cells (data not shown). Therefore, the loss of CD22 had These differences did not result from different densities a selective negative effect on signaling through the anti- of cell surface IgM, since both CD22Ϫ/Ϫ B cells and wild- gen receptor complex, while minimal or positive effects type B cells expressed identical levels of IgM following were observed when the B cells were stimulated with overnight culture (data not shown). Furthermore, anti- other mitogens. body binding to as little as 2%–10% of membrane IgM leads to inactivation of the signal transducing ability of Defective Desensitization of IgM Signaling membrane immunoglobulin (Cambier et al., 1988). B Ϫ/Ϫ 2ϩ in CD22 B Cells cells activated with LPS generated changes in [Ca ]i Since CD22Ϫ/Ϫ mice exhibit a phenotype compatible levels that were similar to those observed for B cells with anergic B cells, it was not clear whether the defec- that had not been activated in culture (Figures 4A and tive proliferative response observed in response to anti- 6). Therefore, even though the antigen receptor complex IgM triggering was a direct consequence of loss of a in CD22Ϫ/Ϫ mice is more efficient at initiating changes CD22 costimulatory function, due to small differences in Ca2ϩ mobilization, signal transduction through the in the activation status of the B cells, or a secondary antigen receptor complex in CD22Ϫ/Ϫ mice was not de- consequence of developmental abnormalities leading to sensitized following surface IgM cross-linking. partial desensitization of the antigen receptor–induced proliferative pathway. This was addressed by examining B Cell Differentiation in Mice that Lack CD22 the ability of membrane IgM ligation to desensitize Ca2ϩ Serum immunoglobulin levels in nonimmunized mice mobilization following Bcell activation. In normal B cells, were determined to assess the influence of the CD22 antigen receptor ligation leads to homologous desensiti- deficiency on B cell differentiation. On average, higher zation of membrane immunoglobulin and heterologous levels of IgM (40%, p < 0.05) and IgA (200%, p < 0.01) desensitization of other cell surface receptors, making were found in CD22-deficient mice (Figure 7). However, them incapable of transducing Ca2ϩ mobilizing signals serum IgG levels of all isotype were not significantly (Lazarus et al., 1991; Rigley et al., 1989; Rijkers et al., affected by the loss of CD22. The elevated serum IgM CD22 Regulates B Lymphocyte Signaling 557

to DNP-KLH that were similar to those of wild-type mice (Table 3). Following secondary challenge with DNP-KLH, germinal centers in the spleens of CD22-deficient mice were similar to those of wild-type littermates in both frequency and size (data not shown). Therefore, CD22 was not required for isotype switching or T cell–B cell interactions in the generation of humoral immune responses.

Discussion

The generation of CD22-deficient mice provides considerable insight into the role of CD22 in vivo and demonstrates directly that CD22 regulates cell surface antigen receptor signaling. Expression levels of cell surface IgM were significantly reduced on mature B cells from the bone marrow, blood, spleen, and lymph nodes of CD22Ϫ/Ϫ mice, but not for immature B cells in the bone marrow (Figure 2; Table 2). Spleen B cells from CD22Ϫ/Ϫ mice were generally IgMloIgDhiCD21/CD35lo-med, with increased expression of MHC class II antigens, CD23, and CD44 (Figure 3; Table 2). Although this distinct phenotype suggests B cell activation, the B cells did not appear to be overtly activated since B cell size was the same as in wild-type mice (data not shown). The IgMlo IgDhiCD21/CD35lo-med phenotype is equivalent to that of anergic B cells that have been chronically exposed to soluble autoantigens in vivo (Cooke et al., 1994; Cyster and Goodnow, 1995; Goodnow et al., 1988). Decreased expression of cell surface IgM is also observed in B cells from mice that overexpress CD19 (S. S. and T. F. T., unpublished data; Engel et al., 1995b; Sato et al., 1995) Figure 5. Proliferation of CD22-Deficient B Cells in Response to and in SHP1-defective B cells from motheaten mice Mitogenic Signals (Cyster and Goodnow, 1995; Pani et al., 1995; Schultz Spleen B cells (2 ϫ 105 per well) from CD22Ϫ/Ϫ mice and wild-type et al., 1993; Sidman et al., 1978; Tsui et al., 1993). Down- littermates were purified by complement-mediated lysis of T cells. regulated surface IgM levels in SHP1-defective moth- B cells were cultured for 72 hr with the indicated amounts of anti- eaten mice is a consequence of augmented transmem- IgM antibodies, LPS, or anti-CD40 MAbs. B cells were also cultured brane signaling through the B cell antigen receptor with anti-IgM antibodies (5 ␮g/ml) plus the indicated concentrations complex (Cyster and Goodnow, 1995; Pani et al., 1995). of IL-4 or IL-4 (100 U/ml) plus the indicated concentrations of anti- Ϫ/Ϫ CD38 or anti-CD40 MAbs. Proliferation was assessed by the incor- The phenotype of mature CD22 B cells may also ex- poration of labeled thymidine (1 ␮Ci per well) added during the last plain their decrease within the bone marrow and circula- 16 hr of culture. Values represent the mean values (Ϯ SD) obtained tion and their commensurate increase within peripheral from triplicate cultures. These results are representative of those lymph nodes (Table 1). Rather than reflecting a defi- obtained in four independent experiments. ciency in maturation, preliminary studies suggest that the activation status of CD22Ϫ/Ϫ B cells promotes their migration into peripheral lymphoid tissues. Therefore, levels in CD22-deficient mice may correlate with the these results demonstrate a strong association between small increase in B-1 cell numbers (Fo¨ rster and Rajew- CD22 function and antigen receptor expression levels sky, 1987). and suggest that B cells from CD22-deficient mice may The influence of CD22 deficiency on the humoral im- be enduring enhanced antigen receptor signaling in the mune response was assessed by immunizing mice with absence of antigen, as occurs in SHP1-defective mice. 2ϩ a T cell–dependent antigen, 2,4-dinitrophenol-conjugated B cells from CD22-deficient mice generated [Ca ]i keyhole limpet hemocyanin (DNP-KLH), a T cell–inde- increases that were dramatically higher than those of pendent type2 antigen, DNP-Ficoll, and a T cell–independ- wild-type littermates in response to cross-linking of sur- 2ϩ ent type 1 antigen, 2,4,6-trinitrophenol (TNP)-conju- face IgM (Figure 4). Exaggerated [Ca ]i responses have gated LPS. Following immunization, CD22-deficient also been observed with SHP1-defective B cells at high mice mounted primary IgM responses that were higher antigen concentrations (Cyster and Goodnow, 1995). than, but not significantly different from, those of wild- However, CD22-deficient B cells appear particularly type mice (Table 3). IgG1, IgG2a, IgG2b, IgG3, and IgA sensitive to antigen receptor signaling because subopti- 2ϩ responses of CD22-deficient mice were not significantly mal antigen receptor cross-linking generated [Ca ]i re- different from those of wild-type littermates. CD22-defi- sponses equivalent to optimal responses in wild-type 2ϩ cient mice generated secondary IgM and IgG responses mice (Figure 4). Since PLC-␥ phosphorylation, [Ca ]i Immunity 558

Figure 6. Antigen Receptor Desensitization in Activated B Cells from CD22-Deficient Mice Splenocytes from wild-type and CD22Ϫ/Ϫ mice were cultured overnight with anti-IgM antibodies or LPS, loaded with indo-1, and stained with FITC-labeled anti-B220 MAb. B 2ϩ cells were examined for relative [Ca ]i levels by flow cytometry after gating on the B220ϩ population of cells as in Figure 4. Anti-IgM antibodies (10 ␮g/ml) were added to the cell mixtures at the indicated times (arrows).

mobilization, and protein kinase C activation in response the absence of CD22 may mimic the absence of SHP1 to antigen receptor ligation are downstream of tyrosine and permit chronic tyrosine phosphorylation of the anti- kinase activation (Hempel et al., 1992; Lane et al., 1991; gen receptor in the absence of antigen. Padeh et al., 1991; Roifman and Wang, 1992), the loss B cell proliferation in CD22-deficient mice was signifi- of CD22 may augment upstream signaling events such cantly diminished in response to IgM ligation over a as antigen receptor or kinase phosphorylation, which in broad range of concentrations (Figure 5). The loss of turn leads to the enhanced mobilization of Ca2ϩ immedi- CD22 appeared specifically to diminish surface IgM– ately following antigen receptor ligation. As a specific induced proliferation, since CD22-deficient B cell re- example, the absence of CD22 may result in augmented sponses to LPS, CD40, and CD38 plus IL-4 were not phosphorylation of PLC-␥1, since CD22 is reported to very different from those of wild-type B cells (Figure 5). serve a central role in the formation of a multimolecular complex of SHP1 with PLC-␥1 and p72syk (Law et al., 1996). Therefore PLC-␥1 phosphorylation may not be negatively regulated by SHP1 in CD22Ϫ/Ϫ B cells. These results emphasize a negative role for CD22 in regulating signaling through the antigen receptor complex. Persistent low level phosphorylation of the antigen receptor complex would explain why B cells from Ϫ/Ϫ 2ϩ CD22 mice generated augmented [Ca ]i responses and why CD22-deficient B cells manifest a phenotype that occurs in B cells chronically exposed to soluble autoantigens in vivo (Cooke et al., 1994; Cyster and Goodnow, 1995; Goodnow et al., 1988). Protein tyrosine kinases that associate with the antigen receptor complex in resting B cells are likely to tyrosine phosphorylate antigen receptor subunits at low levels in the absence of antigen (Burkhardt et al., 1991; Hutchcroft et al., 1992; Yamanashi et al., 1993). ZAP-70 in T cells is also associated with constitutively tyrosine-phosphorylated T cell antigen receptor ␨ chains in vivo (van Oers et al., 1993, 1994). In resting B cells, SHP1 normally inhibits this process and maintains a tyrosine-dephosphorylated antigen receptor complex (Pani et al., 1995). Enzymatically active SHP1 physically and functionally associates with the B cell antigen receptor complex in resting B cells through interactions that are not mediated by SHP1 SH2 domains binding phosphotyrosines of the antigen receptor complex (Pani et al., 1995). A small portion of CD22 also physically associates with the B cell antigen receptor complex, which may be rapidly phosphorylated following antigen receptor signaling (Leprince et al., 1993; Peaker and Neuberger, 1993). Therefore, it is rea- sonable to postulate that spontaneous activation of the Figure 7. Serum Immunoglobulin Levels of 2-Month-Old CD22ϩ/ϩ, B cell antigen receptor complex results in low level tyro- CD22ϩ/Ϫ, and CD22Ϫ/Ϫ Mice sine phosphorylation of proximal CD22 molecules. SHP1 CD22ϩ/ϩ mice are indicated by open circles, CD22ϩ/Ϫ mice by binds to phosphorylated CD22, thereby maintaining an shaded circles, and CD22Ϫ/Ϫ by closed circles. Immunoglobulin lev- enzymatically active phosphatase within the antigen re- els of eight paired littermates were determined by isotype-specific ceptor complex of resting B cells. In CD22Ϫ/Ϫ B cells, ELISA. Bars represent mean serum immunoglobulin levels. CD22 Regulates B Lymphocyte Signaling 559

Table 3. Humoral Immune Responses in CD22-Deficient Mice

Relative Antibody Level (OD Ϯ SEM) Immunoglobulin Mouse 405 Antigen Isotype Genotype Day 0 Day 7 Day 14 Day 28a

TNP-LPS IgM Wild type 26 Ϯ 4 565 Ϯ 117 263 Ϯ 62 CD22ϩ/Ϫ 17 Ϯ 4 600 Ϯ 175 316 Ϯ 109 CD22Ϫ/Ϫ 28 Ϯ 4 633 Ϯ 76 325 Ϯ 44 DNP-Ficoll IgM Wild type 35 Ϯ 8 629 Ϯ 237 696 Ϯ 295 CD22ϩ/Ϫ 17 Ϯ 2 1016 Ϯ 243 753 Ϯ 198 CD22Ϫ/Ϫ 43 Ϯ 6 815 Ϯ 109 905 Ϯ 169

IgG1 Wild type 2 Ϯ 4 126 Ϯ 73 513 Ϯ 325 CD22ϩ/Ϫ 2 Ϯ 2 358 Ϯ 112 763 Ϯ 247 CD22Ϫ/Ϫ 1 Ϯ 2 132 Ϯ 34 426 Ϯ 141 DNP-KLH IgM Wild type 10 Ϯ 1 457 Ϯ 83 501 Ϯ 112 646 Ϯ 135 CD22ϩ/Ϫ 18 Ϯ 9 646 Ϯ 141 785 Ϯ 125 996 Ϯ 112 CD22Ϫ/Ϫ 20 Ϯ 5 559 Ϯ 126 537 Ϯ 158 999 Ϯ 92

IgG1 Wild type 8 Ϯ 2 758 Ϯ 104 1091 Ϯ 27 1415 Ϯ 29 CD22ϩ/Ϫ 9 Ϯ 2 717 Ϯ 59 1058 Ϯ 34 1376 Ϯ 44 CD22Ϫ/Ϫ 4 Ϯ 3 528 Ϯ 115 994 Ϯ 41 1348 Ϯ 19 Relative antibody levels of 2-month-old mice were determined by isotype-specific ELISA. Values represent mean serum antibody levels from four to six paired littermates. These results are representative of those obtained for IgG2a, IgG2b, IgG3, and IgA in immunization with DNP-Ficoll and DNP-KLH and are representative of those obtained for IgG1, IgG2a, IgG2b, IgG3, and IgA for immunization with TNP-LPS. a Mice immunized with DNP-KLH were challenged with antigen again on day 21 so that secondary antibody responses could be assessed on day 28.

Likewise, the overnight activation of CD22Ϫ/Ϫ B cells could be correct, since the presence of multiple phos- through ligation of cell surface IgM did not result in anti- phorylated tyrosine residues as well as TAM and TIM gen receptor desensitization, while signaling through motifs in CD22 suggests that CD22 intersects multiple IgM on wild-type B cells was completely desensitized signaling cascades with the overall outcome depending (Figure 6). These two defects in CD22Ϫ/Ϫ B cell function on the relative strengthof individual signaling pathways. may result from common causes, since both receptor The current studies demonstrate that CD22 function desensitization and proliferation are downstream of ty- is closely linked with antigen receptor expression and rosine kinase activation (Brunswick et al., 1994). These signaling and that CD22 may control basal signal trans- results demonstrate a role for CD22 in the positive regu- duction thresholds initiated through the B lymphocyte lation of signaling events through the antigen receptor in antigen receptor in resting B cells and during B cell response to persistent ligation by antigen. By contrast, proliferation in response to antigen. These studies also SHP1-defective B cells are hyper-responsive to subopti- suggest that CD22 and SHP1 function through overlap- mal concentrations of mitogenic anti-IgM antibodies, ping pathways to regulate B cell signaling. Based on but proliferate normally in response to LPS stimulation the current studies, inherited polymorphisms in CD22 (Pani et al., 1995). This suggests that CD22 and SHP1 or other linked regulatory molecules may vary the strin- play a different role following antigen receptor ligation. gency of tolerance or response thresholds in outbred Antigen receptor ligation disrupts the association be- human populations. This may also be a particularly im- tween SHP1 and the antigen receptor complex, while portant factor for the proliferative capacity of human B inducing tyrosine phosphorylation of SHP1 as well as a cell malignancies that display considerable hetero- large portion of cellular CD22 (Pani et al., 1995; Schulte geneity in expression of CD22. CD22 is only weakly et al., 1992). Two closely related models could therefore expressed by B lineage chronic lymphocytic leuke- explain diminished signaling in CD22-deficient B cells mias, but is expressed by a large percentage of B lin- in response to prolonged incubation with anti-immuno- eage lymphomas and leukemias (Do¨ rken et al., 1986; globulin antibodies. It is possible that SHP1 is recruited Schwarting et al., 1985). In fact, CD22 expression on away from the antigenreceptor by phosphorylated CD22 hairy cells is markedly increased when compared with molecules not associated with the antigen receptor normal B cells (Do¨ rken et al., 1986; Schwarting et al., complex. In CD22-deficient B cells, SHP1 would remain 1985). Thus, alterations in CD22 function or expression associated with the B cell antigen receptor and down- could contribute substantially to autoimmunity or other regulate tyrosine phosphorylation. Alternatively, CD22 diseases in which B cell function is dysregulated. may function as a costimulatory molecule that functions in close and specific association with the antigen recep- Experimental Procedures tor complex. When strong positive signals are generated Production of CD22-Deficient Mice through the antigen receptor complex, positive costimu- DNA encoding the CD22 gene was isolated from a genomic library latory interactions among CD22, surface IgM, p72syk, derived from 129/Sv strain mice using a cDNA probe that encoded p53/p56lyn, PI 3-kinase, and PLC-␥1 may result in prolif- the first two immunoglobulin domains of CD22 as previously de- erative signals that surpass the negative regulatory ef- scribed (Zhou et al., 1991). Fragments of genomic DNA containing the 5Ј end of the CD22 gene were mapped for restriction endonucle- fects of SHP1. This mechanism is consistent with the ase sites, and exons were localized (Figure 1A) as described (Law observation that ligation of CD22 alone can induce B et al., 1993). The targeting vector was constructed using a pBlue- cell proliferation (Tuscano et al., 1996a). Both models script SK (Stratagene, La Jolla, CA) based targeting vector (p594; Immunity 560

provided by Dr. D. Milstone, Brigham and Women’s Hospital, Bos- erythrocytes were lysed after staining using the Coulter whole blood ton, MA). Fragments of genomic DNA containing the CD22 gene Immuno-Lyse kit as detailed by the manufacturer (Coulter, Inc., and flanking sequences were sequentially inserted into the targeting Miami, FL). Cells were washed and analyzed on a FACScan flow vector flanking the neomycin resistance marker from pGK–neo cytometer (Becton Dickinson, San Jose, CA). Cells with the forward poly(A) (Stratagene) that contained the PGK promoter and poly(A) and side light scatter properties of mononuclear cells were analyzed signal sequence. The 5Ј end of the targeting vector was generated for each sample with fluorescence intensity shown on a four-decade from a 1.4 kb HindIII fragment of DNA that contained the 5Ј end of log scale. Fluorescence contours are shown as 50% log density exon 1 and upstream DNA that was inserted proximal to the pMC1- plots. Positive and negative populations of cells were determined HSV–TK gene (Figure 3C). The 3Ј end of the targeting vector was using unreactive isotype-matched MAbs (Coulter, Inc.) as controls obtained from a 3.2 kb fragment of DNA that started at the HincII for background staining. Background levels of staining were deline- site in exon 4 that encodes the first immunoglobulin domain and ated using gates positioned to include 98% of the control cells. also contained exon 5. The vector was linearized using a unique

KpnI restriction site that flanked the 3Ј end of the CD22 gene insert. 2؉ Measurement of [Ca ]i HM-1 ES cells (provided by Dr. D. W. Melton, The University of Spleen cells were isolated at room temperature, washed, and resus- Edinburgh, United Kingdom) were transfected with linearized plas- pended at 107 per milliliter in RPMI 1640 medium containing 5% mid DNA and selected for G418 resistance as described (Selfridge (v/v) bovine calf serum, 10 mM HEPES (all from Sigma, St. Louis, et al., 1992). Genomic DNA from individual selected clones was MO) and loaded with 1 ␮M indo-1-AM ester (Molecular Probes, digested with EcoRI plus KpnI and hybridized with a 900 bp probe Eugene, OR) at 37ЊC for 30 min. The cells were washed and stained external to the targeting vector to screen for homologous recombi- with FITC-conjugated anti-B220 MAb for 30 min at room tempera- nation as described (Zhou et al., 1994). A DNA restriction fragment ture, washed, and resuspended at 2 ϫ 106 cells per milliliter. For of 10 kb corresponded to the wild-type allele, while a new 4.8 kb analysis, the ratio of fluorescence (525/405 nm) of B220ϩ cells (1.5 ϫ fragment resulted from the targeted allele. Two clones containing 106 cells in 0.75 ml samples) was determined using a FACStar flow both alleles were identified out of 1025 G418-resistant ES colonies cytometer (Becton Dickinson). Baseline fluorescence ratios were screened. The structure of the targeted allele was further analyzed collected for 1 min before specific MAbs were added. The final by HincII and BstEII digestion using the 5Ј flanking probe. DNA concentrations of antibodies were as follows: MB19-1, 40 or 80 ␮g/ restriction fragments of 9.6 and 5.4 kb corresponded to the wild- ml; affinity-purified goat F(abЈ)2 antibody fragments to mouse IgM, type HincII allele because of an inefficiently cut HincII site within 10 or 40 ␮g/ml (Cappel, Durham, NC). Fluorescence ratios were exon 4, while a new 3.3 kb fragment resulted from the targeted collected at real time for 7 min following addition of antibody. Results allele. DNA restriction fragments of 9.4 and 2.6 kb corresponded to were plotted as the fluorescence ratio at 20 s intervals with the the wild-type BstEII allele, while 9.4 and 9.6 kb fragments resulted background subtracted. An increase in the fluorescence ratio indi- from the targeted allele. The Southern blot pattern obtained was 2ϩ 2ϩ cates an increase in [Ca ]i. Changes in [Ca ]i levels were also consistent with the appropriate predicted mutation indicating that measured in B cells cultured overnight in culturemedium (RPMI1640 detrimental recombinations did not occur in the vicinity of the de- medium containing 10% fetal bovine serum, 10 mM L-glutamine, sired homologous recombination. Targeted ES cell clones were in- antibiotics, 55 ␮M mercaptoethanol) with 20 ␮g/ml LPS or 20 ␮g/ jected into 3.5-day-old C57BL/6 blastocysts, which were then trans- ml goat F(abЈ)2 anti–mouse IgM antibodies. Cells were washed and ferred to foster mothers. Chimeric mice from one ES cell clone 2ϩ loaded with indo-1-AM ester, and changes in [Ca ]i were measured transmitted the mutation to their progeny. Mice carrying the mutant after treating the cells with 10 ␮g/ml goat F(abЈ)2 anti–mouse IgM CD22 allele were intercrossed and the genotype of the offspring antibody. was determined by Southern blot analysis of DNA obtained from tail biopsies. B Cell Proliferation Spleen B cells were purified (>98% B220ϩ) by complement-medi- Antibodies and Immunofluorescence Analysis ated lysis of T cells with anti–T cell receptor (H57-597; provided by Antibodies used in this study included the following: anti-CD22 Dr. R. Kubo, Denver, CO), anti-Thy1.2 (HO-13-4, ATCC clone TIB99), (CY34.1.2; American Type Culture Collection [ATCC], Bethesda, MD; andanti-CD3 MAbs. B cells werecultured in0.2 ml of culture medium clone TIB163; Symington et al., 1982); anti-CD22 (NIM-R6; provided in 96-well flat-bottom tissue culture plates with LPS (E. coli serotype by Dr. M. Parkhouse, Pirbright, United Kingdom); anti-CD19 0111:B4; Sigma) as indicated for 72 hr. B cells were also stimulated (MB19-1, IgA; Sato et al., 1996); biotinylatedor fluorescein isothiocy- with F(abЈ)2 fragments of goat anti–mouse IgM antibodies (Cappel) anate (FITC)-conjugated anti-B220 (CD45RA, RA3-6B2; Coffman either alone or in the presence of recombinant mouse IL-4 (100 U/ml; and Weissman, 1981); anti-I-A (M5/114.15.2; ATCC clone TIB120); Pharmingen), anti-CD38 MAb (NIMR5; provided by Dr. M. Howard, anti-CD21/CD35 (7E9; provided by Dr. T. Kinoshita, Osaka, Japan); DNAX Corp., Palo Alto, CA) in the presence of IL-4 (100 U/ml), anti- anti-heat-stable antigen (M1/69; Pharmingen, San Diego, CA); anti- CD40 MAb (1C10; provided by Dr. M. Howard) either alone or in CD43 (S7; Pharmingen); anti-CD18 (C71/16; Pharmingen); anti-CD44 the presence of IL-4 (100 U/ml). Proliferation was assessed by the (IM7.8.1; Caltag, South San Francisco, CA); anti-CD5 (53-7.313; incorporation of 3H-labeled thymidine (1 ␮Ci per well) added during ATCC clone TIB104); anti-CD23 (B3B4; provided by Dr. D. Conrad, the last 16 hr of culture, followed by scintillation counting. All treat- Virginia Commonwealth University, Richmond, VA); anti-B7-2 (GL1; ments were carried out in triplicate cultures. Pharmingen); anti-Syndecan-1 (281-2; Pharmingen); anti-Mac-1 (M1/70.15; Caltag);anti-CD3 (145-2C11; ATCC clone CRL1975); anti- CD4 (H129.19; Pharmingen); anti-CD8a (53-6.7; Pharmingen); and Immunization of Mice anti–mouse IgD allotype b (Southern Biotechnology Associates Inc., We intraperitoneally immunized 2-month-old mice with 100 ␮gof DNP-KLH (Calbiochem-Novabiochem Corp., La Jolla, CA) in com- Birmingham, AL) MAbs. Phycoerythrin-conjugated goat F(abЈ)2 anti– rat IgG antibodies absorbed with mouse immunoglobulin (GIBCO plete Freund’s adjuvant and boosted them 21 days later. Also, mice were immunized intraperitoneally with 25 ␮g of DNP-Ficoll (Bio- BRL, Gaithersburg, MD); FITC-conjugated goat anti–mouse IgG1 (Fisher Scientific, Fair Lawn, NJ); and goat anti–mouse IgM isotype– search Technologies, San Rafael, CA) or 50 ␮g of TNP-LPS (Sigma) specific antibodies (Southern Biotechnology Associates Inc.) were in saline. Mice were bled before and after immunization. also used. Phycoerythrin-conjugated streptavidin (Fisher Scientific) was used to reveal biotin-coupled MAb staining. Mouse Immunoglobulin Isotype–Specific ELISAs Single cell suspensions of lymphocytes from the spleen, bone Enzyme-linked immunosorbent assays (ELISAs) were carried out as marrow, peripheral lymph nodes, thymus, and peritonealcavity were described previously using affinity-purified mouse IgM, IgG1, IgG2a, isolated from mice and counted using a hemocytometer prior to IgG2b, IgG3, and IgA MAbs (Southern Biotechnology Associates, Inc.) two-color immunofluorescence analysis. Retroorbital venous plexus to generate a standard curve (Engel et al., 1995b; Sato et al., 1995). puncture was utilized to obtain blood. Leukocytes (0.5 ϫ 106 to 1 ϫ The relative concentration of immunoglobulin in individual samples 106) were stained at 4ЊCusing predeterminedoptimal concentrations was calculated by comparing the mean OD obtained for triplicate of the test MAb for 20 min as described (Zhou et al., 1994). Blood wells to a semi-log standard curve of titrated standard antibody CD22 Regulates B Lymphocyte Signaling 561

using linear regression analysis. DNP- or TNP-specific antibody ti- Kiesel, S., and Nadler, L.M. (1986). HD39 (B3), a B lineage-restricted ters of sera were measured as above, except sample sera were antigen whose cell surface expression is limited to resting and acti- added to ELISA plates coated with DNP–bovine serum albumin (5 vated human B lymphocytes. J. Immunol. 136, 4470–4479. ␮g/ml; Calbiochem-Novabiochem Corp.) or TNP–bovine serum al- Engel, P., Nojima, Y., Rothstein, D., Zhou, L.-J., Wilson, G.L., Kehrl, bumin (Biosearch). J.H., and Tedder, T.F. (1993). The same epitope on CD22 of B lymphocytes mediates the adhesion of erythrocytes, T and B lympho- Statistical Analysis cytes, neutrophils and monocytes. J. Immunol. 150, 4719–4732. All data are shown as mean values Ϯ SD except when otherwise Engel, P., Wagner, N., Miller, A., and Tedder, T.F. (1995a). Identifica- indicated. The Student’s t test was used to determine the level of tion of the ligand binding domains of CD22, a member of the immu- significance of differences in sample means. The paired Student’s noglobulin superfamily that uniquely binds a sialic acid-dependent t test was used to compare mean fluorescence channel numbers ligand. J. Exp. Med. 181, 1581–1586. for gated populations of cells analyzed by flow cytometry. Pairs Engel, P., Zhou, L.-J., Ord, D.C., Sato, S., Koller, B., and Tedder, were between samples from different mice that were stained in T.F. (1995b). Abnormal B lymphocyte development, activation and parallel and analyzed sequentially using the same flow cytometer differentiation in mice that lack or overexpress the CD19 signal parameter settings. transduction molecule. Immunity 3, 39–50.

Acknowledgments Erickson, L.D., Tygrett, L.T., Bhatia, S.K., Grabstein, K.H., and Waldschmidt, T.J. (1996). Differential expression of CD22 (Lyb8) on murine B cells. Int. Immunol. 8, 1121–1129. Correspondence should be addressed to T. F. T. We thank Drs. A. Abbas, Y. Zhuang, J. Kehrl, J. Tuscano, P. Engel, D. Steeber, M. Fo¨ rster, I., andRajewsky, K. (1987). Expansion andfunctional activity Howard, D. Milstone, D. W. Melton, T. Waldschmidt, M. Parkhouse, of Ly-1ϩ B cells upon transfer of peritoneal cells into allotype-con- R. Kubo, T. Kinoshita, and R. Coffman for providing reagents or genic, newborn mice. Eur. J. Immunol. 17, 521–528. helpful discussions. This work was supported by National Institutes Fry, A.M., Lanier, L.L., and Weiss, A. (1996). Phosphotyrosines in of Health grants AI-26872, HL-50985, and CA-54464. T. F. T. is a the killer cell inhibitory receptor motif of NKB1 are required for Stohlman Scholar of the Leukemia Society of America. negative signaling and for association with protein tyrosine phosphatase 1C. J. Exp. Med. 184, 295–300. Received September 10, 1996; revised October 29, 1996. Goodnow, C.C. (1996). Balancing immunity and tolerance: deleting and tuning lymphocyte repertoires. Proc. Natl. Acad. Sci. USA 93, References 2264–2271. Goodnow, C.C., Crosbie, J., Adelstein, S., Lavoie, T.B., Smith-Gill, Aruffo, A., Kanner, S.B., Sgroi, D., Ledbetter, J.A., and Stamenkovic, S.J., Brink, R.A., Pritchard-Briscoe, H., Wotherspoon, J.S., Loblay, I. (1992). CD22-mediated stimulation of T cells regulates T-cell re- R.H., Raphael, K.,Trent, R.J., and Basten, A. (1988). Altered immuno- ceptor/CD3-induced signaling. Proc. Natl. Acad. Sci. USA 89, globulin expression and functional silencing of self-reactive B lym- 10242–10246. phocytes in transgenic mice. Nature 334, 676–682. Brunswick, M., June, C.H., and Mond, J.J. (1994). B lymphocyte Hempel, W.M., Schatzman, R.C., and DeFranco, A.L. (1992). Tyro- immunoglobulin receptor desensitization is downstream of tyrosine sine phosphorylation of phospholipase C-␥2 upon cross-linking of kinase activation. Cell. Immunol. 156, 240–244. membrane Ig on murine B lymphocytes. J. Immunol. 148, 3021–3027. Burkhardt, A.L., Brunswick, M., Bolen, J.B., and Mond, J.J. (1991). Hutchcroft, J.E., Harrison, M.L., and Geahlen, R.L. (1992). Associa- Anti-immunoglobulin stimulation of B lymphocytes activates src- tion of the 72 kDa protein-tyrosine kinase PTK72 with the B cell related protein-tyrosine kinases. Proc. Natl. Acad. Sci. USA 88, antigen receptor. J. Biol. Chem. 267, 8613–8619. 7410–7414. Kelm, S., Pelz, A., Schauer, R., Filbin, M.T., Tang, S., de Bellard, Burshtyn, D.N., Scharenberg, A.M., Wagtmann, N., Rajagopalan, S., M.-E., Schnaar, R.L., Mahoney, J.A., Hartnell, A., Bradfield, P., and Berrada, K., Yi, T., Kinet, J.-P., and Long, E.O. (1996). Recruitment Crocker, P.R. (1994). Sialoadhesin, myelin-associated glycoprotein of tyrosine phosphatase HCP by the killer cell inhibitory receptor. and CD22 define a new family of sialic acid-dependent adhesion Immunity 4, 77–85. molecules of the immunoglobulin superfamily. Curr. Biol. 4, 965–972. Cambier, J.C. (1995). New nomenclature for the Reth motif (or ARH1/ Lane, P.J.L., Ledbetter, J.A., McConnel, F.M., Draves, K., Deans, J., TAM/ARAM/YXXL). Immunol. Today 16, 110. Schieven, G.L., and Clark, E.A. (1991). The role of tyrosine phosphor- Cambier, J., Chen, Z.Z., Pasternak, J., Ransom, J., Sandoval, V., ylation in signal transduction through surface Ig in human B-cells. and Pickles, H. (1988). Ligand-induced desensitization of B-cell J. Immunol. 146, 715–722. 2ϩ membrane immunoglobulin-mediated Ca mobilization and protein Lankester, A.C., van Schijndel, G.M., and van Lier, R.A. (1995). He- kinase C translocation. Proc. Natl. Acad. Sci. USA 85, 6493–6497. matopoietic cell phosphatase is recruited to CD22 following B cell Campbell, M.A., and Klinman, N.R. (1995). Phosphotyrosine-depen- antigen receptor ligation. J. Biol. Chem. 270, 20305–20308. dent association between CD22 and protein tyrosine phosphatase Law, C.-L., Torres, R.M., Sundberg, H.A., Parkhouse, R.M.E., Bran- 1C. Eur. J. Immunol. 25, 1573–1579. nan, C.I., Copeland, N.G., Jenkins, N.A., and Clark, E.A. (1993). Orga- Coffman, R.L., and Weissman, I.L. (1981). B220: a B cell specific nization of the murine Cd22 locus: mapping to chromosome 7 and member of the T200 glycoprotein family. Nature 289, 681–685. characterization of two alleles. J. Immunol. 151, 175–187. Cooke, M.P., Heath, A.W., Shokat, K.M., Zeng, Y., Finkelman, F.D., Law, C.-L., Sidorenko, S.P., and Clark, E.A. (1994). Regulation of Linsley, P.S., Howard, M., and Goodnow, C.C. (1994). Immunoglobu- lymphocyte activation by the cell-surface molecule CD22. Immunol. lin signal transduction guides the specificity of B cell-T cell interac- Today 15, 442–449. tions and is blocked in tolerant self-reactive B cells. J. Exp. Med. Law, C.-L., Aruffo, A., Chandran, K.A., Doty, R.T., and Clark, E.A. 179, 425–438. (1995). Ig domains 1 and 2 of murine CD22 constitute the ligand- Cyster, J.G., and Goodnow, C.C. (1995). Protein tyrosine phospha- binding domain and bind multiple sialylated ligands expressed on tase 1C negatively regulates antigen receptor signaling in B lympho- B and T cells. J. Immunol. 155, 3368–3376. cytes and determines thresholds for negative selection. Immunity Law, C.-L., Sidorenko, S.P., Chandran, K.A., Zhao, Z., Shen, S.-H., 2, 13–24. Fischer, E.H., and Clark, E.A. (1996). CD22 associates with protein

Doody, G.M., Justement, L.B., Delibrias, C.C., Mathews, R.J., Lin, tyrosine phosphatase 1C, Syk, and phospholipase C-␥1 upon B cell J., Thomas, M.L., and Fearon, D.T. (1995). A role in B cell activation activation. J. Exp. Med. 183, 547–560. for CD22 and the protein tyrosine phosphatase SHP. Science 269, Lazarus, A.H., Mills, G.B., Crow, A.R., and Delovitch, T.L. (1991). 242–244. Antigen-induced Fc receptor-dependent and -independent B cell 2ϩ Do¨ rken, B., Moldenhauer, G., Pezzutto, A., Schwartz, R., Feller, A., desensitization: an elevation in [Ca ]i is not sufficient and protein Immunity 562

kinase C activation is not required for these pathways of surface Sgroi, D., Koretzky, G.A., and Stamenkovic, I. (1995). Regulation of IgM-mediated desensitization. J. Immunol. 147, 1739–1745. CD45 engagement by the B-cell receptor CD22. Proc. Natl. Acad. Leprince, C., Draves, K.E., Geahlen, R.L., Ledbetter, J.A., and Clark, Sci. USA 92, 4026–4030. E.A. (1993). CD22 associates with the human surface IgM-B cell Shen, S.H., Bastien, L., Posner, B.I., and Chretien, P. (1991). A pro- antigen receptor complex. Proc. Natl. Acad. Sci. USA 90,3236–3240. tein-tyrosine phosphatase with sequence similarity to the SH2 do- Matthews, R.J., Bowne, D.B., Flores, E., and Thomas, M.L. (1992). main of the protein-tyrosine kinases. Nature 352, 736–739. Characterization of hematopoietic intracellular protein tyrosine Sidman, C.L., Shultz, L.D., and Unanue, E.R. (1978). The mouse phosphatases: description of a phosphatase containing an SH2 do- mutant “Motheaten”: functional studies of the immune system. J. main and another enriched in proline-, glutamic acid-, serine-, and Immunol. 121, 2399–2404. threonine-rich sequences. Mol. Cell. Biol. 12, 2396–2405. Stamenkovic, I., Sgroi, D., Aruffo, A., Sy, M.S., and Anderson, T. Padeh, S., Levitzki, A., Gazit, A., Mills, G.B., and Roifman, C.M. (1991). The B lymphocyte adhesion molecule CD22 interacts with (1991). Activation of phospholipase C in human B cells is dependent leukocyte common antigen CD45RO on T cells and ␣2,6 sialyltrans- on tyrosine phosphorylation. J. Clin. Invest. 87, 1114–1118. ferase, CD75, on B cells. Cell 66, 1133–1144. Pani, G., Kozlowski, M., Cambier, J.C., Mills, G.B., and Siminovitch, Symington, F.W., Subbarao, B., Mosier, D.E., and Sprent, J. (1982). K.A. (1995). Identification of the tyrosine phosphatase PTP1C as a Lyb-8.2: a new B cell antigen defined and characterized with a B cell antigen receptor-associated protein involved in the regulation monoclonal antibody. Immunogenetics 16, 381–391. of B cell signaling. J. Exp. Med. 181, 2077–2084. Tedder, T.F., Tuscano, J., Sato, S., and Kehrl, J.H. (1997). CD22, a Peaker, C.J.G., and Neuberger, M.S. (1993). Association of CD22 B lymphocyte-specific adhesion molecule that regulates antigen with the B cell antigen receptor. Eur. J. Immunol. 23, 1358–1363. receptor signaling. Annu. Rev. Immunol. 15, 481–504. Pezzutto, A., Do¨ rken, B., Moldenhauer, G., and Clark, E.A. (1987). Tsui, H.W., Siminovitch, K.A., de Souza, L., and Tsui, F.W.L. (1993). Amplification of human B cell activation by a monoclonal antibody Motheaten and viable motheaten mice have mutations in the hemo- to the B cell-specific antigen CD22, Bp130/140. J. Immunol. 138, poietic cell phosphatase gene. Nature Genet. 4, 124–129. 98–103. Tuscano, J., Engel, P., Tedder, T.F., and Kehrl, J.H. (1996a). Engage- Pezzutto, A., Rabinovitch, P.S., Do¨ rken, B., Moldenhauer, G., and ment of the adhesion receptor CD22 triggers a potent stimulatory Clark, E.A. (1988). Role of the CD22 human B cell antigen in B cell signal for B cells and blocking CD22/CD22L interactions impairs triggering by anti-immunoglobulin. J. Immunol. 140, 1791–1795. T-cell proliferation. Blood 87, 4723–4730. Plutzky, J., Neel, B.G., and Rosenberg, R.D. (1992). Isolation of a Tuscano, J.M., Engel, P., Tedder, T.F., Agarwal, A., and Kehrl, J.H. src homology 2-containing tyrosine phosphatase. Proc. Natl. Acad. (1996b). Involvement of p72syk kinase, p53/56lyn kinase and phos- Sci. USA 89, 1123–1127. phatidyl inositol-3 kinase in signal transduction via the human B Powell, L.D., Sgroi, D., Sjoberg, E.R., Stamenkovic, I., and Varki, A. lymphocyte antigen CD22. Eur. J. Immunol. 26, 1246–1252. (1993). Natural ligands of the B cell adhesion molecule CD22␤ carry van der Merwe, P.A., Crocker, P.R., Vinson, M., Barclay, A.N., N-linked oligosaccharides with ␣-2,6-linked sialic acids that are re- Schauer, R., and Kelm, S. (1996). Localization of the putative sialic quired for recognition. J. Biol. Chem. 268, 7019–7027. acid-binding site on the immunoglobulin superfamily cell-surface Rigley, K.P., Harnett, M.M., and Klaus, G.G.B. (1989). Co-cross- molecule CD22. J. Biol. Chem. 271, 9273–9280. linking of surface immunoglobulin Fc␥ receptors on B lymphocytes van Oers, N.S.C., Tao, W., Watts, J.D., Johnson, P., Aebersold, R., uncouples the antigen receptors from their associated G protein. and Teh, A.-S. (1993). Constitutive tyrosine phosphorylation of the T Eur. J. Immunol. 19, 481–485. cell receptor (TCR) ␨ subunit: regulation of TCR-associated tyrosine Rijkers, G.T., Griffioen, A.W., Zegers, B.J.M., and Cambier, J.C. kinase activity by TCR ␨. Mol. Cell. Biol. 13, 5771–5780. (1990). Ligation of membrane immunoglobulin leads to inactivation van Oers, N.S.C., Killeen, N., and Weiss, A. (1994). ZAP-70 is consti- of the signal-transducing ability of membrane immunoglobulin, tutively associated with tyrosine-phosphorylated TCR ␨ in murine CD19, CD21, and B-cell gp95. Proc. Natl. Acad. Sci. USA 87, 8766– thymocytes and lymph node T cells. Immunity 1, 675–685. 8770. Weiss, A., and Littman, D.R. (1994). Signal transduction by lympho-

Roifman, C.M., and Wang, G. (1992). Phospholipase C-␥1 and phos- cyte antigen receptors. Cell 76, 263–274. pholipase C-␥2 are substrates of the B cell antigen receptor associ- Wilson, G.L., Fox, C.H., Fauci, A.S., and Kehrl, J.H. (1991). cDNA ated protein tyrosine kinase. Biochem. Biophys. Res. Commun. 183, cloning of the B cell membrane protein CD22: a mediator of B-B 411–416. cell interactions. J. Exp. Med. 173, 137–146. Sato, S., Steeber, D.A., and Tedder, T.F. (1995). The CD19 signal Yamanashi, Y., Fukui, Y., Wongsasant, B., Kinoshita, Y., Ichimori, transduction molecule is a response regulator of B-lymphocyte dif- Y., Toyoshima, K., and Yamamoto, T. (1993). Activation of Src-like ferentiation. Proc. Natl. Acad. Sci. USA 92, 11558–11562. protein-tyrosine kinase Lyn and its association with phosphatidyl- Sato, S., Ono, N., Steeber, D.A., Pisetsky, D.S., and Tedder, T.F. inositol 3-kinase upon B-cell antigen receptor-mediated signaling. (1996). CD19 regulates B lymphocyte signaling thresholds critical for Proc. Natl. Acad. Sci. USA 89, 1118–1122. the development of B-1 lineage cells. J. Immunol. 157, 4371–4378. Zhou, L.-J., Ord, D.C., Hughes, A.L., and Tedder, T.F. (1991). Struc- Schulte, R.J., Campbell, M.-A., Fischer, W.H., and Sefton, B.M. ture and domain organization of the CD19 antigen of human, mouse (1992). Tyrosine phosphorylation of CD22 during B cell activation. and guinea pig B lymphocytes: conservation of the extensive cyto- Science 258, 1001–1004. plasmic domain. J. Immunol. 147, 1424–1432. Schultz, L.D., Schweitzer, P.A., Ranjan, T.V., Yi, T., Ihle, J.N., Mat- Zhou, L.-J., Smith, H.M., Waldschmidt, T.J., Schwarting, R., Daley, thews, R.J., Thomas, M.L., and Beier, D.R. (1993). Mutations at the J., and Tedder, T.F. (1994). Tissue-specific expression of the human murine motheaten locus are within the hematopoietic cell protein- CD19 gene in transgenic mice inhibits antigen-independent B lym- tyrosine phosphatase (Hcph) gene. Cell 73, 1445–1454. phocyte development. Mol. Cell. Biol. 14, 3884–3894. Schwarting, R., Stein, H., and Wang, C.Y. (1985). The monoclonal antibodies ␣S-HCL 1 (␣Leu-14) and ␣S-HCL 3 (␣Leu-M5) allow the diagnosis of hairy cell leukemia. Blood 65, 974–983. Selfridge, J., Pow, A.M., McWhir, J., Magin, T.M., and Melton, D.W. (1992). Gene targeting using a mouse HPRT minigene/HPRT-deficient embryonic stem cell system: inactivation of the mouse ERCC-1 gene. Som. Cell Mol. Genet. 18, 325–336. Sgroi, D., Varki, A., Braesch-Andersen, S., and Stamenkovic, I. (1993). CD22, a B cell-specific immunoglobulin superfamily member, is a sialic acid-binding lectin. J. Biol. Chem. 268, 7011–7018.