Developmental Acquisition of the Lyn-CD22-SHP-1 Inhibitory Pathway Promotes B Cell Tolerance

This information is current as Andrew J. Gross, Julia R. Lyandres, Anil K. Panigrahi, Eline of October 1, 2021. T. Luning Prak and Anthony L. DeFranco J Immunol 2009; 182:5382-5392; ; doi: 10.4049/jimmunol.0803941 http://www.jimmunol.org/content/182/9/5382 Downloaded from

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

Developmental Acquisition of the Lyn-CD22-SHP-1 Inhibitory Pathway Promotes B Cell Tolerance1

Andrew J. Gross,* Julia R. Lyandres,† Anil K. Panigrahi,§ Eline T. Luning Prak,§ and Anthony L. DeFranco2‡

To better understand whether autoimmunity in Lyn-deficient mice arises from compromised central or peripheral B cell tolerance, we examined BCR signaling properties of wild-type and Lyn-deficient B cells at different stages of development. Wild-type mature follicular B cells were less sensitive to BCR stimulation than were immature transitional stage 1 B cells with regard to BCR- induced calcium elevation and ERK MAPK activation. In the absence of Lyn, mature B cell signaling was greatly enhanced, whereas immature B cell signaling was minimally affected. Correspondingly, Lyn deficiency substantially enhanced the sensitivity of mature B cells to activation via the BCR, but minimally affected events associated with tolerance induction at the immature stage. The effects of CD22 deficiency on BCR signaling were very similar in B cells at different stages of maturation. These results Downloaded from indicate that the Lyn-CD22-Src homology region 2 domain-containing phosphatase-1 inhibitory pathway largely becomes oper- ational as B cell mature, and sets a threshold for activation that appears to be critical for the maintenance of tolerance in the B cell compartment. The Journal of Immunology, 2009, 182: 5382–5392.

cell receptor hyperresponsiveness is associated with a would lead to a loss of tolerance in lynϪ/Ϫ mice is not entirely breakdown in tolerance and the development of autoan- self-evident, because whereas BCR signaling drives activation of B tibodies in several genetically modified mouse strains (1, mature B cells, it also promotes tolerance-inducing mechanisms in http://www.jimmunol.org/ 2). A good example of this is the Lyn-deficient mouse (3). Lyn is immature B cells. For example, binding of Ag by immature B cells a Src family kinase (SFK)3 that, like two other SFKs expressed by in the bone marrow (BM) induces expression of RAG 1 and 2, B cells, Blk and Fyn, phosphorylates ITAMs on BCR Ig␣/Ig␤ which can generate L chain rearrangement and thereby change the chains following Ag binding (4–6). Lyn also functions to phos- specificity of the BCR (receptor editing) (16–18). Continued rec- phorylate ITIMs on inhibitory receptors that negatively regulate ognition of Ag by the BCR may induce apoptosis of self-reactive BCR signaling, including CD22 (5, 7–9), Fc␥RIIb (7, 8), and per- B cells (clonal deletion) (16). Therefore, genetic alterations that haps others (10–13). In this way, Lyn facilitates recruitment of the enhance BCR signaling should increase the sensitivity of immature

Src homology region 2 domain-containing phosphatase-1 (SHP-1) B cells to self Ags, causing more thorough removal of autoreactive by guest on October 1, 2021 and SHIP phosphatases to the plasma membrane, which down- BCR specificities from the newly formed B cell repertoire. For this modulate BCR signaling (5, 8, 9). In the absence of Lyn, BCR reason, it seems paradoxical that autoimmunity develops in the signaling is supported by Blk and Fyn, but inhibitory receptors are lynϪ/Ϫ mouse and in other mice with genetic alterations increasing ineffective at down-regulating BCR signaling, thereby leading to the strength of BCR signaling. BCR hyperresponsiveness (3). To account for the break in B cell tolerance observed in Lyn- Lyn-deficient mice exhibit increased plasma cell numbers and deficient mice, we hypothesized that the level of Lyn-mediated serum Ig levels. Surprisingly, these mice also produce autoanti- inhibitory signaling might change during the course of B cell de- bodies to nuclear Ags (4, 14, 15). Why elevated BCR signaling velopment. Interestingly, we found that B cells become less sensitive to BCR engagement as they proceed through development. This reduction in BCR sensitivity was mediated by Lyn and CD22, *Department of Medicine, †Department of Surgery, and ‡Department of Microbiol- because Lyn or CD22 deficiency strongly increased BCR signaling ogy and Immunology, University of California, San Francisco, CA 94143; and §De- partment of Pathology and Laboratory Medicine, University of Pennsylvania, Phila- in follicular B cells, but only weakly increased signaling in tran- delphia, PA 19104 sitional stage 1 (T1) B cells. Consistent with these findings, Lyn Received for publication December 1, 2008. Accepted for publication February deficiency had a modest effect on events associated with receptor 13, 2009. editing, whereas it more strongly enhanced the sensitivity of ma- The costs of publication of this article were defrayed in part by the payment of page ture B cells to BCR-induced proliferation. These results indicate charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. that inhibition of BCR signaling by the Lyn-CD22-SHP-1 pathway is up-regulated as B cells mature in the spleen, and suggest that this 1 This work was supported by National Institutes of Health Grant K08 AI52249 (to A.J.G.), by the Rosalind Russell Medical Research Center for Arthritis, and by Na- developmental change is involved in the maintenance of peripheral tional Institutes of Health Grant R01 AI20038 (to A.L.D.). immune tolerance of B cells. 2 Address correspondence and reprint requests to Dr. Anthony L. DeFranco, Univer- sity of California, 400 Parnassus Avenue, Box 0414, San Francisco, CA 94143. E-mail address: [email protected] Materials and Methods 3 Abbreviations used in this paper: SFK, Src family kinase; BM, bone marrow; Mice [Ca2ϩ] , intracellular Ca2ϩ concentration; HEL, hen egg lysozyme; mIg, membrane- i Mice aged 7–12 wk were used for most experiments. Four-week-old mice bound Ig; NF, immature newly formed; SHP1, Src homology region 2 domain-con- me-v taining phosphatase-1; T1, transitional stage 1; T2, transitional stage 2; T3, transi- were used in experiments using motheaten-viable mice (Ptpn6 ) back- Ϫ/Ϫ tional stage 3; WT, wild type; RS, recombining sequence. crossed onto C57BL/6 background (The Jackson Laboratory). The lyn mice were used as described (4), and backcrossed at least 15 generations Ϫ Ϫ Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 onto C57BL/6 background. The cd22 / mice (19) backcrossed at least 16 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803941 The Journal of Immunology 5383

generations onto C57BL/6 background were obtained from E. Clark (Uni- Proteins were separated on a NuPAGE bis-Tris gel (Invitrogen) with

versity of Washington, Seattle, WA). MD4 transgenic IgHel mice were MOPS buffer and immunoblotted on Immobilon-FL transfer membrane obtained from J. Cyster (University of California, San Francisco, CA). All (Millipore). Abs were directed against phospho-Src family kinase Tyr416 animals were housed in a specific pathogen-free facility at University of (Cell Signaling Technology; 2101s), phospho-Lyn Tyr507 (Abcam California, according to University and National Institutes of Health guide- ab53122), Lyn (Abcam ab1890), and BAP31 (Abcam ab37120) as a load- lines. Animal use was approved by the University of California Institu- ing control. These Abs were detected with fluorescently labeled secondary tional Animal Care and Use Committee. Abs using the Odyssey Infrared Imaging System (LI-COR Biosciences). Abs, immunofluorescence analysis, B cell purification, and cell Real-time RT-PCR sorting Total RNA was isolated from sorted B cells using the RNeasy Micro kit Fluorophore-conjugated Abs directed against the following molecules were (Qiagen), and cDNA was transcribed using the iScript cDNA synthesis kit ␭ used: B220 (RA3-6B2), CD23 (B3B4), IgM (II/41), Ig 1–3 (R26-46), (Bio-Rad), according to the manufacturers’ instructions. Equivalent CD16/CD32 (2.4G2) CD22.2 (Cy34.1), and CD72 (K10.6) all from BD amounts of cDNA were used in quantitative PCR on an ABI Prism 7700 Pharmingen; CD24 (M1/69) from BioLegend; CD93 (AA4.1) and CD5 sequence detection instrument (Taqman; PerkinElmer Applied Biosys- ␬ ␭ (53-7.3) from eBioscience; Ig (187.1) and Ig (JC5-1) from Southern tems). Primer and probe sequences for RAG1 and RAG2 (20), and Biotechnology Associates; and IgM (goat polyclonal Fab monomer, GAPDH (21) were used as published. ␮-chain specific) (Jackson ImmunoResearch Laboratories). Cells were analyzed on an LSR-II or FACSCalibur (both from BD Pharmingen). Lymph Assay of V␬-recombining sequence (RS) rearrangement node B cells were purified by negative selection using CD43 microbeads (Miltenyi Biotec), according to the manufacturer’s protocol, and passage Genomic DNA was isolated from sorted Ig␭Ϫ B cell populations using the through an autoMACS Separator (Miltenyi Biotec). For cell sorting, Gentra PureGene tissue kit (Qiagen). Quantitative PCR was performed, as splenocytes or BM cells were stained for CD23, CD93, and IgM Fab described (22). The amount of V␬-RS product in each sample was nor-

monomer for 30 min and either B220 or a non-B cell mixture (CD4, CD8, malized to the amount of ␤-actin product and compared with the normal- Downloaded from CD11b, Gr1, NK1.1, and Ter119) in HBSS, 1% FCS, and 0.5% BSA. Cells ized target value in wild-type (WT) C57BL/6 follicular B220ϩ IgMϩ ␬ϩ were then sorted on a MoFlo sorter (DakoCytomation). Dead cells were splenocytes to determine a relative quantity (comparative cycle threshold excluded by propidium iodide (BioChemika) uptake. All FACS data were method). analyzed with FlowJo version 6.4.1 (Tree Star software). Analysis of calcium mobilization Statistical analyses Statistical analyses, including unpaired two-tailed t test and four-parameter

For measurements of intracellular-free calcium levels, splenocytes or BM http://www.jimmunol.org/ cells were loaded with Indo-1 AM (Molecular Probes/Invitrogen) and then logistic equation (linear regression analysis), were performed with Prism labeled for CD93 (allophycocyanin), CD23 (PE), IgM (Fab monomer) v.4.0 (GraphPad software). (FITC), and B220 (PE-Cy7), or a mixture of Abs recognizing non-B cells (CD4, CD8, CD11b, Gr1, NK1.1, Ter119) (PE-Cy7). Cells were resus- Results pended in RPMI 1640 supplemented with 1% BSA and 20 mM HEPES and T1 and transitional stage 2 (T2) B cells are more sensitive to warmed to 37°C for 3 min, and analysis was initiated with flow cytometry. After the baseline was established for 30–45 s, cells were stimulated with BCR stimulation than mature follicular B cells ␮ Ј 0.5–50 g/ml goat anti-mouse IgM F(ab )2 (Jackson ImmunoResearch It has previously been suggested that immature B cells are more Laboratories), 5 ng to 100 ␮g of 0.2-␮m filtered hen egg lysozyme (HEL; Sigma-Aldrich), or 16 ␮g/ml ionomycin (Sigma-Aldrich). Median intra- sensitive than mature B cells to limiting amounts of BCR stim- cellular calcium concentration as indicated by the ratio of fluorescence 405 ulation (23, 24), but other studies have not agreed with this by guest on October 1, 2021 nm emission to 530 nm emission was measured over time by flow cytom- conclusion (25, 26). These earlier studies have used various etry. Dead cells were excluded by propidium iodide uptake. manipulations to generate sufficient numbers of immature B Assay of ERK phosphorylation cells to analyze signaling. To better evaluate this issue, we analyzed calcium elevation in unmanipulated B cells taken di- For intracellular measurement of phospho-ERK, 3 ϫ 106 splenocytes or 2 ϫ 106 BM cells were warmed to 37°C for 30 min in RPMI 1640 medium rectly ex vivo from young adult mice. B cells at various devel- with 1% BSA and 20 mM HEPES. During the final 10 min of warming, the opmental stages were identified by a combination of mAbs, as cells were labeled with anti-IgM Fab FITC. Some samples were treated described by Allman et al. (27) (Fig. S1 in the supplemental with 10 ␮M MEK inhibitor U0126 (Sigma-Aldrich) during the warming data).4 We found this Ab combination, which included non- period. Cells were then stimulated with 0–50 ␮g/ml goat anti-mouse IgM Ј ␮ stimulating anti-IgM Fab FITC, had minimal effects on BCR- Fab )2 (Jackson ImmunoResearch Laboratories) or 1 g/ml PMA (Sigma- 4 Aldrich). After the desired period of stimulation, cells were fixed with 4% induced calcium signaling (Fig. S2). When WT splenic B cells paraformaldehyde (Electron Microscopy Sciences) and permeabilized with were stimulated with subsaturating concentrations of anti-IgM ice-cold 100% methanol (Electron Microscopy Sciences). Cells were la- F(abЈ) , T1 and T2 B cells exhibited greater increases in cyto- 202 204 2 beled with phospho-p44/42 (Thr /Tyr ) rabbit mAb (197G2; Cell Sig- plasmic calcium levels (intracellular Ca2ϩ concentration naling Technology) that recognizes phosphorylation sites required for ERK 2ϩ activity. Cells were then labeled with donkey anti-rabbit IgG allophyco- ([Ca ]i)) than did transitional stage 3 (T3) and mature follic- cyanin (Jackson ImmunoResearch Laboratories) as well as Abs to B220 ular B cells (Fig. 1, A–C). All of these B cell subsets had similar (PE-Cy7), CD23 (PE), and CD24 (Pacific blue). (The fixation process pre- maximal calcium responses when stimulated with a saturating vented labeling for CD93.) Cells were analyzed by flow cytometry on an Ј ␮ concentration of anti-IgM F(ab )2 (50 g/ml). Although it has LSR-II. been proposed that T3 B cells represent an anergic population B cell proliferation assays of B cells (28), we observed that T3 B cells responded to anti- 2ϩ Sorted splenic B cells from four mice of each genotype were suspended at IgM by increasing [Ca ]i similarly to follicular B cells. These a concentration of 2 ϫ 106 cells/ml in RPMI 1640 with 10% FCS, 20 mM data indicate that mature follicular B cells require more BCR HEPES, 2 mM glutamine, and 1 mM sodium pyruvate, and stimulated with stimulation than immature T1 and T2 B cells to induce com- ␮ Ј 0–15 g/ml goat anti-mouse IgM F(ab )2 (Jackson ImmunoResearch Lab- parable BCR-induced calcium signaling. oratories) in 96-well tissue culture plates (Corning Glass) at 37°C, 5% As B cells mature, IgM levels are down-regulated and IgD lev- CO2. For proliferation assay, cells were incubated for 48 h. A total of 1 ␮Ci/well [3H]thymidine (Amersham) was added during the final4hof els are up-regulated (29). We therefore wondered whether treat- culture, and incorporation was measured by scintillation counting. ment of B cells with anti-IgM accurately measured the sensitivity of B cells to BCR engagement at each stage of development. To Western blotting Whole-cell lysates were prepared in SDS lysis buffer from sorted splenic B cells or lymph node B cells purified by autoMACS negative selection. 4 The online version of this article contains supplemental data. 5384 THE ROLE OF Lyn IN B CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. Immature T1 B cells are more sensitive than mature follicular B cells to anti-IgM stimulation. A, Cytoplasmic-free calcium levels indicated 2ϩ ϩ ϩ Ϫ 2ϩ ϩ ϩ by indo-1 fluorescence emission ratio at 405 and 530 nm ([Ca ]i F405/F530) in WT splenic B220 T1 (AA4.1 , CD23 , IgM ), T2 (AA4.1 , CD23 , IgM2ϩ), T3 (AA4.1ϩ, CD23ϩ, IgMϩ), and mature follicular B cells (AA4.1Ϫ, CD23ϩ, IgMϩ) (as described by Allman et al. (27) and shown in Fig. S1)4 ␮ Ј ϭ stimulated with 5 g/ml goat anti-mouse IgM F(ab )2. Arrows indicate time at which cells were stimulated with anti-IgM. Red line at F405/F530 0.39 indicates a baseline threshold level, in which 90% of unstimulated WT follicular B cells were below this F405/F530 value. B, Calcium response in WT ␮ Ј T1 (green), T2 (blue), T3 (gold), and follicular (red) splenic B cells stimulated with 5 or 50 g/ml anti-IgM F(ab )2. Left panels, Depict median cytoplasmic calcium level as indicated by indo-1 F405/F530 ratio. Right panels, Indicate percentage of cells with indo-1 F405/F530 ratio above baseline (0.39). C, Dose Ј response of WT B cell populations to anti-IgM F(ab )2 stimulation, as measured by peak median F405/F530 elevation (left panel) or maximal percentage of cells responding above baseline (F405/F530 Ͼ 0.39) (right panel). Responses to no stimulation (none) and 16 ␮g/ml ionomycin (iono) are indicated. n ϭ 2; similar data obtained from four additional experiments. The sigmoidal dose-response curves were generated by nonlinear regression; R2 Ͼ 0.96 for each B cell population analyzed (four-parameter logistic equation). D, Calcium response in WT B220ϩ BM NF (AA4.1ϩ, CD23Ϫ, IgM2ϩ) (black dashed line) and mature recirculating (AA4.1Ϫ, CD23ϩ, IgMϩ) (purple dashed line) B cells, as well as splenic T1 (green solid line) and follicular (red solid line) ␮ Ј B cells stimulated with 5 g/ml anti-IgM F(ab )2. address this issue, we therefore gated on the subpopulation of fol- using Ig transgenic B cells specific for hen-egg lysozyme (HEL). licular B cells with levels of membrane-bound Ig (mIg)M compa- Similar to the results obtained when an anti-IgM reagent was used, rable to the level of mIgM on T1 B cells. These cells still exhibited immature transitional B cells exhibited greater increases in cyto- decreased sensitivity to anti-IgM for the elevation of intracellular- plasmic calcium levels than did follicular B cells when treated with free calcium compared with the T1 B cells (data not shown). To subsaturating concentrations of HEL, whereas their responses to investigate whether reduction in mature B cell sensitivity to Ag is higher doses of HEL were similar (Fig. 2). These data are consis- averted during development through up-regulation of surface IgD tent with those reported in another BCR transgenic system (23) levels, we measured the sensitivity of B cells to Ag engagement and confirm that immature T1 B cells are in fact more sensitive to The Journal of Immunology 5385

FIGURE 2. Immature B cells are more sensitive than mature follicular B cells to stimulation with Ag. A, Cy- toplasmic-free calcium levels, indicated by indo-1 ﬂuorescence emission ratio as in Fig. 1A,ofIghel transgenic B cells stimulated with 100 ng/ml HEL. Arrows indicate time at which cells were stimulated with HEL. Red line at F405/F530 ϭ 0.88 indicates a baseline threshold level, in which 90% of unstimulated WT follicular B cells were below this F405/ F530 value. Note, T2 and T3 B cells cannot be distinguished in these mice because transgenic expression of Ig causes uniformly high expression levels of surface IgM. Difference in y- axis scale between data shown in this

ﬁgure and Fig. 1 was due to change in Downloaded from ﬂow cytometer equipment used. B, Ag stimulation of WT B cells transgenic for Ighel. Calcium release by T1, T2–3, and mature follicular B cells stimulated with various concentrations of HEL. C, Dose response of http://www.jimmunol.org/ WT Ighel B cell populations to HEL stimulation, as measured by the peak calcium response seen in B. n ϭ 2; similar data obtained from three additional experiments.

BCR stimulation than mature follicular B cells. These data also signaling reactions, we examined BCR-induced ERK activation as by guest on October 1, 2021 show that stimulation of B cells with anti-IgM accurately reflects assessed by flow cytometry. This assay was largely specific be- the relative responses of different B cell populations to BCR en- cause the response was nearly completely blocked by the MEK gagement by Ag. inhibitor U0126 (Fig. S3).4 Stimulation of WT spleen cells with

ϩ anti-IgM led to significant increases in the levels of ERK phos- Immature IgM B cells in the BM are also more sensitive to phorylation in the various B cell subpopulations. Peak levels of BCR stimulation than mature B cells phospho-ERK were detected in B cells after 2–3 min of stim- Because the first events associated with the establishment of B cell ulation (Fig. S3).4 As with calcium elevation, ERK activation tolerance occur in the BM, it was important to determine whether after 2.5 min was induced by lower doses of anti-IgM in T1 B B cells at various developmental stages in the BM respond to BCR cells than in mature follicular B cells (Fig. 3, A–C). Interest- stimulation in a manner similar to their splenic counterparts. We ingly, even at high doses of anti-IgM (50 ␮g/ml), follicular B applied the same gating strategy for identifying B cell populations cells activated ERK to a lesser degree than did immature T1 B in the spleen to BM cells (30) (Fig. S1).4 Calcium responses to cells. In contrast, stimulation with the diacylglycerol mimetic, anti-IgM stimulation of BM B cell subpopulations were remark- PMA, induced higher levels of ERK phosphorylation in follic- ably similar to those of corresponding splenic B cell populations ular B cells than in T1 B cells (Fig. 3B). Phospho-ERK levels (Fig. 1D, and data not shown for 20 ␮g/ml anti-IgM). Splenic T1 in stimulated BM B cell populations were similar to those in the cells responded nearly identically to immature newly formed (NF) corresponding stimulated splenic B cell populations (Fig. 3C). cells, splenic T2 cells responded identically to BM-T2-like cells Thus, data for both elevation of intracellular-free calcium and (data not shown), and splenic follicular B cells responded identi- activation of ERK MAPKs showed that WT immature B cells cally to mature naive B cells recirculating in the BM. As expected, are more sensitive to BCR stimulation than are WT follicular B the pro-B and pre-B cell populations had minimal or no response cells. to anti-IgM (data not shown). These data indicate that WT B cells become less sensitive to low levels of BCR stimulation as they transition from immaturity to maturity, regardless of whether they Lyn deficiency greatly magnifies calcium response by follicular reside in the BM or spleen. B cells, but only slightly enhances response by T1 B cells Although Lyn-deficient B cells are known to have elevated sig- Immature B cells are more sensitive to BCR stimulation than naling responses to BCR engagement (5, 7), it was not known mature B cells for BCR-induced ERK phosphorylation whether this hypersensitivity is dependent on the maturation stage To determine whether the changes in calcium signaling during B of the B cell. Spleens from lynϪ/Ϫ mice contained T1, T2, T3, and cell development were representative of changes in other BCR follicular B cell populations (Fig. S1),4 as previously reported (27). 5386 THE ROLE OF Lyn IN B CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Lyn deficiency strongly enhances activation of ERK in follicular B cells, but has a lesser effect on T1 B cells. A, ERK activation, as measured by flow cytometry analysis of intracellular staining with a mAb recognizing the activating phosphorylations of ERK1 and ERK2, in B cell populations identified by B220, IgM, CD24, and CD23 expression (see Fig. S3).4 Phospho-ERK histograms of unstimulated WT splenic B220ϩ T1 (CD242ϩ, CD23Ϫ, IgM2ϩ)(left panel) or follicular (CD24ϩ, CD23ϩ, IgMϩ)(right panel) B cells (filled-in gray histograms) or of these cells stimulated ␮ ␮ Ј ␮ with 1 g/ml (light blue) or 50 g/ml (purple) goat anti-mouse IgM F(ab )2 or with 1 g/ml PMA (orange dashed) for 2.5 min, as shown. B, ERK activation Ј ␮ ϭ dose response of WT B cell populations after 2.5 min of anti-IgM F(ab )2 stimulation. Response to 1 g/ml PMA is indicated. n 4; data points represent the mean Ϯ SD; R2 Ͼ 0.98 for each sigmoidal dose-response curve (four-parameter logistic equation); similar data were obtained in two additional experiments. C, Comparison of ERK activation in BM and spleen B cell populations. n ϭ 4; bars represent means Ϯ SD; one of three experiments with similar findings. D, ERK activation in WT (blue lines) and lynϪ/Ϫ (red lines) T1 (left panel) and follicular B cells (right panel). Phospho-ERK geometric mean fluorescent intensity levels of unstimulated B cell populations (filled-in gray histogram for WT, dashed red histogram for lynϪ/Ϫ) and B cells ␮ Ј Ϫ/Ϫ stimulated with 50 g/ml anti-IgM F(ab )2 for 2.5 min. E, Comparison of ERK activation dose response of WT (solid lines, solid symbols) and lyn (dashed lines, open symbols) splenic T1 (green lines) and follicular B cells (red lines). n ϭ 4 per genotype; data points represent the mean Ϯ SD; R2 Ͼ 0.97 for each sigmoidal dose-response curve (four-parameter logistic equation); similar data were obtained in three additional experiments.

We found that the calcium responses of lynϪ/Ϫ follicular B cells tude of BCR signaling as well as dampening the sensitivity of were far more sensitive to IgM stimulation than were WT mature B cells to IgM stimulation. follicular B cells (Fig. 4, A–C), and their ability to respond to low In contrast to the large effect of Lyn deﬁciency on mature follicular doses of anti-IgM resembled WT immature B cells. Additionally, B cells, Lyn deﬁciency had a relatively minor effect on calcium re- the peak amplitude of the calcium response by lynϪ/Ϫ follicular B sponses by immature T1 B cells (Fig. 4, A–C). The peak amplitude as 2ϩ cells was greatly exaggerated over that of WT follicular B cells well as the sustained level of [Ca ]i following BCR stimulation was even at saturating doses of anti-IgM (Fig. 4, A–C). These data only slightly higher in lynϪ/Ϫ T1 cells compared with WT T1 B cells. indicate that Lyn serves an important role in limiting the magni- Similarly, lynϪ/Ϫ newly formed BM B cells had only slightly higher The Journal of Immunology 5387 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. Lyn deficiency has a major effect on calcium responses in follicular B cells, but a lesser effect on those in T1 B cells. A, Cytoplasmic- 2ϩ Ϫ/Ϫ ␮ free calcium levels ([Ca ]i F405/F530) in splenic T1, T2, T3, and follicular (Fo) B cells from lyn mice stimulated with 5 g/ml goat anti-mouse Ј ϭ IgM F(ab )2. Red line at indo-1 ratio F405/F530 0.39 was the 90th percentile for the fluorescence ratio of unstimulated WT follicular B cells. B, Calcium release in WT (blue lines) and lynϪ/Ϫ (red lines) T1 and follicular splenic B cells after stimulation with 5 ␮g/ml (dashed lines) and 50 ␮g/ml Ј 2ϩ (solid lines) anti-IgM F(ab )2. Left panels, Depict median [Ca ]i as measured by fluorescence ratio (F405/F530). Right panels, Indicate percentage of cells with indo-1 F405/F530 ratio above baseline (0.39). C, Dose response by WT (solid lines, solid symbols) and lynϪ/Ϫ (dashed lines, open Ј ␮ symbols) T1 and follicular (Fo) B cells to anti-IgM F(ab )2 stimulation. Responses to no stimulation (none) and 16 g/ml ionomycin (iono) are indicated. R2 Ͼ 0.95 for each sigmoidal dose-response curve (four-parameter logistic equation). n ϭ 2 for WT, n ϭ 3 for lynϪ/Ϫ; similar data obtained in four additional experiments. D, Calcium response in WT (solid lines) and lynϪ/Ϫ (dashed lines) BM NF (black lines) and mature ␮ Ј 2ϩ Ϫ/Ϫ recirculating (purple lines) B cells stimulated with 5 g/ml anti-IgM F(ab )2. (Note: the serrated line representing [Ca ]i in lyn BM mature recirculating B cells reflects the low numbers of cells in this population.)

2ϩ elevations of [Ca ]i than their WT counterparts (Fig. 4D). These data pared with WT T1 B cells (Fig. 3, D and E). Thus, BCR-induced indicate that Lyn greatly inhibits BCR calcium signaling by follicular ERK activation and calcium elevation were both strongly en- B cells, but surprisingly provides much less restraint on BCR calcium hanced in follicular B cells lacking Lyn, whereas these signaling signaling by newly formed or T1 B cells. events were not greatly affected in T1 B cells.

BCR-induced ERK phosphorylation is greatly enhanced by Lyn deficiency in follicular B cells, but not in T1 B cells Surface expression levels of inhibitory receptors during B cell To confirm the effects of Lyn deficiency on BCR signaling, we development examined ERK phosphorylation in lynϪ/Ϫ B cells. Anti-IgM-stim- These data collectively indicate that Lyn has a stronger effect on ulated lynϪ/Ϫ follicular B cells had much higher levels of phospho- modulating BCR signaling in mature B cells than in immature ERK than stimulated WT follicular B cells (Fig. 3, D and E). In B cells. Because Lyn can negatively regulate BCR signaling contrast, lynϪ/Ϫ T1 B cells had either slightly lower or similar through the recruitment of ITIM-bearing inhibitory proteins, we levels of ERK phosphorylation following BCR stimulation com- wondered whether the strong effect of Lyn on mature B cell 5388 THE ROLE OF Lyn IN B CELL DEVELOPMENT

FIGURE 5. Greater effect of CD22 on follicular B cells than on T1 B cells. A, Surface levels of CD22, CD16/CD32 (which on B cells is primarily Fc␥RIIb), CD5, and CD72 on WT splenic T1 (dashed lines) and follicular B cells (solid lines). Isotype control shown as filled-in gray histograms. See Table I for mean fluorescent intensity quantification. B, Cyto- 2ϩ plasmic-free calcium levels ([Ca ]i F405/ F530) in splenic T1 and follicular B cells from cd22Ϫ/Ϫ (green lines), WT (blue lines), and lynϪ/Ϫ mice (red lines) stimulated with 2, 5, and 50 ␮g/ml goat anti- Ј mouse IgM F(ab )2. Arrows indicate time at which cells were stimulated with anti- IgM. Similar data were obtained in multiple experiments. The delayed calcium response in lynϪ/Ϫ B cells compared with cd22Ϫ/Ϫ B cells most likely reflects the

positive signaling function of Lyn in phos- Downloaded from phorylating BCR ITAMs. C, Cytoplas- 2ϩ mic-free calcium levels ([Ca ]i F405/ F530) in BM NF from WT (blue lines), cd22Ϫ/Ϫ (green lines), lynϪ/Ϫ mice (red lines), and motheaten-viable mice that have a loss of function mutation in the me-v/me-v SHP-1 gene, ptpn6 (shp-1 ) (or- http://www.jimmunol.org/ ange lines), that were stimulated with 2, 5, and 50 ␮g/ml goat anti-mouse IgM Ј F(ab )2. Arrows indicate time at which cells were stimulated with anti-IgM. Similar data were obtained in multiple experiments. Analysis of mature B cell responses was not possible because of the extremely limited size of this population in motheaten-viable mice. D, Sur- face levels of CD22 in splenic follicular by guest on October 1, 2021 (Fo) and T1 B cells from cd22ϩ/Ϫ and WT mice. E, Cytoplasmic-free calcium levels in splenic T1 (solid lines) and follicular B cells (dashed lines) from cd22ϩ/Ϫ (green lines) and WT mice (blue lines). signaling was due to increased expression of ITIM-bearing in- magnitude to the responses of lynϪ/Ϫ B cells in both mature and hibitory receptors during B cell development. Consistent with immature B cells (Fig. 5B). Additionally, calcium responses of BM this idea, we found that CD22 expression on the surface of B NF B cells from motheaten-viable mice, which have a partial loss cells was increased 2.5-fold from the T1 stage to the follicular of function mutation in the ptpn6 gene that encodes the tyrosine stage (Fig. 5A; Table I) in agreement with earlier studies (31, phosphatase SHP-1 (shp-1me-v/me-v) (33), were slightly reduced 32). However, expression of CD16/32, which on B cells is pri- compared with WT NF B cells (Fig. 5C). These data indicate that marily Fc␥RIIb, was largely unchanged, and two other negative the Lyn-CD22-SHP-1 inhibitory pathway has minor effects on lim- regulators of BCR signaling, CD72 and CD5, were down-reg- iting BCR signaling in immature B cells, but as B cells mature Lyn ulated during B cell maturation in the spleen (levels reduced by functions in conjunction with CD22 to mediate reduction in BCR 35 and 37%, respectively) (Fig. 5A; Table I). sensitivity. Because CD22 expression increases during B cell development, CD22 deficiency leads to greatly enhanced calcium responses we investigated whether reduction of CD22 levels in mature fol- by follicular B cells, but only slightly increased responses licular B cells could cause these cells to signal in a manner more by T1 B cells similar to immature B cells. To test this, we studied follicular B Because CD22 expression increases substantially during B cell cells from cd22ϩ/Ϫ mice, whose CD22 surface levels are similar to development, we hypothesized that CD22 deficiency would have those of WT T1 B cells (Fig. 5D). We found that calcium release disproportionate effects on mature B cells as compared with im- by cd22ϩ/Ϫ follicular B cells was enhanced compared with WT, mature B cells. Indeed, cd22Ϫ/Ϫ follicular B cells were much more although it was not as robust as the calcium release of WT T1 B sensitive to low-dose BCR stimulation and had much higher cells (Fig. 5E). These data indicate that up-regulation of CD22 2ϩ [Ca ]i than WT follicular B cells (Fig. 5B). In contrast, CD22 during B cell development has an appreciable role in reducing deficiency had relatively minor effects on T1 B cells. Surprisingly, BCR sensitivity, but that additional factors are also likely to the calcium responses of cd22Ϫ/Ϫ B cells were very similar in contribute. The Journal of Immunology 5389

Table I. Surface levels of negative regulators during splenic B cell developmenta

T1 T2 T3 Follicular T1 to Follicular Inductionb

CD22 617 Ϯ 36 1123 Ϯ 66 1119 Ϯ 45 1523 Ϯ 47 2.5 CD16/CD32 1612 Ϯ 35 1687 Ϯ 37 1366 Ϯ 32 1516 Ϯ 21 0.9 CD5 156 Ϯ 15 178 Ϯ 16 143 Ϯ 12 101 Ϯ 10 0.6 CD72 854 Ϯ 33 684 Ϯ 22 759 Ϯ 22 541 Ϯ 6 0.6

a Data indicate the geometric mean fluorescent intensity. Data represent the mean Ϯ SD of five WT mice; the experiment was repeated three times with similar results. b Represents the geometric mean fluorescent intensity at the follicular stage divided by the geometric mean fluorescent intensity at the T1 stage.

Tyrosine phosphorylation of Lyn changes during B cell of development and did not exhibit a substantial change upon BCR development and is regulated by CD22 stimulation (Fig. 6A). Interestingly, Y397 phosphorylation was el- We next investigated whether Lyn levels or function might change evated in unstimulated follicular B cells compared with immature during B cell development to contribute to the acquisition by fol- transitional B cells. However, upon BCR stimulation, Y397 phos- licular B cells of the Lyn-CD22-SHP-1 inhibitory pathway. We phorylation increased to similar levels in each of the B cell subsets sorted T1, T2, T3, and follicular B cells from spleens of WT mice analyzed (Fig. 6A). These ﬁndings suggest that Lyn activity is and measured Lyn protein levels and levels of tyrosine phosphor- enhanced in unstimulated follicular B cells, compared with imma- Downloaded from ylation at its activating and inhibitory regulatory sites by immu- ture B cells. Although these data do not directly assess Lyn func- noblotting of the cell lysates. Lyn protein levels did not change tion inside the B cell, they suggest that Lyn is in a more activated substantially throughout B cell maturation in the spleen (Fig. 6A, state in resting follicular B cells, potentially explaining in part the and data not shown), which is consistent with results from pub- enhanced activity of the Lyn-CD22-SHP-1 inhibitory pathway in lished mRNA microarray analysis of B cell developmental stages these cells compared with immature T1 B cells. Although CD22 is generally considered to be downstream of in the BM (34). Like other src family kinases, Lyn kinase activity http://www.jimmunol.org/ is regulated by phosphorylation of two tyrosine sites (3). Lyn as- Lyn, in that Lyn appears to be responsible for phosphorylating the sumes an inactive conformational state when the C-terminal Y507 ITIM of CD22 leading to SHP-1 recruitment to the plasma mem- site is phosphorylated, whereas kinase activity is enhanced follow- brane, we observed that CD22 expression also affected the activity Ϫ/Ϫ ϳ ing phosphorylation of Y397 in the kinase domain. We found that of Lyn. Lymph node B cells from CD22 mice had a 3- to Y507 phosphorylation was similar in splenic B cells at each stage 4-fold reduction in Lyn Y397 phosphorylation levels, particularly in unstimulated B cells (Fig. 6B). The levels of phosphorylation of Y507 were either slightly increased or unchanged (Fig. 6B). These results suggest that whereas CD22 acts downstream of Lyn to re- T1 T2 T3Fo Lyn-/- T1 T2 FoT3Lyn-/-

A by guest on October 1, 2021 U S U S U S U S U S U S U S U S cruit phosphatases to the plasma membrane and inhibit BCR sig- pY397 pY507 naling, it also acts upstream of Lyn to positively regulate Lyn activity. Lyn Lyn

Bap31 Bap31

pY397:Lyn 1.0 3.4 1.9 4.41.1 2.6 3.2 3.8 1.7 1.0 1.1 1.2 1.0 0.7 1.0 1.1 pY507:Lyn Lyn deficiency minimally affects receptor editing Genetic deficiency in Lyn, CD22, or SHP-1 results in spontaneous B WT cd22-/- WT cd22-/- U S U S U S U S autoantibody production (4, 14, 15, 35, 36), but it is not known pY397 pY507 whether this failure of tolerance occurs primarily during B cell development in the BM or later in the periphery. Because lynϪ/Ϫ Lyn Lyn T1 B cells in the spleen and BM had signaling responses to BCR stimulation that were only slightly greater than those of WT cells, Bap31 Bap31 we hypothesized that Lyn deficiency would either have no effect pY397:Lyn 1.0 1.9 0.3 0.8 1.0 1.4 1.2 1.8 pY507:Lyn on events related to the establishment of B cell tolerance in the BM FIGURE 6. Lyn tyrosine-phosphorylation status in B cells of different or would slightly exaggerate these events. It is thought that BCR developmental stages. A, Sorted WT splenic B cell populations were stim- ␮ Ј signaling above a certain threshold in pre-B and immature B cells ulated with 50 g/ml anti-IgM F(ab )2 for 2 min (S) or with medium alone (U) and were lysed in SDS-PAGE buffer. Lysates were analyzed by si- causes the induction of RAG-mediated recombination events at Ig ␬ ␭ multaneously immunoblotting with Ab to total Lyn protein (53- and 56- L chain loci, leading to the expression of a new or L chain kDa isoforms) and site-specific Abs against either C-terminal inhibitory (receptor editing), which may result in loss of self-reactivity and phosphotyrosine of Lyn (Lyn pY507) or the phosphorylated activation loop thereby allow subsequent maturation (16). Therefore, to determine of SFK (SFK pY416), which identifies Lyn pY397 (this Ab cross-reacts whether receptor editing is affected by Lyn deficiency, we mea- with multiple other SFKs, including Fyn (59 kDa) and Blk (55 kDa)). sured the level of rag1 and rag2 mRNA in BM B cells. We found Ϫ Ϫ BAP31 served as a protein-loading control. Unstimulated lyn / splenic B that there was little difference in the levels of rag1 and rag2 Ϫ/Ϫ cells are also shown (Lyn ). Similar results were obtained in three ad- mRNA between lynϪ/Ϫ and WT small pre-B cells, but lynϪ/Ϫ NF ditional experiments. The ratio of phosphorylated Lyn to total Lyn, quan- B cells expressed several-fold higher levels of RAG1 and RAG2 tified by densitometry, is provided. Ratios are normalized to unstimulated than did their WT counterparts (Fig. 7A). Because RAG induction T1 B cells for the pY397:Lyn blot and to unstimulated T3 B cells for the ␬ pY507:Lyn blot. B, Purified lymph node B cells (which are primarily fol- causes recombination events that can lead to a switch from Ig L Ϫ Ϫ ␭ licular B cells) from WT and cd22 / mice were stimulated, lysed, im- chain expression to expression of Ig , we also investigated munoblotted, and probed with specific Abs, as in A. Similar results were whether Lyn deficiency affected the frequency of Ig␭-expressing B Ϫ/Ϫ ϩ obtained in one additional experiment. As in A, the ratio of phosphorylated cells. The lyn mice had slightly higher frequencies of Ig␭ Lyn to total Lyn, normalized to unstimulated WT B cells, is provided. newly formed cells than WT mice, although the frequencies of 5390 THE ROLE OF Lyn IN B CELL DEVELOPMENT

recombination in these populations were measured by quantitative PCR of genomic DNA (22). This assay therefore measures the overall degree of L chain rearrangement in a population of B cells. The RS frequencies were similar in lynϪ/Ϫ newly formed and T1 B cells compared with WT (Fig. 7C). Collectively, these data indicate that the mechanisms causing the removal of autoreactive BCRs in immature B cells are intact or slightly enhanced in lynϪ/Ϫ mice. This suggests that autoantibody production in lynϪ/Ϫ mice is not a consequence of impaired central tolerance.

Lyn-deficient follicular B cells have enhanced sensitivity for BCR-induced proliferation We next asked whether Lyn-deficient mature follicular B cells are more easily activated through their BCR than their WT counterparts, as suggested by the signaling data. It has been reported that lynϪ/Ϫ B cells are hypersensitive to BCR-induced proliferation, although in those studies B cell populations were not fractionated (4, 14, 15, 38). To differentiate between B cells at different stages of development, we sorted splenic B cell populations and measured their ability to proliferate in response to different levels of Downloaded from BCR engagement. Not surprisingly, BCR stimulation did not induce WT or lynϪ/Ϫ T1 B cells to proliferate (Fig. 7D), in agreement with previous reports (27, 39, 40). In contrast, lynϪ/Ϫ follicular B cells proliferated much more vigorously than did WT follicular B cells when stimulated with low doses of anti-IgM (2.5 ␮g/ml) (Fig. 7E). However, lynϪ/Ϫ and WT follicular B cells in- http://www.jimmunol.org/ corporated similar amounts of [3H]thymidine when stimulated with a higher dose of anti-IgM (15 ␮g/ml). These data show that lynϪ/Ϫ follicular B cells are indeed more sensitive to anti-IgM- induced proliferation than WT follicular B cells, consistent with FIGURE 7. The effect of Lyn deficiency on receptor editing and pro- their increased sensitivity to anti-IgM-induced calcium release and liferative responses. A, Relative RAG1 and RAG2 mRNA levels in sorted lynϪ/Ϫ Ϫ/Ϫ ERK activation. These findings demonstrate that mature B populations of WT and lyn immature BM B cells. RAG transcripts were cells have a reduced threshold of activation, which therefore could normalized to GAPDH transcript levels in each sample. Pre-B represents contribute to the breakdown in tolerance to self Ags in the periph- cells from the pro-pre B cell BM gate (Fig. S1)4 with small lymphocyte by guest on October 1, 2021 size on forward scatter. Each data point represents data from one mouse; ery of these mice. results from two experiments are shown. B, Frequency of Ig␭ϩ cells in B cell populations from the BM and spleen of WT (u) and lynϪ/Ϫ (f) mice. Discussion n ϭ 11 mice for WT, n ϭ 10 for lynϪ/Ϫ; bars represent mean Ϯ SD; Central to the ability of B cells to discriminate between foreign and p ϭ 0.04 (unpaired t test). C, Frequency of RS self Ags is the difference in the responses to Ag of immature vs ,ءء ;indicates p ϭ 0.01 ,ء recombination events in genomic DNA of B cell subpopulations. The mature B cells. Engagement of the BCR of immature B cells in the Ϫ amount of RS recombinations in sorted Ig␭ B cell populations was de- BM induces expression of the RAG1 and RAG2 genes and sub- termined by quantitative PCR of genomic DNA and standardized to the sequent Ig L chain rearrangements with the goal of changing spec- number of actin genes. Shown is the frequency of RS recombination nor- ificity away from self-reactivity, a process called receptor editing malized to the RS recombination frequency in WT follicular B cells. Each (16). If this loss of self-reactivity is not achieved or if self Ag data point represents data from one mouse; results from two experiments are shown. D, Proliferative responses of T1, combined T2-T3, or follicular triggers BCR signaling of T1 B cells in the spleen, then the cell (Fo) B cells sorted from spleens of WT (u)orlynϪ/Ϫ (f) mice and then undergoes apoptosis, resulting in clonal deletion. In contrast, ␮ Ј strong BCR engagement of mature B cells induces proliferation, stimulated for 48 h with 15 g/ml anti-IgM F(ab )2. E, Proliferative responses of WT and lynϪ/Ϫ follicular B cells to stimulation with various and in combination with cytokines or other signals can facilitate a Ј 3 doses of goat anti-mouse IgM F(ab )2, as measured by [ H]thymidine in- productive immune response. A variety of studies has indicated corporation during the last4hofstimulation. Bars represent data from two that immature B cells are more sensitive to Ag than are mature B sets of pooled spleen cells per genotype; each set of pooled cells from two cells. We have used newly improved methods for measuring sig- to three mice; each sample tested in duplicate or triplicate; bars indicate naling reactions in individual cells from immune organs to com- means, and error bars indicate SEM. pare the sensitivity of the various developmental stages of B cells in the spleen and BM for two key BCR-induced signaling responses, the elevation of intracellular-free calcium, and the acti- Ig␭ϩ T1 B cells in the spleen were similar in WT and lynϪ/Ϫ mice vation of the ERK MAPK. These results confirm that T1 B cells (Fig. 7B). are highly sensitive to BCR engagement and that follicular B cells Finally, we also measured the frequency of B cells that had RS are distinctly less sensitive. Moreover, we have shown that this recombinations at the Ig␬ locus as an indication of receptor edit- change in signaling sensitivity of the BCR is due to a maturation- ing. Recombination at the RS sequence inactivates a functional Ig␬ associated increase in the ability of the Lyn-CD22-SHP-1 pathway locus, after which another L chain locus can be rearranged to gen- to attenuate BCR signaling. We propose that the low level of func- erate a new L chain (16, 37). Purified Ig␭Ϫ small pre-B cells, tion of this inhibitory mechanism in immature B cells serves to newly formed, splenic T1, and follicular B cells were isolated by permit efficient engagement of tolerance-promoting programs in cell sorting from lynϪ/Ϫ and WT mice. The frequencies of RS these cells, whereas its greater activity in mature follicular B cells The Journal of Immunology 5391 serves to create a threshold for activation that minimizes the acti- that increasing CD22 levels during B cell development promotes vation of B cells in response to self Ags encountered in the Lyn activity, in addition to providing more substrate for Lyn. periphery. Mice deficient in Lyn or CD22, as well as mice with a B cell- Previous studies have also compared the BCR responsiveness of specific deletion of SHP-1 (36), are known to spontaneously pro- immature and mature B cells, but have disagreed as to whether duce autoantibodies against nuclear Ags, but the mechanism lead- immature B cells are more sensitive (23, 24, 41), equally sensitive ing to this autoantibody production is unknown. We found that (25), or less sensitive (26, 28) to BCR stimulation relative to ma- lynϪ/Ϫ mice had increased rag1 and rag2 mRNA expression in ture B cells. Our studies simultaneously analyzed immature and newly formed B cells, more modest increases in Ig␭ϩ B cell fre- mature B cell populations taken immediately ex vivo from the BM quencies, and no significant changes in RS rearrangements com- or spleen of unmanipulated mice and used staining conditions that pared with their WT counterparts. These data suggest that Lyn did not perturb signaling, and thus, we feel they are likely to rep- deficiency does not compromise tolerance-related responses of im- resent the responses of these cell types in a reliable way. The mature B cells to self Ag and may even enhance them. Consistent difference in sensitivity of BCR signaling that we observed did not with this conclusion, it has been shown that Lyn deficiency does ␭ϩ appear to result primarily from changes in the expression of mIgM not cause a significant change in the number of VH3H9 Ig and mIgD that occur as B cells mature in the spleen. T1 cells have DNA-reactive B cells in the spleen (46). Another study demon- high levels of mIgM and low levels of mIgD, whereas mature B strated that Lyn deficiency minimally affected the number of anti- cells have high levels of mIgD and diverse levels of mIgM, rang- HEL transgenic B cells in the BM of HEL-expressing mice, but did ing from low levels to the high levels seen in T1 B cells. We found enhance the loss of anti-HEL transgenic mature B cells in the that there was still a considerable difference in sensitivity to anti- spleens of these mice (5). The implication of these results is that IgM treatment between T1 B cells and a gated subpopulation of the Lyn-CD22-SHP-1 inhibitory pathway is not necessary for the Downloaded from follicular B cells that had levels of mIgM comparable to the ex- establishment of tolerance to self during B cell development, in pression level on T1 cells (data not shown). Moreover, when we agreement with the slightly enhanced level of BCR signaling seen used the Ag lysozyme to engage the BCR on MD4 antilysozyme in immature B cells from mice genetically missing this pathway. In Ig transgenic T1 and follicular B cells, we again found substan- contrast, genetic mutations causing reductions in BCR signaling, tially greater sensitivity of the former compared with the latter. and therefore reduced sensitivity to Ag, lead to enrichment of the Similarly, Wen et al. (23) demonstrated that immature anti-Thy1 B cell repertoire with autoreactive specificities in mice (47) and http://www.jimmunol.org/ Ig transgenic B cells were more sensitive to Thy1 Ag than their humans (48). Thus, it appears that Lyn deficiency leads to auto- mature B cell counterparts using techniques similar to ours. These immunity not through a defect in central tolerance induction, but findings indicate that mechanisms other than changes in surface Ig rather due to a defect in the maintenance of tolerance in the pe- expression regulate B cell sensitivity to BCR engagement during riphery. In agreement with this idea, lynϪ/Ϫ mature follicular B maturation. cells exhibited substantially enhanced sensitivity to BCR-induced Genetic evidence indicates that this reduction in BCR sensitivity calcium release, ERK activation, and proliferation relative to their during B cell maturation is due to an increase in the efficacy of the WT counterparts, which indicates that there is a reduction in the inhibitory pathway mediated by Lyn, CD22, and SHP-1. BCR sig- threshold of B cell activation in the absence of effective inhibition by guest on October 1, 2021 naling of T1 and T2 B cells was only modestly affected by Lyn by the Lyn-CD22-SHP-1 pathway. This reduced threshold of ac- deficiency, CD22 deficiency, or SHP-1 mutation, indicating that tivation could allow the recruitment of autoreactive B cells into an this inhibitory pathway is relatively inactive in immature B cells of immune response, and perhaps in combination with abnormal my- the BM or spleen. In contrast, there was a dramatic enhancement eloid cell function from Lyn deficiency, result in autoantibody in BCR signaling of mature B cells by Lyn and CD22 deficiencies, production. demonstrating the importance of this inhibitory pathway in limit- In summary, we have found that during maturation of B cells in ing BCR sensitivity once B cells mature. One contributor to this the spleen, the sensitivity of the BCR to Ag stimulation was down- developmental change in the ability of the Lyn-CD22-SHP-1 path- regulated. This developmentally acquired down-regulation in BCR way to inhibit BCR signaling is likely to be the 2.5-fold up-regu- sensitivity required Lyn and CD22, which act together with SHP-1 lation of CD22 during B cell maturation in the spleen. Follicular B in an inhibitory pathway to suppress BCR signaling selectively in cells from cd22ϩ/Ϫ mice, which express CD22 levels similar to mature follicular B cells. Surprisingly, the Lyn-CD22-SHP-1 in- those of WT T1 B cells, exhibited enhanced calcium responses hibitory pathway had a minor role in the regulation of BCR sig- compared with WT follicular B cells, but were distinctly less sen- naling in immature B cells. In agreement with the signaling results, sitive than WT T1 B cells. This indicates that other factors are Lyn deficiency had little effect on receptor editing-related events in present that mediate the developmental reduction in BCR sensi- immature B cells, which suggests that central tolerance mecha- tivity, besides up-regulation of CD22. Regulation of SHP-1, nisms do not require this inhibitory pathway. On the basis of these CD22, or Lyn activity could contribute to the developmental observations, we propose that modulation of B cell sensitivity to change in inhibitory signaling. For example, sialic acid acetyles- Ag by the Lyn-CD22-SHP-1 inhibitory pathway during the course terase has recently been shown to facilitate CD22 suppression of of B cell maturation is a critical factor in the maintenance of pe- BCR signaling (42) and is up-regulated during B cell development ripheral B cell tolerance to self Ags. (43). In contrast, Lyn levels did not change significantly between T1 and mature follicular B cells. However, we did see a higher level of Lyn activity in unstimulated mature B cells compared with Acknowledgments unstimulated T1 B cells, as evidenced by phosphorylation of the We thank Shuwei Jiang for cell sorting, and Arthur Weiss, Jason Cyster, active site Y397 residue. Although regulation of the Lyn Y397 site Michelle Hermiston, and Clifford Lowell (all at University of California) is not well understood, phosphorylation of Y397 might be en- and Ed Clark (University of Washington) for helpful discussions. hanced in a lipid raft environment (44), and mature B cells appear to have a higher lipid raft content than transitional B cells (45). Interestingly, Lyn phosphorylation of Y397 was substantially de- Disclosures Ϫ Ϫ creased in unstimulated cd22 / mature B cells, which suggests The authors have no financial conflict of interest. 5392 THE ROLE OF Lyn IN B CELL DEVELOPMENT

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