CD22 regulates B lymphocyte function in vivo through both ... - Nature

© 2004 Nature Publishing Group http://www.nature.com/natureimmunology

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CD22 regulates B lymphocyte function in vivo through both ligand-dependent and ligand-independent mechanisms Jonathan C Poe1,3, Yoko Fujimoto1,3, Minoru Hasegawa1,2, Karen M Haas1, Ann S Miller1, Isaac G Sanford1, Cheryl B Bock1, Manabu Fujimoto1,2 & Thomas F Tedder1 The interaction of CD22 with a2,6-linked sialic acid ligands has been widely proposed to regulate B lymphocyte function and migration. Here, we generated gene-targeted mice that express mutant CD22 molecules that do not interact with these ligands. CD22 ligand binding regulated the expression of cell surface CD22, immunoglobulin M and major histocompatibility complex class II on mature B cells, maintenance of the marginal zone B cell population, optimal B cell antigen receptor–induced proliferation, and B cell turnover rates. However, CD22 negative regulation of calcium mobilization after B cell antigen receptor ligation, CD22 phosphorylation, recruitment of SHP-1 to CD22 and B cell migration did not require CD22 ligand engagement. These observations resolve longstanding questions regarding the physiological importance of CD22 ligand binding in the regulation of B cell function in vivo.

CD22 is a B cell–specific glycoprotein of the immunoglobulin superfamily expressed in the cytoplasm of pro–B and pre–B cells and on the cell surface as B cells mature to express immunoglobulin D (IgD)1,2. CD22 has seven extracellular immunoglobulin-like domains3,4, of which the two N-terminal (membrane-distal) domains act as a mammalian lectin that binds a2,6-linked sialic acid–bearing ligands containing a specific N-glycolylneuraminic acid (NeuGc), NeuGca2,6Galb1-4Glc(NAc), linkage in mice3,5–7. Like other members of the sialoadhesin family of glycoproteins, CD22 can serve as an adhesion receptor by binding ligands expressed on the surfaces of various hematopoietic and nonhematopoietic cells5,8,9. Potential CD22 ligands identified in vitro include CD45, IgM, members of the Ly-6 family of glycoproteins and other structurally diverse proteins and lipids1,10–13. After either CD22 or B cell antigen receptor (BCR) ligation, highly conserved tyrosine motifs in the CD22 cytoplasmic domain become phosphorylated4,14. Tyrosine-phosphorylated CD22 subsequently recruits the SHP-1 phosphotyrosine phosphatase, the SHIP phosphoinositide phosphatase and other effector molecules that regulate BCR and CD19 signaling15–20. The outcome of CD22 engagement and phosphorylation is generally believed to be negative regulation of B cell activation, as B cells from CD22 knockout (CD22KO) mice generate augmented calcium responses after BCR crosslinking and have an IgMlo, major histocompatibility complex (MHC) class II–high phenotype that is characteristic of stimulated B cells21–24. However,

the physiological importance and in vivo relevance of CD22 ligand engagement and adhesion remain undefined1,25. Although the physiological importance of CD22-mediated ligand binding and adhesion in vivo is unknown, many studies have suggested potential functions for CD22 binding of endogenous ligands in regulating B cell function. Monoclonal antibodies (mAbs) that block human CD22 ligand binding either induce or inhibit B cell signal transduction and survival in vitro26–29. Other in vitro analyses have suggested that CD22 binding to sialic acid–bearing ligands is required for CD22 to negatively influence BCR signal transduction and dampen self reactivity. In one study, CD22 expression suppressed B cell activation by antigen-expressing target cells that coexpressed a2,6sialoglycoconjugates30. In other studies, B cell lines with impaired CD22 ligand-binding activity demonstrated augmented calcium responses after BCR engagement, which was proposed to result from decreased CD22 tyrosine phosphorylation and decreased SHP-1 recruitment to CD22 (refs. 31,32). In contrast, analysis of ST6Gal sialyltransferase (ST6Gal I) knockout mice, which fail to generate CD22 ligands, has suggested that CD22 exerts stronger negative regulation of B cell signaling in the absence of ligand engagement33,34. Whereas CD22-mediated adhesion is readily visualized during in vitro assays when CD22 is overexpressed in cell lines5,6,8, cis interactions between CD22 and other B cell surface sialoglycoproteins have been suggested to result in the occupancy or ‘masking’ of CD22 on most primary

1Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710, USA. 2Present addresses: Department of Dermatology, Kanazawa University Graduate School of Medical Science, 13-1 Takaramachi, Kanazawa, Ishikawa, Japan 920-8641 (M.H.) and Department of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan 113-8655 (M.F.). 3These authors contributed equally to this work. Correspondence should be addressed to T.F.T. ([email protected]).

Published online 19 September 2004; doi:10.1038/ni1121

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mature B cells31,32,35. CD22 masking is proposed to downregulate positive signaling through cell surface receptors such as IgM and CD45, preventing B cell hyperactivation. However, CD22 masking is a reversible process, occurs independently of CD45 expression and does not prevent the recruitment of CD22 to sites of cell-cell contact36. ‘Unmasking’ of some CD22 molecules also occurs after B cell sialidase treatment or costimulation via CD40, which may relieve CD22 negative regulation of BCR signaling in germinal centers or other microenvironments35. CD22 unmasking is also proposed to regulate the migration of mature recirculating B cells to the bone marrow by promoting trans interactions with ligands expressed on bone marrow endothelium37,38. Studies of CD22 adhesion have been forced to rely on ectopic CD22 overexpression or enzymatic modification to show ligand-binding

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Figure 1 Generation of CD22D1-2 and CD22AA mice. (a) COS cells expressing CD22D1-2 and CD22AA do not bind CD22 ligands on Raji cells. COS cells were transiently transfected with vector alone (Control) or with vector containing CD22 wild-type, CD22D1-2 or CD22AA cDNA. Transfected cells were incubated with Raji cells before extensive washing; cellular rosettes were analyzed by light microscopy and digital imaging. Results are representative of those obtained in three independent experiments. (b,c) Genetargeting strategies for the generation of CD22D12 mice (b) and CD22AA mice (c), indicating Cd22 organization, the sites for targeted point mutations or exon deletions and structure of the targeting vectors, including neor and the gene encoding thymidine kinase (tk), and positions of the loxP sites flanking neor, before and after germline neor removal. Below diagrams, restriction fragment length changes after genomic DNA digestion (double-headed arrows), sources of DNA probes for Southern hybridizations, and DNA primers used for PCR analysis of gene structure (inward-facing arrows). 5¢UT, 5¢ untranslated region; L, leader; Ig1–7, immunoglobulin-like domains 1–7; TM, transmembrane; Cyto., cytoplasmic; RR, wild-type arginines; AA, mutant alanines. (d) Cd22 targeting in CD22AA mice, assessed by Southern hybridization of KpnIdigested genomic DNA with DNA probe B. Genomic DNA was from wild-type mice and from CD22AA heterozygous (Cd22 AA/+) or homozygous (Cd22 AA/AA) littermates that had (– neo) or had not (+ neo) been bred with CMV-Cre transgenic mice. (e) Confirmation of genomic neor removal in homozygous CD22AA mice by PCR analysis. Primer sequences are available in the Supplementary Methods online. Targeting of Cd22 and subsequent neor removal was similarly confirmed in CD22D1-2 mice (data not shown). (f) Immunoblot analysis of CD22 protein expression in wild-type, CD22D1-2, CD22AA and CD22KO B cells. Whole-cell lysates of splenic B cells were analyzed by SDS-PAGE with subsequent immunoblot analysis with the MB221 mAb. Filled arrowheads, CD22AA and wild-type CD22 (140 kDa), and CD22D1-2 (120 kDa); open arrowheads, lower-molecular-weight form of CD22. Bottom, the membrane was stripped and reprobed with anti-SHP-1 to confirm equivalent protein loading. (g) Cell surface CD22 expression by CD22D1-2 and CD22AA B cells. Splenocytes from wild-type, CD22AA, CD22D1-2 and CD22KO mice were stained with the MB22-8 mAb followed by FITC-conjugated anti-mouse IgG1 (left), or with FITC-conjugated Cy34 mAb (right), and were analyzed by flow cytometry.

activity. It has therefore been difficult to formulate a unified perspective as to whether and how CD22 ligand binding is physiologically relevant. To directly assess the function of CD22 ligand-binding activity in vivo, we have generated and characterized two lines of mice expressing mutant CD22 molecules that lack ligand-binding activity. These mice demonstrate that interactions of CD22 with its ligands are important for normal B cell physiology. Moreover, whereas ligand binding regulated some functions of CD22, other functions were dependent only on expression of the non-ligand-binding ectodomains of CD22 and its cytoplasmic tail.

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Figure 2 B cell phenotypes in CD22D1-2 and CD22AA mice. (a,b) Cell surface CD22 expression on bone marrow, spleen, blood and lymph node B cells of CD22D1-2 and CD22AA mice. (a) Two-color flow cytometry of lymphocytes stained with the MB22-8 mAb and FITC-conjugated anti-mouse IgG1 in combination with phycoerythrin-conjugated anti-IgM. Gates delineate the CD22loIgMhi (immature-transitional) and CD22hiIgMlo (mature) B cell subsets with mean linear fluorescence intensity (MFI) of CD22 expression in plots. Similar results were obtained from at least four mice of each genotype. (b) MFI (mean 7 s.e.m.) of CD22 expression by tissue B cells from at least four mice of each genotype. BM, bone marrow. Right, CD22 expression by blood B cells from CD22D1-2 and CD22AA mice that were back-crossed with B6 mice for six generations, relative to that of their wild-type littermates (three or more mice each). (c) CD22 transcript abundance in spleen B cells from CD22D1-2 and CD22AA mice. Values represent the mean ratios of CD22/CD20 transcripts in CD22D1-2 or CD22AA mice relative to CD22/CD20 transcripts in wild-type littermates (value, 1.00), as determined by independent real-time PCR assays with RNA from three littermates of each genotype. An internal control is real-time PCR reactions with 50% of wild-type cDNA (filled bar) for CD22 amplification relative to CD20 amplification. * (b,c), P o 0.01, mean values compared with wild-type mice. (d,e) IgM and MHC class II antigen expression on B cells from CD22D1-2 and CD22AA mice. Blood or splenocytes from wild-type, CD22D1-2, CD22AA and CD22KO mice were stained with optimal concentrations of FITC-conjugated anti-IgM (d) or MHC class II mAb (e) in combination with phycoerythrin-conjugated B220 mAb, with gating on the lymphocyte populations. In e, flow cytometry histograms represent B220+-gated cells. Values represent mean MFI obtained from at least four mice of each genotype. *, P o 0.05, mean values, compared with wild-type B cells. Ctl, control (staining of cells with isotype-matched, nonspecific control antibodies). (f) Two-color flow cytometry of splenocytes from wild-type, CD22D1-2, CD22AA and CD22KO mice after staining with FITC-conjugated CD21 mAb in combination with phycoerythrin-conjugated anti-IgM or CD1d mAb. Numbers and gates (ovals) indicate the percentage of lymphocytes with a CD21hiIgMhi or CD21hiCD1dhi marginal zone phenotype. (g) Spleen sections stained with a combination of FITC-conjugated B220 mAb (top; green) and phycoerythrinconjugated CD1d mAb (middle; red). Bottom, fluorescence microscopy images digitized and merged to show the marginal zone regions containing B220+CD1dhi B cells (yellow). Results similar to those in f and g were obtained with three mice of each genotype.

RESULTS Generation of CD22D1-2 and CD22AA mutant mice The first immunoglobulin-like domain of CD22 contains conserved positively charged amino acid residues, arginine 130 (R130) and arginine 137 (R137), that are necessary for interactions with

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sialoglycoconjugates in vitro1,39. Therefore, we used two strategies to generate independent CD22 cDNA constructs and mouse lines with mutant CD22 molecules incapable of interacting with sialoglycoconjugate ligands. We engineered CD22 cDNA and mice to either generate a full-length CD22 containing point mutations of R130 and R137 to

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ARTICLES Table 1 Frequency and numbers of B lymphocytes in CD22D1-2 and CD22AA mice Cell number (106)


Percentage of lymphocytes Tissue

Phenotype

Wild-type

CD22D1-2

Bone marrow

HSAloB220hi HSAloIgM+

24 7 3.9 24 7 4.2

B220hiIgM+ B220loIgM+ Blood Spleen

Lymph nodes Peritoneum

CD22AA

CD22KO

Wild-type

CD22D1-2

CD22AA

CD22KO

22 7 1.5 21 7 1.2

9.8 7 1.2** 9.1 7 1.8**

9.8 7 1.1** 10 7 1.4**

25 7 2.9 31 7 1.9

23 7 2.9 34 7 1.2

14 7 2.2** 38 7 4.0

14 7 2.4** 38 7 3.3

B220+IgM+ B220+IgM+

47 7 4.8 68 7 1.7

39 7 4.6 70 7 1.5

31 7 2.6* 76 7 1.7

18 7 5.7* 68 7 1.3

1.7 7 0.2 44 7 4.1

0.9 7 0.1* 51 7 6.6

0.8 7 0.1** 51 7 5.0

0.4 7 0.1** 40 7 4.6

IgMhiCD21hi B220loCD5+

5.6 7 1.3 1.5 7 0.2

1.6 7 0.2** 1.3 7 0.4

1.5 7 0.3** 1.4 7 0.4

1.2 7 0.2** 1.7 7 0.5

3.4 7 0.6 1.0 7 0.2

1.1 7 0.1** 1.1 7 0.4

1.0 7 0.3** 1.0 7 0.4

0.7 7 0.1** 1.0 7 0.4

B220+IgM+ B220+IgM+

26 7 3.4 65 7 10

37 7 2.7* 68 7 4.5

32 7 4.5 63 7 2.2

36 7 2.8* 62 7 7.1

2.2 7 0.2

3.7 7 0.7*

3.3 7 0.5*

3.9 7 0.5**

B220loCD5+ B220hiIgDhi

38 7 7.5 14 7 1.5

37 7 3.6 25 7 4.8*

32 7 4.4 25 7 3.0*

38 7 6.3 21 7 2.4*

Date are mean 7 s.e.m. values from at least five littermates of each genotype. *, P o 0.05 and **, P o 0.01, compared with wild-type littermates.

uncharged alanine residues (CD22AA) or to express a truncated form of CD22 lacking immunoglobulin-like domains 1 and 2 (CD22D1-2), which mediate ligand binding3,6. COS cells transfected with cDNA encoding wild-type CD22, CD22D1-2 and CD22AA expressed similar amounts of cell surface CD22, as determined by immunofluorescence staining with the MB22-8 mAb, which binds a membrane-proximal epitope of CD22 (Supplementary Note online). Wild-type CD22

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Figure 3 Calcium mobilization, CD22 tyrosine phosphorylation and CD22 recruitment of SHP-1 are normal in CD22D1-2 and CD22AA B cells. (a) BCR-induced calcium mobilization. Splenocytes from wild-type, CD22D1-2, CD22AA and CD22KO mice were loaded with Indo-1 AM ester and stained with FITC-labeled B220 mAb, and relative calcium concentrations were assessed by flow cytometry after gating on the B220+ population of cells (presented as the ratio of fluorescence at 405 nm versus 525 nm). F(ab¢)2 anti-IgM or mAb to CD19 (concentration, above graph) was added to the cells after 1 min (upward arrows). Data represent those obtained in three independent experiments with similar results. (b) BCR-induced CD22 tyrosine phosphorylation and recruitment of SHP-1. Purified splenic B cells from wild-type, CD22D1-2, CD22AA and CD22KO mice were left unstimulated (0) or were stimulated with F(ab¢)2 anti-IgM for 3 or 10 min (above lanes). Cell lysates were immunoprecipitated (IP) with the MB22-8 mAb (CD22 extracellular domain), followed by SDS-PAGE and subsequent immunoblotting (Probe) with the 4G10 mAb for evaluation of CD22 tyrosine phosphorylation and with anti-SHP-1 for evaluation of CD22-associated SHP-1. The blots were subsequently stripped and reprobed with the MB22-1 mAb (CD22 cytoplasmic domain). pTyr, phosphotyrosine. (c) Tyrosine phosphorylation of cellular proteins after BCR ligation. Purified splenic B cells from wild-type, CD22D1-2, CD22AA and CD22KO mice were activated as described in b, lysed and separated by SDS-PAGE, with immunoblotting with the 4G10 mAb. The blots were subsequently stripped and reprobed with anti-SHP-1 to confirm equivalent protein loading between samples. Data in b and c are representative of three independent experiments producing similar results.

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expressed by COS cells bound Raji cells (a human B cell line), human and mouse lymphocytes, and human red blood cells, all of which express CD22 ligands, as described5. In contrast, COS cells expressing CD22D1-2 or CD22AA did not mediate adhesion of the same target cells (Fig. 1a and data not shown). Therefore, both the CD22D1-2 and CD22AA mutations abrogated CD22 ligand binding during in vitro assays.

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Figure 4 Altered proliferative responses of B cells from CD22D1-2 and CD22AA mice. (a) Purified splenic B cells were stimulated with F(ab¢)2 anti-IgM, mAb to CD40 (Anti-CD40) or LPS during 72 h proliferation assays. Data represent mean counts per minute (c.p.m.) of triplicate cultures after [3H]thymidine uptake. *, P o 0.05 and **, P o 0.01, compared with wildtype B cells. Similar results were obtained in three independent experiments. (b) B cells were labeled with 1 mM CFSE before being stimulated with F(ab¢)2 anti-IgM (40 mg/ml) or mAb to CD40 (1 mg/ml). Proliferation was assessed by flow cytometry for determination of the relative number of viable cells and CFSE fluorescence intensity after culture. Histograms indicated by arrow (Unstim; top), unstimulated CD22D1-2 or CD22AA B cells; vertical dashed lines (middle and bottom), positions of nonproliferated B cells. Similar results were obtained in two independent experiments.

We used gene targeting to generate mice expressing mutant CD22D1-2 (Fig. 1b) and CD22AA (Fig. 1c) molecules. The targeting vectors contained loxP recombination sites flanking the neomycinresistance gene (neor), which allowed deletion of neor from the genomes of heterozygous ‘CD22AAneo ’ and ‘CD22D1-2neo ’ mice bred with CMV-Cre transgenic mice, to ensure that neor insertion did not interfere with Cd22 transcription. We interbred heterozygous offspring and screened them by Southern blot and PCR analysis to confirm appropriate gene targeting and neor deletion in CD22AA mice (Fig. 1d,e and Supplementary Fig. 1 online) and CD22D1-2 mice (data not shown). Homozygous CD22AA and CD22D1-2 offspring demonstrated no obvious defects or susceptibility to disease. We confirmed CD22 protein expression by B cells from CD22AA and CD22D1-2 mice using immunoblot analysis of lysates from spleen B cells (Fig. 1f) and flow cytometry (Fig. 1g). CD22AA and wild-type CD22 migrated as 140–kilodalton (kDa) proteins (Fig. 1f). CD22D1-2 migrated as a 120 kDa protein, the predicted size of CD22 lacking two immunoglobulin-like domains (Fig. 1f). In all genotypes, a lowermolecular-weight form of CD22 was also apparent in immunoblots with variable intensity (Fig. 1f), as described15,31. The MB22-8 mAb stained B cells expressing wild-type, CD22AA and CD22D1-2 proteins (Fig. 1g). In contrast, the Cy34 mAb, which reacts with an epitope in the two membrane-distal immunoglobulin-like domains, reacted with wild-type and CD22AA B cells but not CD22D1-2 B cells (Fig. 1g). Thus, B cells from gene-targeted mice expressed cell surface CD22AA and CD22D1-2 appropriately.

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CD22 ligand binding regulates receptor expression Cell surface CD22 was expressed at a similar intensity by immaturetransitional (IgMhiCD22lo) B cells in the bone marrow and spleens of wild-type, CD22AA and CD22D1-2 littermates (Fig. 2a,b). However, CD22 expression was decreased by 40–50% on mature (IgMloCD22hi) B cells in the bone marrow, spleen and lymph nodes of CD22AA and CD22D1-2 mice. CD22 expression was reduced on CD22AA blood B cells but was normal on CD22D1-2 blood B cells (Fig. 2b). However, CD22 expression was reduced by 20–44% on blood B cells from both CD22AA and CD22D1-2 mice that were back-crossed onto the C57BL/ 6 (B6) background for six generations (Fig. 2b, right). Real-time RTPCR analysis of Cd22 expression in splenic B cells with Cd20 expression as an internal reference showed that CD22 mRNA abundance was modestly augmented in CD22D1-2 B cells and was only modestly decreased in CD22AA B cells (Fig. 2c), indicating that the main regulatory mechanism for decreased CD22 surface expression was at the protein level. B cells from CD22D1-2, CD22AA and CD22KO mice also expressed significantly less surface IgM (P o 0.05; Fig. 2d) and significantly more MHC class II (P o 0.05; Fig. 2e) than did B cells from their wild-type littermates. We noted similar decreases in surface IgM and increases in MHC class II expression on B cells from CD22AA and CD22D1-2 mice that were back-crossed onto the B6 background for six generations (data not shown). Therefore, mutation of the CD22 ligand-binding domains resulted in reduced CD22 surface expression by mature B cells and constitutive B cell activation characterized by altered IgM and MHC class II antigen expression.

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Figure 5 Altered B cell turnover in CD22D1-2 and CD22AA mice. (a) Mice (n ¼ 3 for each genotype) were fed BrdU for 7 d and killed, and the percentage of BrdU+ B cells in each tissue (horizontal axis) was determined by flow cytometry. Values represent mean percentages of BrdU+ B220hi B cells in the indicated tissues. (b) Mice (n ¼ 4 for each genotype) back-crossed onto the B6 background for six generations were fed BrdU for 7 d and were analyzed as described in a. *, P o 0.05, compared with wild-type mice.

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ARTICLES B cell development in CD22D1-2 and CD22AA mice The percentage of recirculating (heat-stable antigen–low (HSAlo)B220hiIgM+) B cells in the bone marrow was reduced in CD22AA and CD22KO mice but was normal in CD22D1-2 mice (Table 1). However, recirculating bone marrow B cells were reduced similarly in CD22AA mice (59%; P o 0.01) and CD22D1-2 mice (53%; P o 0.01) that were back-crossed onto the B6 background for six generations, relative to those of their wild-type littermates (data not shown). CD22D1-2, CD22AA and CD22KO mice also had substantially reduced numbers of blood B cells but substantially increased numbers of lymph node B cells relative to those of their wild-type littermates. CD22D1-2, CD22AA and CD22KO mice had normal numbers of spleen B cells, despite a specific reduction in the spleen marginal zone B cell subpopulation that was either IgMhiCD21hi (Table 1) or CD21hiIgMhiCD1dhi (Fig. 2f,g), as described for CD22KO mice24,40. The frequency of marginal zone B cells was also reduced in CD22AA mice (68%; P o 0.01) and CD22D1-2 mice (47%; P o 0.01) that were back-crossed onto the B6 background for six generations (data not shown). Also, the percentage of conventional B cells (B220hiIgDhi) in the peritoneum was augmented in all three mouse lines (Table 1). T cell numbers were not altered in CD22D1-2, CD22AA or CD22KO mice (data not shown). Thus, B cell development was similar in mice with mutations in the CD22 ligand-binding domains that disrupted the generation and/or maintenance of the marginal zone B cell subpopulation.

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Normal BCR signaling in CD22D1-2 and CD22AA B cells We evaluated BCR-induced calcium mobilization, CD22 tyrosine phosphorylation and SHP-1 recruitment to CD22 in CD22D1-2 and CD22AA B cells after BCR ligation. CD22D1-2 and CD22AA B cells had calcium flux identical to that of B cells from their wild-type littermates after treatment with both optimal (40 mg/ml) and suboptimal (10 mg/ml) concentrations of F(ab¢)2 antibody to IgM (antiIgM; Fig. 3a). In contrast, CD22KO B cells had heightened calcium responses after treatment with both optimal and suboptimal concentrations of F(ab¢)2 anti-IgM, as described21–24. Calcium responses after CD19 ligation were similar for all four genotypes. CD22 tyrosine phosphorylation and its subsequent association with SHP-1 were not impaired in CD22D1-2 or CD22AA B cells after BCR ligation (Fig. 3b). We confirmed similar total protein abundance of the higher-molecular-weight, tyrosine-phosphorylated form of CD22 on immunoblots between genotypes by reprobing the membranes with the MB22-1 mAb. We noted the lower-molecular-weight, nonphosphorylated form of CD22 with varying intensities in the blots, as reported15,31. Total protein tyrosine phosphorylation after BCR ligation was also similar for B cells from CD22D1-2, CD22AA or CD22KO mice versus those of their wild-type littermates (Fig. 3c). Although the phosphorylation of some proteins varied between individual mice in individual experiments, we found no consistent changes in protein tyrosine phosphorylation. Therefore, mutation of the CD22 ligand-binding domains did not influence BCR-induced calcium responses, CD22 tyrosine phosphorylation or the recruitment of SHP-1 to CD22.

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CD22 ligand binding regulates B cell proliferation BCR-induced proliferation by CD22D1-2 and CD22AA B cells after 72 h in culture was considerably reduced over a range of F(ab¢)2 antiIgM concentrations compared with that of wild-type B cells, as evaluated by [3H]thymidine uptake (Fig. 4a). This result was similar to the decreased proliferation of CD22KO B cells, as described23,24,41. Reduced proliferation of CD22D1-2 and CD22AA B cells after BCR

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Figure 6 CD22 ligand binding does not promote B cell migration. Purified splenic B cells from wild-type, CD22D1-2, CD22AA or CD22KO mice were labeled with calcein and injected into wild-type recipient mice along with an equal number of PKH-26-labeled wild-type B cells. After 1.5 h or 16 h, single-cell leukocyte suspensions from the bone marrow, spleen and lymph nodes of recipient mice were analyzed by flow cytometry for determination of the ratios of calcein- to PKH-26-labeled B cells. (a) Representative results from one of three 16-hour migration experiments. Top right corners, preinjected cell ratios (Ri) and cell ratios after migration to the bone marrow (Ro); right margins, calculated Ro/Ri values. (b) Pooled results from one 1.5-hour and three independent 16-hour migration experiments for various tissues (horizontal axes), all of which produced similar results. Data represent mean Ro/Ri ratios for each genotype (key).

ligation at 72 h (Fig. 4) reflected a substantial delay in the kinetics of early clonal expansion, as the clonal expansion of CD22D1-2 and CD22AA B cells stained with carboxyfluorescein succinimidyl ester (CFSE) was similar to that of wild-type B cells at 96–120 h (Fig. 4b). CD22D1-2 and CD22AA B cells were also hyperproliferative after stimulation with a pentameric mAb to CD40 (Fig. 4a,b), as described for CD22KO B cells41. Lipopolysaccharide (LPS)–induced proliferation was similar among all genotypes over a range of concentrations (Fig. 4a). Thus, mutation of the CD22 ligand-binding domains substantially influenced the proliferative potential of B cells after BCR- or CD40-induced signals.

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ARTICLES CD22 ligand binding regulates B cell turnover CD22KO mice have high rates of B cell turnover in vivo as measured by bromodeoxyuridine (BrdU) uptake21,23. Therefore, we assessed BrdU uptake in CD22D1-2 and CD22AA mice to determine whether CD22 ligand binding regulates B cell turnover. B cells in both CD22AA and CD22KO mice had augmented rates of BrdU uptake for all tissues examined (Fig. 5a). Decreased CD22 expression does not explain high turnover by CD22AA B cells, as wild-type and CD22D1-2 B cells had similar turnover rates. Moreover, B cells from mice heterozygous for CD22 (Cd22+/ mice), which have a 50% reduction in CD22 surface expression24, had wild-type turnover rates. B cell turnover was higher in both CD22AA and CD22D1-2 mice that were back-crossed onto the B6 background for six generations (Fig. 5b). Therefore, mutation of the CD22 ligand binding domains altered B cell survival in CD22AA and CD22D1-2 mice. Normal migration of CD22D1-2 and CD22AA B cells It has been suggested that CD22 binding to endogenous ligands is required for the migration of mature recirculating B cells to the bone marrow21,38. As CD22AA and CD22KO mice had considerably reduced numbers of recirculating (HSAloB220hiIgM+) B cells in the bone marrow, we assessed the function of CD22 ligand binding in B cell migration in vivo directly with two-color B cell migration assays. We labeled purified splenic B cells from wild-type, CD22D1-2, CD22AA and CD22KO mice with the intravital fluorochrome calcein and mixed the cells with an equivalent number of wild-type B cells labeled with the intravital fluorochrome PKH-26, followed by adoptive transfer into wild-type recipient mice. We assessed B cell migration by comparing the ratio of calcein- to PKH-26-labeled cells collected from each organ (Ro) with the ratio of injected calcein- to PKH-26-labeled cells (Ri). Cells with equivalent migratory properties distribute evenly and generate Ro/Ri ratios approaching 1 (Fig. 6a). At either 1.5 h or 16 h after injection, wild-type, CD22D1-2, CD22AA and CD22KO B cells migrated to the bone marrow, spleen and lymph nodes at similar frequencies (Fig. 6). Therefore, mutation of the CD22 ligand binding domains did not substantially influence B cell migration to lymphoid tissues. DISCUSSION Gene-targeted mice with CD22 mutations that prevent ligand engagement demonstrated that CD22 carries out physiologically relevant ligand-dependent and -independent functions that have distinct effects on B cell biology. For example, a notable feature of CD22AA and CD22D1-2 B cells was their reduced expression of CD22. This suggests that cell surface CD22 on mature B cells is mainly engaged by ligands, which stabilizes its cell surface expression. As a consequence, agents or CD22-directed therapies that block ligand binding may considerably enhance CD22 internalization and enhance the therapeutic benefit of CD22-directed immunotoxins. Regulating the density or retention of CD22 at the cell surface by ligand engagement may also influence B cell signaling thresholds. Elimination of CD22 ligand binding resulted in an activated B cell phenotype characterized by reduced IgM expression and augmented MHC class II expression, similar to the phenotype of CD22KO B cells21–24,42. Reduced CD22 expression alone does not explain increased B cell activation, as IgM and MHC class II expression are only modestly altered on B cells from Cd22+/ mice, in contrast to the more severe defect on CD22KO B cells24. Thus, CD22 ligand engagement may not only transduce important signals but also retain this important regulator of signal transduction at the cell surface, where it is functionally active.

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Whereas some CD22 functions required ligand binding, other functions required only the expression of CD22 ectodomains with an intact cytoplasmic domain. For example, CD22D1-2 and CD22AA B cells regulated BCR-induced calcium responses normally. CD22 tyrosine phosphorylation and recruitment of SHP-1 were also normal in CD22D1-2 and CD22AA B cells after BCR ligation. These results contrast with in vitro studies using B cell lines in which inhibition of CD22 ligand binding was proposed to reduce CD22 phosphorylation and SHP-1 recruitment to CD22, leading to augmented calcium mobilization. In one study, BCR-induced calcium responses were augmented in a transformed CD22KO B cell line transfected with mutant CD22 molecules lacking ligand-binding activity when antiIgM was used at a concentration of 1 mg/ml (ref. 31), a very low dose for BCR stimulation21–24. In a second study, a synthetic inhibitor of CD22 ligand binding augmented calcium responses induced by antiIgM (2 mg/ml) and decreased CD22 phosphorylation and SHP-1 recruitment32. As suggested, this CD22 ligand mimetic may have affected the membrane distribution of CD22 on target cells32 or it could have crosslinked or generated signals through CD22 that induced CD22 internalization or other alterations, leading to augmented calcium responses. Regardless, calcium responses alone do not regulate the proliferative capacity of B cells, as CD22KO, CD22D1-2 and CD22AA B cells were similarly hypoproliferative to BCR stimulation. Also, CD22D1-2, CD22AA and CD22KO B cells were hyperproliferative to CD40 stimulation, indicating that CD22 ligand binding negatively regulates CD40 signaling, even though CD40 ligation has been reported to unmask CD22 on the cell surface35. The fact that CD22 ligand binding was required for optimal BCR-induced proliferation and inhibition of CD40-induced proliferation may relate to the observation that CD22 regulates Myc transcription factor–induced expression of the ubiquitin ligase complex protein Cullin 1 after BCR ligation, which promotes ubiquitin-dependent cell cycle progression41. CD22D1-2 and CD22AA mice demonstrate that the marginal zone B cell subpopulation requires CD22 interactions with in vivo ligands. CD22KO mice have a similar deficiency in marginal zone B cells24,40. Thus, the generation or homeostasis of this unique subpopulation is regulated by CD22 interactions with endogenous ligands that may be ubiquitously expressed by multiple cell types in vivo1. CD22 ligand binding may also be important in the regulation of overall B cell homeostasis in vivo, as B cell turnover was substantially increased in CD22D1-2, CD22AA and CD22KO mice. Constitutively augmented apoptosis of peripheral B cells has been noted in CD22KO mice in vivo41. Reduced CD22 expression alone in CD22D1-2 and CD22AA mice does not explain increased B cell turnover rates and decreased marginal zone and recirculating B cell populations, as these characteristics are normal in mice heterozygous for CD22 expression23,24. Instead, these collective findings indicate that CD22 ligand binding may be required for normal follicular B cell survival in the periphery, in addition to its function in marginal zone B cell generation/survival. As a2,6-linked sialic acid motifs are essential components of CD22 ligands5,7, lymphocytes from ST6Gal I knockout mice show substantially reduced binding to CD22 fusion proteins33. Cell surface CD22 and IgM densities are reduced on ST6Gal I knockout B cells, with CD22 density reduced by 62%, compared with the 40–50% on CD22D1-2 and CD22AA B cells. However, the ST6Gal I deficiency results in a phenotype that is distinct from that of CD22D1-2, CD22AA and CD22KO mice. For example, MHC class II densities and mature B cell numbers in the bone marrow of ST6Gal I knockout mice are normal. ST6Gal I knockout B cells are also substantially impaired in their ability to proliferate in response to anti-CD40 and LPS, in addition to anti-IgM, and generate modest calcium responses

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ARTICLES after BCR ligation. Moreover, ST6Gal I knockout mice are severely immunodeficient, with weak humoral immune responses and serum IgM concentrations that are reduced by 63%. Based on these functional properties and the fact that B cells from ST6Gal I knockout mice express unmasked cell surface CD22 molecules, it was proposed that CD22 may exert stronger negative regulation of B cell signaling in the absence of ligand binding34. However, the functional properties of CD22D1-2 and CD22AA B cells ‘argue against’ this possibility, but nonetheless indicate an important function for ligand binding in the regulation of cell surface CD22 expression. Instead, the comparison of our results with those of ST6Gal I knockout mice suggests that functionally important regulatory receptors, in addition to CD22, require ST6Gal I–dependent ligands33,34,43. B cells and mice bearing the CD22D1-2 and CD22AA mutations shared either CD22KO or wild-type phenotypic properties and functioned similarly in most assays. Despite this, the percentage of mature (HSAloB220hiIgM+) bone marrow B cells, CD22 density on blood B cells and B cell turnover were each normal in CD22D1-2 mice with a mixed B6/129 genetic background, which contrasted with those of CD22AA and CD22KO mice on the same mixed background. However, genetic differences are likely to exist between CD22D1-2, CD22AA and CD22KO mice on mixed B6/129 genetic backgrounds, as backcrossing CD22D1-2 mice onto a B6 background for six generations substantially reduced or eliminated their differences with CD22AA mice. This demonstrates the known influence of genetic background on CD22 function41 and emphasizes the importance of generating and characterizing two lines of mice using different targeting strategies to provide a more comprehensive assessment of the importance of CD22 ligand binding. Structural differences between the ligand-binding domains of CD22D1-2 and CD22AA could also influence CD22 function. Several strains of autoimmune mice have alterations in their CD22 ligand-binding domains44, although these receptors mediated normal amounts of adhesion during in vitro assays (data not shown). This suggests that amino acid substitutions and/or insertions in the CD22 ligand-binding domains of some autoimmune mouse lines may be insufficient on their own to induce the phenotypic characteristics of CD22D1-2, CD22AA or CD22KO B cells, but may nevertheless may have unappreciated functional consequences that contribute substantially to autoimmunity through altered CD22 function. Binding of CD22 to endogenous ligands is proposed to regulate mature recirculating B cell migration to the bone marrow. In one report, CD22KO B cell migration to the bone marrow after adoptive transfer into mice deficient in recombination activating gene 2 was decreased by 50% compared with that of wild-type B cells21. In another study, a CD22 fusion protein preferentially bound to ligands expressed on the surface of endothelial cells of bone marrow sinusoids, and mice treated with the CD22 fusion protein reportedly had a 50% reduction in B cells recirculating to the bone marrow38. Like CD22KO mice, CD22AA and back-crossed CD22D1-2 mice had substantially reduced numbers of recirculating (HSAloB220hiIgM+) B cells in the bone marrow. However, B cells from CD22D1-2, CD22AA and CD22KO mice migrated normally to the bone marrow, spleen and lymph nodes when transferred into wild-type recipient mice. Likewise, lymphocytes from ST6Gal I knockout mice migrate normally33. It is therefore possible that soluble CD22 fusion proteins bind ligands for other adhesion receptors rather than selectively blocking CD22 interactions with bone marrow stromal cells38. Moreover, CD22 deficiency or the absence of CD22 ligand binding accelerates B cell turnover, which would decrease the number of recirculating cells in the bone marrow of CD22KO, CD22AA and CD22D1-2 mice.

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Our results obtained with CD22D1-2 and CD22AA mice provide new insight into the mechanisms through which CD22 regulates B cell function and indicate distinct roles for intrinsic CD22 functions and CD22 functions that are mediated by ligand binding. Thus, these animal models provide a context for future studies examining the involvement of CD22 ligation by in vivo ligands in the regulation of B cell function during immune responses to pathogenic organisms, as well as in suppression of the onset of B cell–dependent autoimmune diseases such as systemic lupus erythematosus. METHODS Adhesion assays. The cDNA encoding CD22AA or CD22D1-2 was generated by site-directed or deletion mutagenesis, respectively, with the appropriate forward and reverse PCR primers (primer sequences, Supplementary Methods online). For CD22AA, the arginine codons R130 and R137 were changed to alanine codons. For CD22D1-2, exons 4 and 5 were deleted while an in-frame CD22-encoding sequence was retained. COS cells were transfected with wildtype CD22, CD22D1-2 or CD22AA cDNA in the pcDNA 3.1 vector or with vector alone using the DEAE-dextran method. Cell surface CD22 expression was confirmed 24 h later by fluorescence microscopy with the MB22-8 mAb. The cells were then trypsinized, transferred to 35-mm tissue culture dishes and cultured overnight. Raji cells (1 106 cells/dish) in DMEM (Gibco-BRL) were then incubated with the transfected COS cells for 30 min at 25 1C. Unbound Raji cells were removed by extensive washing with DMEM before the cellular rosettes were fixed in DMEM containing 2% formaldehyde. Digital images were captured with an Olympus IX-70 inverted microscope with an Optronics MagnaFire imaging system. Generation of CD22AA mutant mice. DNA comprising Cd22 was isolated from a 129/Sv mouse genomic library as described24. The R130 and R137 codons (AGG and CGA, respectively) in exon 4 were targeted by site-directed PCR mutagenesis to generate codons encoding alanine residues (GCG and GCA, respectively). Primer sequences are available in the Supplementary Methods online. A KpnI cleavage site was introduced between the alanine residues without a change in the amino acid sequence. The neor containing flanking loxP sites to facilitate removal of neor by the Cre-loxP recombination system was inserted between exons 4 and 5. A gene encoding thymidine kinase (pMC1-HSVTK) was ligated to the 5¢ end of the targeting vector into a BstEII restriction site in exon 2. The 3¢ end of the targeting vector consisted of a 6– kilobase (kb) DNA region retaining exons 6 and 7 and their flanking intron sequences. The vector was linearized with a unique XhoI site in the genomic DNA. R1 129/Sv embryonic stem cells45 were electroporated with the linearized DNA and selected for G418 resistance. Correctly targeted embryonic stem cell clones were identified by Southern blot analysis of genomic DNA and were injected into 3.5-day-old B6 blastocysts before being transferred into pseudopregnant female mice. Chimeric offspring from one embryonic stem clone were bred with B6 mice (The Jackson Laboratory) to provide germline transmission of the disrupted allele, generating CD22AAneo heterozygous offspring. Heterozygous CD22AAneo mice were then crossed with CMV-Cre transgenic mice on the B6 background to facilitate removal of neor, which was confirmed by Southern blot analysis of DNA obtained from tail tissue. Heterozygous offspring on a mixed B6/129 genetic background were interbred to generate homozygous CD22AA and wild-type littermates lacking both neor and the gene encoding Cre. Generation of CD22D1-2 mutant mice. With 129/Sv strain mouse genomic DNA, a 2.1-kb fragment containing exons 4 and 5, which encodes the first two immunoglobulin domains of CD22 was replaced with neor containing flanking loxP sites. As part of the leader sequence is included in the first 33 bp of exon 4, this region was retained and fused in-frame with exon 3, with preservation of an appropriate 3¢ mRNA splice junction of exon 3. An XhoI cleavage site was also introduced in exon 3 without a change in the amino acid sequence. Primer sequences used for targeting are available in the Supplementary Methods online. A gene encoding thymidine kinase was ligated to the 5¢ end of the targeting vector into a BstEII restriction site in exon 2. The 3¢ end of the targeting vector consisted of a 5-kb DNA region retaining exons 6 and 7 and their flanking intron sequences. The construct was linearized with a unique

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ARTICLES ApaI site in the genomic DNA. R1 129/Sv embryonic stem cells45 were electroporated with the linearized DNA, selected for G418 resistance and used to generate chimeric mice. Chimeric offspring from one embryonic stem clone were bred with B6 mice to provide germline transmission of the disrupted allele, generating CD22D1-2neo heterozygous offspring. Heterozygous CD22D1-2neo mice were then crossed with CMV-Cre transgenic mice, as described above for CD22AA mice, to facilitate removal of neor. Heterozygous offspring on a mixed B6/129 genetic background were interbred to generate homozygous CD22D1-2 and wild-type littermates lacking both neor and the gene encoding Cre. We found no differences in the phenotypic characteristics of wild-type littermate mice generated from heterozygous breeding of either CD22D1-2 or CD22AA mice, and thus we pooled them in this study. B6/129 CD22KO mice were as described24. B6/129 Cd22+/ mice were generated by crossing of wildtype littermate mice (described above) with B6/129 CD22KO mice. In some experiments, CD22D1-2 and CD22AA mice back-crossed onto a B6 background for six generations and their wild-type littermates were used. All mice were housed in a specific pathogen–free barrier facility and were 8–12 weeks of age when used. All procedures were approved by the Duke University Institutional Animal Care and Use Committee (Durham, North Carolina). Antibodies and immunohistochemistry. The MB22-8 mAb was generated by the fusion of NS-1 myeloma cells with spleen cells from CD22KO mice immunized with the baby hamster kidney (BHK) cell line, stably transfected with a full-length mouse CD22 cDNA. Hybridomas producing mAbs specifically reactive with CD22-transfected mouse L cells, but not with untransfected cells, were subcloned twice with isotypes determined with a Mouse Monoclonal Antibody Isotyping kit (Amersham). Reactivity of the MB22-8 mAb with membrane-proximal CD22 immunoglobulin domains was verified with COS cells transfected with CD22AA or CD22D1-2 cDNA and immunofluorescence staining. Other antibodies used in this study included the following: the MB221 mouse IgG1 anti–mouse mAb to CD22, reactive with the CD22 cytoplasmic tail15; fluorescein isothiocyanate (FITC)–conjugated anti–mouse CD22 N terminus (Cy34, TIB163; American Type Culture Collection); FITC-conjugated I-A/I-E MHC class II (clone M5/114.15.2, TIB120; American Type Culture Collection); phycoerythrin-conjugated rat anti–mouse HSA (clone M1/69), FITC-conjugated rat anti–mouse CD21/35 (clone 7G6), phycoerythrin-conjugated rat anti-mouse CD1d (clone 1B1), FITC-conjugated rat anti–mouse CD5 (clone 53-7.3) and hamster IgM anti-mouse CD40 (clone HM40-3, no azide and low endotoxin format), all from BD Pharmingen; FITC-conjugated polyclonal goat anti-mouse IgM, phycoerythrin-conjugated rat anti-mouse IgM (clone 1B4B1), phycoerythrin-conjugated rat anti-mouse IgD (clone 11-26) and FITC-conjugated polyclonal goat anti-mouse IgG1, all from Southern Biotechnology Associates; FITC- and phycoerythrin-conjugated rat anti–mouse B220 (clone RA3-6B2), from Caltag Laboratories; purified rabbit polyclonal anti-SHP-1 and mouse phosphotyrosine mAb (clone 4G10), from Upstate Biotechnology; goat F(ab¢)2 anti-mouse IgM, from Cappel; and mouse IgA anti–mouse CD19 (clone MB19-1)46. For immunohistochemistry, spleen sections 5 mm in thickness were fixed in acetone, blocked with 5% rabbit serum and then stained with FITC-conjugated mAb to B220 and phycoerythrinconjugated mAb to CD1d. Digital images were merged to indicate marginal zone location. B cell purification and flow cytometry. Single-cell leukocyte suspensions from spleen, bone marrow (bilateral femur), peritoneal lavage and peripheral lymph nodes (bilateral inguinal and brachial) were isolated and erythrocytes were lysed in Tris-buffered 100 mM ammonium chloride solution. For splenic B cell purification, single-cell splenocyte suspensions were depleted of T cells with anti-Thy1.2 magnetic beads (Dynal). Leukocytes were then stained for 30 min at 4 1C with predetermined optimal concentrations of antibodies. For whole blood, erythrocytes were lysed after being stained with FACS Lysing Solution (Becton Dickinson). Antibody binding was analyzed on a FACScan flow cytometer (Becton Dickinson) by gating on cells with the forward and side light-scatter properties of lymphocytes. Nonreactive, isotype-matched antibodies (Caltag) were used as controls for background staining. RNA quantification. Total RNA was isolated from 1 107 purified spleen B cells with a RNeasy kit (Qiagen). Random hexamer primers (Promega) and

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SuperScript II RNase H Reverse Transcriptase (Invitrogen) were used to generate cDNA as described5. Forward and reverse primers specific for Cd22 exons 10 and 11 and Cd20 exons 3 and 5 were used for PCR amplification (primer sequences, Supplementary Methods online), with annealing temperatures and cycle times optimized to generate a single cDNA product of the appropriate size without genomic DNA amplification. Melting curve analysis was done to ensure amplification of appropriate PCR products. Relative Cd22 and Cd20 expression was determined by real-time PCR on a LightCycler Instrument with analysis software Version 3 and a LightCycler FastStart DNA MasterPLUS SYBR Green I kit (all from Roche). A standard curve was generated with tenfold serial dilutions of wild-type cDNA. As a control, 50% of wild-type cDNA was used for CD22 transcript amplifications relative to the amount of cDNA used to amplify CD20 transcripts. Pairwise comparisons between CD22, CD22D1-2 or CD22AA cDNA abundance relative to CD20 cDNA abundance were made with the relative expression software program for groupwise comparison and statistical analysis (http://www.bioinformatics.gene-quantification. info/). Results are expressed as the ratio between CD22 and CD20 transcript abundance relative to the ratio observed in wild-type littermates. Calcium response measurements. Splenocytes were isolated at 25 1C and were resuspended (1 107 cells/ml) in RPMI 1640 medium (Sigma) containing 5% FCS and 10 mM HEPES (Gibco BRL). The cells were loaded for 30 min at 37 1C with 1 mM Indo-1 AM ester (Molecular Probes), labeled for an additional 15 min with FITC-conjugated mAb to B220, washed and resuspended in warm medium at a density of 2 106 cells/ml for flow cytometry. Baseline emission fluorescence ratios (405:525 nm) of B220+-gated cells were collected for 1 min before the addition of F(ab¢)2 anti-IgM (10 or 40 mg/ml) or mAb to CD19 (40 mg/ml), with fluorescence ratios plotted at 20-second intervals. Increases in calcium mobilization are presented as increased ratios of fluorescence intensity after antibody treatment relative to the fluorescence intensity of untreated cells. Immunoprecipitation and immunoblotting. Purified splenic B cells were resuspended (2 107 cells/ml) in RPMI 1640 medium containing 5% FCS and 10 mM HEPES. After incubation for 5 min at 37 1C, the cells were left resting or were stimulated with F(ab¢)2 anti-IgM (40 mg/ml) for the times indicated in Figure 3b,c at 37 1C. The cells were then suspended in cold Dulbecco’s PBS containing 400 mM EDTA and 100 mM Na orthovanadate, were pelleted and were lysed in buffer containing 1% Nonidet-P40, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1 mM Na orthovanadate, 2 mM EDTA, 50 mM NaF and 1 mM phenylmethyl sulfonyl fluoride. For analysis of CD22 phosphorylation and SHP-1 coassociation, B cell lysate volumes were adjusted to achieve equivalent CD22 protein content between wild-type, CD22D1-2 and CD22DRR samples based on CD22 cell surface expression, and then lysates were immunoprecipitated with the MB22-8 mAb, followed by SDS-PAGE and electrophoretic transfer to nitrocellulose membranes. Direct immunoblotting for CD22 tyrosine phosphorylation used horseradish peroxidase–conjugated 4G10 mAb and indirect immunoblotting for coassociated SHP-1 with anti-SHP-1 followed by horseradish peroxidase–conjugated anti-rabbit IgG secondary antibodies. The membranes were subsequently stripped and reprobed with the MB22-1 mAb to confirm equivalent CD22 protein abundance between samples. For analysis of total tyrosine phosphorylation, whole-cell lysates were directly separated by SDS-PAGE with direct immunoblotting with horseradish peroxidase–conjugated 4G10 mAb. The blots were subsequently stripped and reprobed with anti-SHP-1 as a control to confirm equivalent total protein between samples. Immunoblots were developed with an enhanced chemiluminescence kit (Pierce). B cell proliferation assays. Purified splenic B cells were cultured for 72 h (2 105 cells per 0.2 ml of RPMI 1640 medium containing 10% FCS, 10 mM HEPES and 55 mM 2-mercaptoethanol) in triplicate wells of 96-well flatbottomed tissue culture plates either alone or in the presence of F(ab¢)2 antiIgM, mAb to CD40 or LPS (E. coli serotype 0111:B4, Sigma). Proliferation was measured by incorporation of [3H]thymidine added during the final 18 h of culture, followed by scintillation counting. Alternatively, B cells were labeled with 1 mM CFSE with a Vybrant CFDA SE Cell Tracer Kit (Molecular Probes) before cell culture. After 72 h, 96 h and 120 h, the relative number of viable cells (from 4 104 events recorded) and intensity of CFSE staining were assessed by flow cytometry.

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ARTICLES B cell migration assays. Migration assays were as described47. Purified splenic B cells (2 107) from wild-type, CD22D1-2, CD22AA or CD22KO mice were labeled with calcein-AM (Molecular Probes) and were mixed with an equal number of wild-type B cells labeled with PKH-26 (Sigma). Preinjection ratios of calcein- to PKH-26-labeled cells were determined by flow cytometry of a sample of the mixed cells before injection. The cells were then injected through the tail veins of wild-type recipient mice. After 1.5 or 16 h, single-cell leukocyte suspensions were prepared from the bone marrow, spleen and lymph nodes of recipient mice and were analyzed by flow cytometry to determine the ratios of calcein- to PKH-26-labeled cells. Statistical analysis. All values are means 7 s.e.m. unless indicated otherwise. Analysis of variance was used along with the Student’s t-test to determine the significance between sample means. Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTS We thank A. Jackson, A. Meade and I. Dzhagalov for assistance with real-time PCR assays. Supported by the National Institutes of Health (CA96547, CA81776 and AI56363) and a Biomedical Science Grant from the Arthritis Foundation (to T.F.T.). COMPETING INTERESTS STATEMENT The authors declare competing financial interests (see the Nature Immunology website for details). Received 31 March; accepted 25 August 2004 Published online at http://www.nature.com/natureimmunology/

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