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

doi:10.4049/jimmunol.0803941

J Immunol. Author manuscript; available in PMC 2010 May 1.

Published in final edited form as:

J Immunol. 2009 May 1; 182(9): 5382–5392.

doi: 10.4049/jimmunol.0803941

PMCID: PMC2840041

NIHMSID: NIHMS183254

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

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

^*Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA

^†Department of Surgery, University of California San Francisco, San Francisco, CA 94143, USA

^§Department of Microbiology & Immunology, University of California San Francisco, San Francisco, CA 94143, USA

^‡Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA

²Address correspondence and reprint requests to Dr. Anthony DeFranco, University of California San Francisco, 400 Parnassus Ave, Box 0414, San Francisco, CA 94143, USA. Email: anthony.defranco/at/ucsf.edu, tel. (415) 476-5488, fax. (415) 502-8424

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Abstract

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 T1 B cells with regard to BCR-induced calcium elevation and Erk MAP kinase 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 indicate that the Lyn–CD22–SHP-1 inhibitory pathway largely becomes operational 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.

Introduction

B cell receptor (BCR) hyperresponsiveness is associated with a breakdown in tolerance and the development of autoantibodies in several genetically modified mouse strains(1, 2). A good example of this is the Lyn-deficient mouse(3). Lyn is a Src-family-kinase (SFK) that, like two other SFKs expressed by B cells, Blk and Fyn, phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMs) on B cell receptor Igα/Igβ chains following antigen binding(4–6). Lyn also functions to phosphorylate immunoreceptor tyrosine-based inhibitory motifs (ITIMs) on inhibitory receptors that negatively regulate BCR signaling, including CD22(5, 7–9), FcγRIIb(7, 8) and perhaps others(10–13). In this way, Lyn facilitates recruitment of the SHP-1 and SHIP phosphatases to the plasma membrane, which down-modulate BCR signaling(5, 8, 9). In the absence of Lyn, BCR signaling is supported by Blk and Fyn, but inhibitory receptors are ineffective at downregulating BCR signaling, thereby leading to BCR hyperresponsiveness(3).

Lyn-deficient mice exhibit increased plasma cell numbers and serum immunoglobulin levels. Surprisingly, these mice also produce autoantibodies to nuclear antigens(4, 14, 15). Why elevated BCR signaling would lead to a loss of tolerance in lyn^−/− mice is not entirely self-evident, because while BCR signaling drives activation of mature B cells, it also promotes tolerance-inducing mechanisms in immature B cells. For example, binding of antigen by immature B cells in the bone marrow (BM) induces expression of recombination activating genes (RAG 1 and 2), which can generate light-chain rearrangement and thereby change the specificity of the BCR (receptor editing) (16–18). Continued recognition of antigen by the BCR may induce apoptosis of self-reactive B cells (clonal deletion)(16). Therefore, genetic alterations that enhance BCR signaling should increase the sensitivity of immature B cells to self-antigens, causing more thorough removal of autoreactive BCR specificities from the newly formed B cell repertoire. For this reason, it seems paradoxical that autoimmunity develops in the lyn^−/− mouse and in other mice with genetic alterations increasing the strength of BCR signaling.

In order to account for the break in B cell tolerance observed in Lyn-deficient mice, we hypothesized that the level of Lyn-mediated inhibitory signaling might change during the course of B cell development. 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, because Lyn or CD22 deficiency strongly increased BCR signaling in follicular B cells, but only weakly increased signaling in T1 B cells. Consistent with these findings, Lyn-deficiency had a modest effect on events associated with receptor editing, whereas it more strongly enhanced in the sensitivity of mature B cells to BCR-induced proliferation. These results indicate that inhibition of BCR signaling by the Lyn–CD22–SHP-1 pathway is upregulated as B cells mature in the spleen, and suggests that this developmental change is involved in the maintenance of peripheral immune tolerance of B cells.

Materials and Methods

Mice

Mice aged 7–12 weeks were used for most experiments. Four week-old mice were used in experiments using motheaten-viable mice (Ptpn6^me-v) backcrossed onto C57BL/6 background (Jackson Laboratory). lyn^−/− mice were used as described(4), and backcrossed at least fifteen generations onto C57BL/6 background. cd22^−/− mice(19) backcrossed at least sixteen generations onto C57BL/6 background were obtained from E. Clark (University of Washington). MD4 transgenic Ig_Hel mice were obtained from J. Cyster (University of California, San Francisco). All animals were housed in a specific pathogen-free facility at UCSF according to University and National Institutes of Health (NIH) guidelines. Animal use was approved by the UCSF Institutional Animal Care and Use Committee.

Antibodies, Immunofluorescence Analysis, B cell purification and Cell Sorting

Fluorophore conjugated antibodies directed against the following molecules were used: B220 (RA3-6B2), CD23 (B3B4), IgM (II/41), Igλ_1–3 (R26-46), CD16/CD32 (2.4G2) CD22.2 (Cy34.1), and CD72 (K10.6) all from BD Pharmingen; CD24 (M1/69) from BioLegend; CD93 (AA4.1) and CD5 (53–7.3) from eBioscience; Igκ (187.1) and Igλ (JC5-1) from Southern Biotech; IgM (goat polyclonal F(ab') monomer, μ-chain specific) (Jackson Immuoresearch). Cells were analyzed on an LSR-II or FACSCalibur (both from BD Pharmingen). Lymph node B cell were purified by negative selection using CD43 microbeads (Miltenyi Biotech) according to manufacturer's protocol and passage through an autoMACS Separator (Miltenyi Biotech). For cell sorting, splenocytes or BM cells were stained for CD23, CD93, and IgM F(ab') monomer for 30 min and either B220 or a non-B cell cocktail (CD4, CD8, CD11b, Gr1, NK1.1, Ter119) in HBSS, 1% fetal calf serum (FCS), 0.5% BSA. Cells were then sorted on a MoFlo sorter (Dako-Cytomation). Dead cells were excluded by propidium iodide (PI)(BioChemika) uptake. All FACS data were analyzed with FlowJo v. 6.4.1 (TreeStar software).

Analysis of Calcium Mobilization

For measurements of intracellular-free-calcium levels, splenocytes or BM cells were loaded with Indo-1 AM (MolecularProbes/Invitrogen) and then labeled for CD93 (APC), CD23 (PE), IgM (Fab' monomer)(FITC) and B220 (PE-Cy7) or a cocktail of antibodies recognizing non-B cells (CD4, CD8, CD11b, Gr1, NK1.1, Ter119) (PE-Cy7). Cells were resuspended in RPMI 1640 supplemented with 1% BSA and 20mM HEPES, warmed to 37°C for 3 minutes and analysis was initiated with flow cytometry. After the baseline was established for 30–45 seconds, cells were stimulated with 0.5–50μg/ml goat anti-mouse IgM F(ab')₂ (Jackson Immunoresearch) or 16μg/ml ionomycin (Sigma-Aldrich). Median intracellular calcium concentration as indicated by the ratio of fluorescence 405nm emission to 530nm emission was measured over time by flow cytometry. Dead cells were excluded by PI uptake.

Assay of Erk Phosphorylation

For intracellular measurement of phospho-Erk, 3×10⁶ splenocytes or 2×10⁶ BM cells were warmed to 37°C for 30 minutes in RPMI 1640 media with 1% BSA and 20mM HEPES. During the final 10 minutes of warming, the cells were labeled with anti-IgM F(ab') FITC. Some samples were treated with 10μM of the MEK inhibitor U0126 (Sigma Aldrich) during the warming period. Cells were then stimulated with 0–50μg/ml goat anti-mouse IgM F(ab')₂ (Jackson ImmunoResearch) or 1μg/ml of PMA (Sigma Aldrich). After the desired period of stimulation, cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) and permeabilized with ice cold 100% methanol (Electron Microscopy Sciences). Cells were labeled with phospho-p44/42 (Thr202/Tyr204) rabbit monoclonal antibody (197G2, Cell Signaling) that recognizes phosphorylation sites required for Erk activity. Cells were then labeled with donkey anti-rabbit IgG APC (Jackson Immuoresearch) as well as antibodies to B220 (PE-Cy7), CD23 (PE) and CD24 (pacific blue). (The fixation process prevented labeling for CD93.) Cells were analyzed by flow cytometry on an LSR-II.

B Cell Proliferation Assays

Sorted splenic B cells from 4 mice of each genotype were suspended at a concentration of 2×10⁶ cells/ml in RPMI 1640 with 10% FCS, 20mM HEPES, 2mM glutamine, and 1mM sodium pyruvate and stimulated with 0–15μg/ml of goat anti-mouse IgM F(ab')₂ (Jackson ImmunoResearch) in 96-well tissue culture plates (Corning) at 37°C, 5% CO₂. For proliferation assay, cells were incubated for 48 hours. 1 μCi per well of [³H]thymidine (Amersham) was added during the final 4 hr of culture, and incorporation was measured by scintillation counting.

Western blotting

Whole-cell lysates were prepared in SDS lysis buffer from sorted splenic B cells or lymph node B cells purified by auto-MACS negative selection. Proteins were separated on a NuPAGE Bis-Tris gel (Invitrogen) with MOPS buffer and immunoblotted on Immobilon–FL transfer membrane (Millipore). Antibodies were directed against phospho-Src family kinase Tyr416 (Cell Signaling 2101s), phospho-Lyn Tyr507 (Abcam ab53122), Lyn (Abcam ab1890), and BAP31 (Abcam ab37120) as a loading control. These antibodies were detected with fluorescently-labeled secondary antibodies using the Odyssey Infrared Imaging System (LI-COR Biosciences).

Real time RT-PCR

Total RNA was isolated from sorted B cells using the RNeasy Micro Kit (Qiagen) and cDNA was transcribed using the iScript cDNA Synthesis Kit (Biorad) according to the manufacturers' instructions. Equivalent amounts of cDNA were used in quantitative PCR on an ABI Prism 7700 Sequence detection instrument (Taqman; PerkinElmer Applied Biosystems). Primer and probe sequences for RAG1 & RAG2(20), and GAPDH(21) were used as published.

Assay of Vκ-RS rearrangement

Genomic DNA was isolated from sorted Igλ⁻ B cell populations using the Gentra PureGene Tissue Kit (Qiagen). Quantitative PCR was performed as described(22). The amount of Vκ-RS product in each sample was normalized to the amount of β-actin product and compared to the normalized target value in WT C57Bl/6 follicular B220⁺ IgM⁺ κ⁺ splenocytes to determine a relative quantity (comparative C_T (DDC_T) method).

Statistical Analyses

Statistical analyses, including unpaired two-tailed t-test and four parameter logistic equation (linear regression analysis) were performed with Prism v.4.0 (GraphPad software).

Results

Transitional (T1 & T2) B Cells are More Sensitive to BCR Stimulation than Mature Follicular B Cells

It has previously been suggested that immature B cells are more sensitive than mature B cells to limiting amounts of BCR stimulation (23, 24), but other studies have not agreed with this conclusion (25, 26). These earlier studies have used various manipulations in order to generate sufficient numbers of immature B cells to analyze signaling. To better evaluate this issue, we analyzed calcium elevation in unmanipulated B cells taken directly ex-vivo from young adult mice. B cells at various developmental stages were identified by a combination of monoclonal antibodies, as described by Allman et. al.(27) (Figure S1 in the Supplemental Data). We found this antibody combination, which included non-stimulating anti-IgM F(ab') FITC, had minimal effects on BCR-induced calcium signaling (Figure S2). When WT splenic B cells were stimulated with subsaturating concentrations of anti-IgM F(ab')₂, transitional T1 and T2 B cells exhibited greater increases in cytoplasmic calcium levels ([Ca²⁺]_i) than did T3 and mature follicular B cells (Figure 1A–C). All of these B cell subsets had similar maximal calcium responses when stimulated with a saturating concentration of anti-IgM F(ab')₂ (50μg/ml). Although it has been proposed that T3 B cells represent an anergic population of B cells(28), we observed that T3 B cells responded to anti-IgM by increasing [Ca²⁺]_i similarly to follicular B cells. These data indicate that mature follicular B cells require more BCR stimulation than immature T1 and T2 B cells to induce comparable BCR induced calcium signaling.

FIGURE 1

Immature T1 B cells are More Sensitive than Mature Follicular B Cells to anti-IgM Stimulation. (A) Cytoplasmic free calcium levels indicated by indo-1 fluorescence emission ratio at 405nm and 530nm ([Ca²⁺]_i F405/F530) in WT splenic B220⁺ T1 (AA4.1⁺, CD23 (more ...)

As B cells mature, IgM levels are downregulated and IgD levels are upregulated(29). We therefore wondered whether treatment of B cells with anti-IgM accurately measured the sensitivity of B cells to BCR engagement at each stage of development. To address this issue, we therefore gated on the subpopulation of follicular B cells with levels of mIgM comparable to the level of mIgM on T1 B cells. These cells still exhibited decreased sensitivity to anti-IgM for the elevation of intracellular free calcium compared to the T1 B cells (data not shown). To investigate whether reduction in mature B cell sensitivity to antigen is averted during development through up-regulation of surface IgD levels, we measured the sensitivity of B cells to antigen engagement using Ig-transgenic B cells specific for hen-egg lysozyme (HEL). Similar to the results obtained when an anti-IgM reagent was used, immature transitional B cells exhibited greater increases in cytoplasmic calcium levels than did follicular B cells when treated with subsaturating concentrations of HEL, whereas their responses to higher doses of HEL were similar (Figure 2). These data are consistent with those reported in another BCR transgenic system(23) and confirm that immature T1 B cells are in-fact more sensitive to BCR stimulation than mature follicular B cells. These data also show that stimulation of B cells with anti-IgM accurately reflects the relative responses of different B cell populations to BCR engagement by antigen.

FIGURE 2

Immature B cells are More Sensitive than Mature Follicular B Cells to Stimulation with Antigen.

Immature IgM⁺ B Cells in the Bone Marrow are also More Sensitive to BCR Stimulation than Mature B Cells

Because the first events associated with the establishment of B cell tolerance occur in the bone marrow, it was important to determine whether B cells at various developmental stages in the BM respond to BCR stimulation in a manner similar to their splenic counterparts. We applied the same gating strategy for identifying B cell populations in the spleen to BM cells(30) (Figure S1). Calcium responses to anti-IgM stimulation of BM B cell subpopulations were remarkably similar to those of corresponding splenic B cell populations (Figure 1D and data not shown for 20μg/ml anti-IgM). Splenic T1 cells responded nearly identically to immature “Newly Formed” (NF) cells, splenic T2 cells responded identically to “BM-T2” cells (data not shown), and splenic follicular B cells responded identically to mature naive B cells recirculating in the BM. As expected, the pro-B and pre-B cell populations had minimal or no response to anti-IgM (data not shown). These data indicate that wild-type B cells become less sensitive to low levels of BCR stimulation as they transition from immaturity to maturity, regardless of whether they reside in the BM or spleen.

Immature B Cells are More Sensitive to BCR Stimulation than Mature B Cells for BCR-Induced Erk Phosphorylation

To determine whether the changes in calcium signaling during B cell development were representative of changes in other BCR signaling reactions, we examined BCR-induced Erk activation as assessed by flow cytometry. This assay was largely specific since the response was nearly completely blocked by the MEK inhibitor U0126 (Figure S3). Stimulation of wild-type spleen cells with anti-IgM led to significant increases in the levels of Erk phosphorylation in the various B cell subpopulations. Peak levels of phospho-Erk were detected in B cells after 2–3 minutes of stimulation (Figure S3). As with calcium elevation, Erk activation after 2.5 minutes was induced by lower doses of anti-IgM in T1 B cells than in mature follicular B cells (Figure 3A–C). Interestingly, even at high doses of anti-IgM (50μg/ml), follicular B cells activated Erk to a lesser degree than did immature T1 B cells. In contrast, stimulation with the diacylglycerol mimetic, phorbol 12-myristate 13-acetate (PMA), induced higher levels of Erk phosphorylation in follicular B cells than in T1 B cells (Figure 3B). Phospho-Erk levels in stimulated BM B cell populations were similar to those in the corresponding stimulated splenic B cell populations (Figure 3C). Thus, data for both elevation of intracellular free calcium and activation of Erk MAP kinases showed that WT immature B cells are more sensitive to BCR stimulation than are WT follicular B cells.

FIGURE 3

Lyn-deficiency has a Major Effect on Calcium Responses in Follicular B Cells but a Lesser Effect on those in T1 B Cells.

Lyn-Deficiency Greatly Magnifies Calcium Response by Follicular B Cells but Only Slightly Enhances Response by T1 B Cells

Although Lyn-deficient B cells are known to have elevated signaling responses to BCR engagement (5, 7), it was not known whether this hypersensitivity is dependent on the maturation stage of the B cell. Spleens from lyn^−/− mice contained T1, T2, T3 and follicular B cell populations (Figure S1), as previously reported(27). We found that the calcium responses of lyn^−/− follicular B cells were far more sensitive to IgM stimulation than were WT follicular B cells (Figure 4A–C), and their ability to respond to low doses of anti-IgM resembled WT immature B cells. Additionally, the peak amplitude of the calcium response by lyn^−/− follicular B cells was greatly exaggerated over that of wild type follicular B cells even at saturating doses of anti-IgM (Figure 4A–C). These data indicate that Lyn serves an important role in limiting the magnitude of BCR signaling as well as dampening the sensitivity of mature B cells to IgM stimulation.

FIGURE 4

Lyn Deficiency Strongly Enhances Activation of Erk in Follicular B Cells but has a Lesser Effect on T1 B Cells.

In contrast to the large effect of Lyn-deficiency on mature follicular B cells, Lyn-deficiency had a relatively minor effect on calcium responses by immature T1 B cells (Figure 4A–C). The peak amplitude as well as the sustained level of [Ca²⁺]_i following BCR stimulation was only slightly higher in lyn^−/− T1 cells compared to WT T1 B cells. Similarly, lyn^−/− newly formed bone marrow B cells had only slightly higher elevations of [Ca²⁺]_i than their WT counterparts (Figure 4D). These data indicate that Lyn greatly inhibits BCR calcium signaling by follicular B cells, but surprisingly provides much less restraint on BCR calcium signaling by newly formed or T1 B cells.

BCR-Induced Erk Phosphorylation is Greatly Enhanced by Lyn Deficiency in Follicular B Cells but Not in T1 B Cells

To confirm the effects of Lyn-deficiency on BCR signaling, we examined Erk-phosphorylation in lyn^−/− B cells. Anti-IgM stimulated lyn^−/− follicular B cells had much higher levels of phospho-Erk than stimulated wild type follicular B cells (Figure 3D,E). In contrast, lyn^−/− T1 B cells had either slightly lower or similar levels of Erk phosphorylation following BCR stimulation compared to WT T1 B cells (Figure 3D,E). Thus, BCR-induced Erk activation and calcium elevation were both strongly enhanced in follicular B cells lacking Lyn, whereas these signaling events were not greatly affected in T1 B cells.

Surface Expression Levels of Inhibitory Receptors During B Cell Development

These data collectively indicate that Lyn has a stronger effect on modulating BCR signaling in mature B cells than in immature B cells. Because Lyn can negatively regulate BCR signaling through the recruitment of ITIM bearing inhibitory proteins, we wondered whether the strong effect of Lyn on mature B cell signaling was due to increased expression of ITIM-bearing inhibitory receptors during B cell development. Consistent with this idea, we found that CD22 expression on the surface of B cells was increased 2.5 fold from the T1 stage to the follicular stage (Figure 5A, Table 1) in agreement with earlier studies(31, 32). However, expression of CD16/32, which on B cells is primarily FcγRIIb, was largely unchanged, and two other negative regulators of BCR signaling, CD72 and CD5, were downregulated during B cell maturation in the spleen (levels reduced by 35% and 37%, respectively)(Figure 5A, Table 1)

FIGURE 5

Greater Effect of CD22 on Follicular B Cells Than on T1 B Cells.

Table I

Surface levels of negative regulators during splenic B cell development^a

CD22-Deficiency Leads to Greatly Enhanced Calcium Responses by Follicular B Cells but Only Slightly Increased Responses by T1 B Cells

Because CD22 expression increases substantially during B cell development, we hypothesized that CD22-deficiency would have disproportionate effects on mature B cells as compared to immature B cells. Indeed, cd22^−/− follicular B cells were much more sensitive to low-dose BCR stimulation and had much higher [Ca²⁺]_i than WT follicular B cells (Figure 5B). In contrast, CD22-deficiency had relatively minor effects on T1 B cells. Surprisingly, the calcium responses of cd22^−/− B cells were very similar in magnitude to the responses of lyn^−/− B cells in both mature and immature B cells (Figure 5B). Additionally, calcium responses of bone marrow NF B cells from motheaten viable mice, which have a partial loss of function mutation in the ptpn6 gene that encodes the tyrosine phosphatase SHP-1 (shp-1^me-v/me-v)(33), were slightly reduced compared to WT NF B cells (Figure 5C). These data indicates that the Lyn–CD22–SHP-1 inhibitory pathway has minor effects on limiting BCR signaling in immature B cells, but as B cells mature Lyn functions in conjunction with CD22 to mediate reduction in BCR sensitivity.

Since CD22 expression increases during B cell development, we investigated whether reduction of CD22 levels in mature follicular B cells could cause these cells to signal in a manner more similar to immature B cells. To test this, we studied follicular B cells from cd22^+/− mice, whose CD22 surface levels are similar to those of wild-type T1 B cells (figure 5D). We found that calcium release by cd22^+/− follicular B cells was enhanced compared to wild-type, although it was not as robust as the calcium release of WT T1 B cells (Figure 5E). This data indicates that up-regulation of CD22 during B cell development has an appreciable role in reducing BCR sensitivity, but that additional factors are also likely to contribute.

Tyrosine phosphorylation of Lyn changes during B cell development and is regulated by CD22

We next investigated whether Lyn levels or function might change during B cell development to contribute to the acquisition by follicular B cells of the Lyn-CD22-SHP-1 inhibitory pathway. We sorted T1, T2, T3 and follicular B cells from spleens of WT mice and measured Lyn protein levels and levels of tyrosine phosphorylation at its activating and inhibitory regulatory sites by immunoblotting of the cell lysates. Lyn protein levels did not change substantially throughout B cell maturation in the spleen (Figure 6A and data not shown), which is consistent with results from published mRNA microarray analysis of B cell developmental stages in the bone marrow(34). Like other src-family-kinases, Lyn kinase activity is regulated by phosphorylation of two tyrosine sites(3). Lyn assumes an inactive conformational state when the C-terminal Y507 site is phosphorylated, whereas kinase activity is enhanced following phosphorylation of Y397 in the kinase domain. We found that Y507 phosphorylation was similar in splenic B cells at each stage of development and did not exhibit a substantial change upon BCR stimulation (Figure 6A). Interestingly, Y397 phosphorylation was elevated in unstimulated follicular B cells compared to immature transitional B cells. However, upon BCR stimulation, Y397 phosphorylation increased to similar levels in each of the B cell subsets analyzed (Figure 6A). These findings suggest that Lyn activity is enhanced in unstimulated follicular B cells, compared to immature B cells. Although these data do not directly assess Lyn function inside the B cell, they suggest that Lyn is in a more activated state in resting follicular B cells, potentially explaining in part the enhanced activity of the Lyn-CD22-SHP-1 inhibitory pathway in these cells compared to immature T1 B cells.

FIGURE 6

Lyn Tyrosine-Phosphorylation Status in B Cells of Different Developmental Stages.

Although CD22 is generally considered to be downstream of Lyn, in that Lyn appears to be responsible for phosphorylating the ITIM of CD22 leading to SHP-1 recruitment to the plasma membrane, we observed that CD22 expression also affected the activity of Lyn. Lymph node B cells from CD22^−/− mice had an approximately 3–4 fold reduction in Lyn Y397 phosphorylation levels, particularly in unstimulated B cells (Figure 6B). The levels of phosphorylation of Y507 were either slightly increased or unchanged (Figure 6B). These results suggest that while CD22 acts downstream of Lyn to recruit phosphatases to the plasma membrane and inhibit BCR signaling, it also acts upstream of Lyn to positively regulate Lyn activity.

Lyn-Deficiency Minimally Affects Receptor Editing

Genetic deficiency in Lyn, CD22 or SHP-1 results in spontaneous autoantibody production(4, 14, 15, 35, 36), but it is not known whether this failure of tolerance occurs primarily during B cell development in the bone marrow or later in the periphery. Because 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, we hypothesized that Lyn-deficiency would either have no effect on events related to the establishment of B cell tolerance in the bone marrow or would slightly exaggerate these events. It is thought that BCR signaling above a certain threshold in pre-B and immature B cells causes the induction of RAG-mediated recombination events at Ig light chain loci, leading to the expression of a new κ or λ light chain (receptor editing), which may result in loss of self-reactivity and thereby allow subsequent maturation(16). Therefore, to determine whether receptor editing is affected by Lyn-deficiency, we measured the level of rag1 and rag2 mRNA in BM B cells. We found that there was little difference in the levels of rag1 and rag2 mRNA between lyn^−/− and WT small pre-B cells, but lyn^−/− newly formed immature B cells expressed several-fold higher levels of RAG1 and RAG2 than did their WT counterparts (Figure 7A). Because RAG induction causes recombination events that can lead to a switch from Igκ light chain expression to expression of Igλ, we also investigated whether Lyn-deficiency affected the frequency of Igλ expressing B cells. lyn^−/− mice had slightly higher frequencies of Igλ⁺ newly formed cells than wild-type mice, although the frequencies of Igλ⁺ T1 B cells in the spleen were similar in WT and lyn^−/− mice (Figure 7B).

FIGURE 7

The Effect of Lyn-Deficiency on Receptor Editing and Proliferative Responses.

Finally, we also measured the frequency of B cells that had RS recombinations at the Igκ locus as an indication of receptor editing. Recombination at the RS sequence inactivates a functional Igκ locus, after which another light chain locus can be rearranged to generate a new light chain(16, 37). Purified Igλ⁻ small pre-B cells, newly formed, splenic T1, and follicular B cells were isolated by cell sorting from lyn^−/− and WT mice. The frequencies of RS recombination in these populations were measured by quantitative PCR of genomic DNA(22). This assay therefore measures the overall degree of light chain rearrangement in a population of B cells. Consistent with the Igλ frequency data, the RS frequencies tended to be higher in lyn^−/− newly formed and T1 B cells compared to WT, although these differences were relatively small (Figure 7C). Collectively, these data indicate that the mechanisms causing the removal of autoreactive BCRs in immature B cells are intact or slightly enhanced in lyn^−/− mice. This suggests that autoantibody production in lyn^−/− 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 wild-type counterparts as suggested by the signaling data. It has been reported that lyn^−/− 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 BCR engagement. Not surprisingly, BCR stimulation did not induce WT or lyn^−/− T1 B cells to proliferate (Figure 7D), in agreement with previous reports(27, 39, 40). In contrast, lyn^−/− follicular B cells proliferated much more vigorously than did wild type follicular B cells when stimulated with low doses of anti-IgM (2.5μg/ml) (Figure 7E). However, lyn^−/− and WT follicular B cells incorporated similar amounts of ³H-thymidine when stimulated with a higher dose of anti-IgM (15μg/ml). These data show that lyn^−/− follicular B cells are indeed more sensitive to anti-IgM-induced proliferation than wild type follicular B cells, consistent with their increased sensitivity to anti-IgM induced calcium release and Erk activation. These findings demonstrate that lyn^−/− mature B cells have a reduced threshold of activation, which therefore could contribute to the breakdown in tolerance to self-antigens in the periphery of these mice.

Discussion

Central to the ability of B cells to discriminate between foreign and self antigens is the difference in the responses to antigen of immature vs. mature B cells. Engagement of the BCR of immature B cells in the bone marrow induces expression of the RAG1 and RAG2 genes and subsequent Ig light chain rearrangements with the goal of changing specificity away from self-reactivity, a process called receptor editing(16). If this loss of self-reactivity is not achieved or if self-antigen triggers BCR signaling of T1 B cells in the spleen, then the cell undergoes apoptosis, resulting in clonal deletion. In contrast, strong BCR engagement of mature B cells induces proliferation and in combination with cytokines or other signals can facilitate a productive immune response. A variety of studies have indicated that immature B cells are more sensitive to antigen than are mature B cells. We have used newly improved methods for measuring signaling reactions in individual cells from immune organs to compare the sensitivity of the various developmental stages of B cells in the spleen and bone marrow for two key BCR-induced signaling responses, the elevation of intracellular free calcium and the activation of the Erk MAP kinase. These results confirm that T1 B cells are highly sensitive to BCR engagement and that follicular B cells are distinctly less sensitive. Moreover, we have shown that this change in signaling sensitivity of the BCR is due to a maturation-associated increase in the ability of the Lyn-CD22-SHP-1 pathway to attenuate BCR signaling. We propose that the low level of function of this inhibitory mechanism in immature B cells serves to permit efficient engagement of tolerance-promoting programs in these cells whereas its greater activity in mature follicular B cells serves to create a threshold for activation that minimizes the activation of B cells in response to self-antigens encountered in the periphery.

Previous studies have also compared the BCR responsiveness of immature and mature B cells, but have disagreed as to whether immature B cells are more sensitive(23, 24, 41), equally sensitive(25) or less sensitive(26, 28) to BCR stimulation relative to mature B cells. Our studies simultaneously analyzed immature and mature B cell populations taken immediately ex-vivo from the BM or spleen of unmanipulated mice and used staining conditions that did not perturb signaling, and thus, we feel they are likely to represent the responses of these cell types in a reliable way. The difference in sensitivity of BCR signaling that we observed did not appear to result primarily from changes in the expression of mIgM and mIgD that occur as B cells mature in the spleen. T1 cells have high levels of mIgM and low levels of mIgD, whereas mature B cells have high levels of mIgD and diverse levels of mIgM, ranging from low levels to the high levels seen in T1 B cells. We found that there was still a considerable difference in sensitivity to anti-IgM treatment between T1 B cells and a gated subpopulation of follicular B cells that had levels of mIgM comparable to the expression level on T1 cells (data not shown). Moreover, when we used the antigen lysozyme to engage the BCR on MD4 anti-lysozyme Ig transgenic T1 and follicular B cells, we again found substantially greater sensitivity of the former compared to the latter. Similarly, Wen et al(23), demonstrated that immature anti-Thy1 Ig transgenic B cells were more sensitive to Thy1 antigen than their mature B cell counterparts using techniques similar to ours. These findings indicate that mechanisms other than changes in surface Ig expression regulate B cell sensitivity to BCR engagement during maturation.

Genetic evidence indicates that this reduction in BCR sensitivity during B cell maturation is due to an increase in the efficacy of the inhibitory pathway mediated by Lyn, CD22 and SHP-1. BCR signaling of T1 and T2 B cells was only modestly affected by Lyn-deficiency, CD22-deficiency, or SHP-1 mutation, indicating that this inhibitory pathway is relatively inactive in immature B cells of the bone marrow or spleen. In contrast, there was a dramatic enhancement in BCR signaling of mature B cells by Lyn- and CD22-deficiencies demonstrating the importance of this inhibitory pathway in limiting BCR sensitivity once B cells mature. One contributor to this developmental change in the ability of the Lyn-CD22-SHP-1 pathway to inhibit BCR signaling is likely to be the 2.5-fold upregulation of CD22 during B cell maturation in the spleen. Follicular B cells from cd22^+/− mice, which express CD22 levels similar to those of wild type T1 B cells, exhibited enhanced calcium responses compared to WT follicular B cells but were distinctly less sensitive than WT T1 B cells. This indicates that other factors are present that mediate the developmental reduction in BCR sensitivity, besides upregulation of CD22. Regulation of SHP-1, CD22 or Lyn activity could contribute to the developmental change in inhibitory signaling. For example, Sialic acid acetylesterase has recently been shown to facilitate CD22 suppression of BCR signaling(42) and is upregulated during B cell development(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 to unstimulated T1 B cells, as evidenced by phosphorylation of the active site Y397 residue. Although regulation of the Lyn Y397 site is not well understood, phosphorylation of Y397 might be enhanced 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 decreased in unstimulated cd22^−/− mature B cells, which suggests that increasing CD22 levels during B cell development promotes Lyn activity, in addition to providing more substrate for Lyn.

Mice deficient in Lyn or CD22, as well as mice with a B cell-specific deletion of SHP-1 (36), are known to spontaneously produce autoantibodies against nuclear antigens, but the mechanism leading to this autoantibody production is unknown. We found that lyn^−/− mice had increased rag1 and rag2 mRNA expression in newly formed B cells and more modest increases in Igλ+ B cell frequencies and RS rearrangements compared to their wild-type counterparts. These data suggest that Lyn deficiency does not compromise tolerance-related responses of immature B cells to self-antigen and may even enhance them. Consistent with this conclusion, it has been shown that Lyn-deficiency does not cause a significant change in the number of V_H3H9 Igλ⁺ DNA-reactive B cells in the spleen(46). Another study demonstrated that Lyn-deficiency minimally affected the number of anti-HEL transgenic B cells in the bone-marrow of HEL-expressing mice, but did enhance the loss of anti-HEL transgenic mature B cells in the spleens of these mice(5). The implication of these results is that the Lyn–CD22–SHP-1 inhibitory pathway is not necessary for the establishment of tolerance to self during B cell development, in agreement with the slightly enhanced level of BCR signaling seen in immature B cells from mice genetically missing this pathway. In contrast, genetic mutations causing reductions in BCR signaling, and therefore reduced sensitivity to antigen, lead to enrichment of the B cell repertoire with autoreactive specificities in mice(47) and humans(48). Thus, it appears that Lyn-deficiency leads to autoimmunity not through a defect in central tolerance induction but rather due to a defect in the maintenance of tolerance in the periphery. In agreement with this idea, lyn^−/− mature follicular B cells exhibited substantially enhanced sensitivity to BCR-induced calcium release, Erk activation and proliferation relative to their wild type counterparts, which indicates that there is a reduction in the threshold of B cell activation in the absence of effective inhibition by the Lyn–CD22–SHP-1 pathway. This reduced threshold of activation could allow the recruitment of autoreactive B cells into an immune response, and perhaps in combination with abnormal myeloid cell function from Lyn-deficiency, result in autoantibody production.

In summary, we have found that during maturation of B cells in the spleen, the sensitivity of the BCR to antigen stimulation was down-regulated. This developmentally acquired down-regulation in BCR sensitivity required Lyn and CD22, which act together with SHP-1 in an inhibitory pathway to suppress BCR signaling selectively in mature follicular B cells. Surprisingly, the Lyn–CD22–SHP-1 inhibitory pathway had a minor role in the regulation of BCR signaling in immature B cells. In agreement with the signaling results, Lyn-deficiency had little effect on receptor editing-related events in immature B cells, which suggests that central tolerance mechanisms do not require this inhibitory pathway. On the basis of these observations, we propose that modulation of B cell sensitivity to antigen by the Lyn–CD22–SHP-1 inhibitory pathway during the course of B cell maturation is a critical factor in the maintenance of peripheral B cell tolerance to self-antigens.

Supplementary Material

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Acknowledgements

We thank Shuwei Jiang for cell sorting and Arthur Weiss, Jason Cyster, Michelle Hermiston, and Clifford Lowell (all at UCSF) and Ed Clark (U. Washington) for helpful discussions.

This work was supported by NIH K08 AI52249 and the Rosalind Russell Medical Research Center for Arthritis (AJG) and by NIH RO1 AI20038 (ALD).

Abbreviations used in this paper


BCR	B-cell receptor

BM	bone marrow

Fo	follicular B cell

HEL	hen-egg lysozyme

ITAM	immunoreceptor tyrosine-based activation motif

ITIM	immunoreceptor tyrosine-based inhibitory motif

mIgD and mIgM	membrane bound IgD and IgM

NF	newly formed immature B cell

BM-T2	bone marrow T2-like celll

RAG	recombinase activating gene

T1, T2, T3	transitional stage 1, 2 or 3 B cell

Tg	transgenic

Footnotes

Supplemental Data Three figures are available.

Disclosures The authors have no financial conflict of interest.

References

1. Zouali M, Sarmay G. B lymphocyte signaling pathways in systemic autoimmunity: implications for pathogenesis and treatment. Arthritis Rheum. 2004;50 [PubMed]

2. Lipsky PE. Systemic lupus erythematosus: an autoimmune disease of B cell hyperactivity. Nat Immunol. 2001;2 [PubMed]

3. Xu Y, Harder KW, Huntington ND, Hibbs ML, Tarlinton DM. Lyn tyrosine kinase: accentuating the positive and the negative. Immunity. 2005;22 [PubMed]

4. Chan VW, Meng F, Soriano P, DeFranco AL, Lowell CA. Characterization of the B lymphocyte populations in Lyn-deficient mice and the role of Lyn in signal initiation and down-regulation. Immunity. 1997;7 [PubMed]

5. Cornall RJ, Cyster JG, Hibbs ML, Dunn AR, Otipoby KL, Clark EA, Goodnow CC. Polygenic autoimmune traits: Lyn, CD22, and SHP-1 are limiting elements of a biochemical pathway regulating BCR signaling and selection. Immunity. 1998;8 [PubMed]

6. Saijo K, Schmedt C, Su IH, Karasuyama H, Lowell CA, Reth M, Adachi T, Patke A, Santana A, Tarakhovsky A. Essential role of Src-family protein tyrosine kinases in NF-kappaB activation during B cell development. Nat Immunol. 2003;4 [PubMed]

7. Chan VW, Lowell CA, DeFranco AL. Defective negative regulation of antigen receptor signaling in Lyn-deficient B lymphocytes. Curr Biol. 1998;8 [PubMed]

8. Nishizumi H, Horikawa K, Mlinaric-Rascan I, Yamamoto T. A double-edged kinase Lyn: a positive and negative regulator for antigen receptor-mediated signals. J Exp Med. 1998;187 [PMC free article] [PubMed]

9. Smith KG, Tarlinton DM, Doody GM, Hibbs ML, Fearon DT. Inhibition of the B cell by CD22: a requirement for Lyn. J Exp Med. 1998;187 [PMC free article] [PubMed]

10. Ochi H, Watanabe T. Negative regulation of B cell receptor-mediated signaling in B-1 cells through CD5 and Ly49 co-receptors via Lyn kinase activity. Int Immunol. 2000;12 [PubMed]

11. Ho LH, Uehara T, Chen CC, Kubagawa H, Cooper MD. Constitutive tyrosine phosphorylation of the inhibitory paired Ig-like receptor PIRB. Proc Natl Acad Sci U S A. 1999;96 [PubMed]

12. Adachi T, Flaswinkel H, Yakura H, Reth M, Tsubata T. The B cell surface protein CD72 recruits the tyrosine phosphatase SHP-1 upon tyrosine phosphorylation. J Immunol. 1998;160 [PubMed]

13. Ravetch JV, Lanier LL. Immune inhibitory receptors. Science. 2000;290 [PubMed]

14. Hibbs ML, Tarlinton DM, Armes J, Grail D, Hodgson G, Maglitto R, Stacker SA, Dunn AR. Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease. Cell. 1995;83 [PubMed]

15. Nishizumi H, Taniuchi I, Yamanashi Y, Kitamura D, Ilic D, Mori S, Watanabe T, Yamamoto T. Impaired proliferation of peripheral B cells and indication of autoimmune disease in lyn-deficient mice. Immunity. 1995;3 [PubMed]

16. Nemazee D. Receptor editing in lymphocyte development and central tolerance. Nat Rev Immunol. 2006;6 [PubMed]

17. Tiegs SL, Russell DM, Nemazee D. Receptor editing in self-reactive bone marrow B cells. J Exp Med. 1993;177 [PMC free article] [PubMed]

18. Gay D, Saunders T, Camper S, Weigert M. Receptor editing: an approach by autoreactive B cells to escape tolerance. J Exp Med. 1993;177 [PMC free article] [PubMed]

19. Otipoby KL, Andersson KB, Draves KE, Klaus SJ, Farr AG, Kerner JD, Perlmutter RM, Law CL, Clark EA. CD22 regulates thymus-independent responses and the lifespan of B cells. Nature. 1996;384 [PubMed]

20. Hsu LY, Lauring J, Liang HE, Greenbaum S, Cado D, Zhuang Y, Schlissel MS. A conserved transcriptional enhancer regulates RAG gene expression in developing B cells. Immunity. 2003;19 [PubMed]

21. Allen CD, Ansel KM, Low C, Lesley R, Tamamura H, Fujii N, Cyster JG. Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5. Nat Immunol. 2004;5 [PubMed]

22. Panigrahi AK, Goodman NG, Eisenberg RA, Rickels MR, Naji A, Luning Prak ET. RS rearrangement frequency as a marker of receptor editing in lupus and type 1 diabetes. J Exp Med. 2008;205 [PMC free article] [PubMed]

23. Wen L, Brill-Dashoff J, Shinton SA, Asano M, Hardy RR, Hayakawa K. Evidence of marginal-zone B cell-positive selection in spleen. Immunity. 2005;23 [PubMed]

24. Benschop RJ, Brandl E, Chan AC, Cambier JC. Unique signaling properties of B cell antigen receptor in mature and immature B cells: implications for tolerance and activation. J Immunol. 2001;167 [PubMed]

25. Hoek KL, Antony P, Lowe J, Shinners N, Sarmah B, Wente SR, Wang D, Gerstein RM, Khan WN. Transitional B cell fate is associated with developmental stage-specific regulation of diacylglycerol and calcium signaling upon B cell receptor engagement. J Immunol. 2006;177 [PubMed]

26. Koncz G, Bodor C, Kovesdi D, Gati R, Sarmay G. BCR mediated signal transduction in immature and mature B cells. Immunol Lett. 2002;82 [PubMed]

27. Allman D, Lindsley RC, DeMuth W, Rudd K, Shinton SA, Hardy RR. Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during peripheral B cell maturation. J Immunol. 2001;167 [PubMed]

28. Merrell KT, Benschop RJ, Gauld SB, Aviszus K, Decote-Ricardo D, Wysocki LJ, Cambier JC. Identification of anergic B cells within a wild-type repertoire. Immunity. 2006;25 [PubMed]

29. Allman DM, Ferguson SE, Cancro MP. Peripheral B cell maturation. I. Immature peripheral B cells in adults are heat-stable antigenhi and exhibit unique signaling characteristics. J Immunol. 1992;149 [PubMed]

30. Lindsley RC, Thomas M, Srivastava B, Allman D. Generation of peripheral B cells occurs via two spatially and temporally distinct pathways. Blood. 2007;109 [PubMed]

31. Erickson LD, Tygrett LT, Bhatia SK, Grabstein KH, Waldschmidt TJ. Differential expression of CD22 (Lyb8) on murine B cells. Int Immunol. 1996;8 [PMC free article] [PubMed]

32. Andersson KB, Draves KE, Magaletti DM, Fujioka S, Holmes KL, Law CL, Clark EA. Characterization of the expression and gene promoter of CD22 in murine B cells. Eur J Immunol. 1996;26 [PubMed]

33. Shultz LD, Schweitzer PA, Rajan TV, Yi T, Ihle JN, Matthews RJ, Thomas ML, Beier DR. Mutations at the murine motheaten locus are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene. Cell. 1993;73 [PubMed]

34. Hoffmann R, Seidl T, Neeb M, Rolink A, Melchers F. Changes in gene expression profiles in developing B cells of murine bone marrow. Genome Res. 2002;12 [PubMed]

35. O'Keefe TL, Williams GT, Batista FD, Neuberger MS. Deficiency in CD22, a B cell-specific inhibitory receptor, is sufficient to predispose to development of high affinity autoantibodies. J Exp Med. 1999;189 [PMC free article] [PubMed]

36. Pao LI, Lam KP, Henderson JM, Kutok JL, Alimzhanov M, Nitschke L, Thomas ML, Neel BG, Rajewsky K. B cell-specific deletion of protein-tyrosine phosphatase Shp1 promotes B-1a cell development and causes systemic autoimmunity. Immunity. 2007;27 [PubMed]

37. Casellas R, Shih TA, Kleinewietfeld M, Rakonjac J, Nemazee D, Rajewsky K, Nussenzweig MC. Contribution of receptor editing to the antibody repertoire. Science. 2001;291 [PubMed]

38. Wang J, Koizumi T, Watanabe T. Altered antigen receptor signaling and impaired Fas-mediated apoptosis of B cells in Lyn-deficient mice. J Exp Med. 1996;184 [PMC free article] [PubMed]

39. Loder F, Mutschler B, Ray RJ, Paige CJ, Sideras P, Torres R, Lamers MC, Carsetti R. B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals. J Exp Med. 1999;190 [PMC free article] [PubMed]

40. Su TT, Rawlings DJ. Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development. J Immunol. 2002;168 [PubMed]

41. Yellen AJ, Glenn W, Sukhatme VP, Cao XM, Monroe JG. Signaling through surface IgM in tolerance-susceptible immature murine B lymphocytes. Developmentally regulated differences in transmembrane signaling in splenic B cells from adult and neonatal mice. J Immunol. 1991;146 [PubMed]

42. Cariappa A, Takematsu H, Liu H, Diaz S, Haider K, Boboila C, Kalloo G, Connole M, Shi HN, Varki N, Varki A, Pillai S. B cell antigen receptor signal strength and peripheral B cell development are regulated by a 9-O-acetyl sialic acid esterase. J Exp Med. 2008 [PMC free article] [PubMed]

43. Stoddart A, Zhang Y, Paige CJ. Molecular cloning of the cDNA encoding a murine sialic acid-specific 9-O-acetylesterase and RNA expression in cells of hematopoietic and non-hematopoietic origin. Nucleic Acids Res. 1996;24 [PMC free article] [PubMed]

44. Young RM, Holowka D, Baird B. A lipid raft environment enhances Lyn kinase activity by protecting the active site tyrosine from dephosphorylation. J Biol Chem. 2003;278 [PubMed]

45. Karnell FG, Brezski RJ, King LB, Silverman MA, Monroe JG. Membrane cholesterol content accounts for developmental differences in surface B cell receptor compartmentalization and signaling. J Biol Chem. 2005;280 [PubMed]

46. Seo S, Buckler J, Erikson J. Novel roles for Lyn in B cell migration and lipopolysaccharide responsiveness revealed using anti-double-stranded DNA Ig transgenic mice. J Immunol. 2001;166 [PubMed]

47. Hayashi K, Nojima T, Goitsuka R, Kitamura D. Impaired receptor editing in the primary B cell repertoire of BASH-deficient mice. J Immunol. 2004;173 [PubMed]

48. Ng YS, Wardemann H, Chelnis J, Cunningham-Rundles C, Meffre E. Bruton's tyrosine kinase is essential for human B cell tolerance. J Exp Med. 2004;200 [PMC free article] [PubMed]