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The Journal of Immunology, 2002, 168: 5465-5476.
Copyright © 2002 by The American Association of Immunologists

Complementary Roles for CD19 and Bruton’s Tyrosine Kinase in B Lymphocyte Signal Transduction1

Manabu Fujimoto*, Jonathan C. Poe*, Anne B. Satterthwaite{dagger}, Matthew I. Wahl{dagger}, Owen N. Witte{dagger},{ddagger} and Thomas F. Tedder2,*

* Department of Immunology, Duke University Medical Center, Durham, NC 27710; and {dagger} Department of Microbiology and Molecular Genetics and {ddagger} Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD19 and Bruton’s tyrosine kinase (Btk) may function along common signaling pathways in regulating intrinsic and B cell Ag receptor (BCR)-induced signals. To identify physical and functional interactions between CD19 and Btk, a CD19-negative variant of the A20 B cell line was isolated, and CD19-deficient (CD19-/-) and CD19-overexpressing mice with the X-linked immunodeficient (Xid; Btk) mutation were generated. In A20 cells, Btk physically associated with CD19 following BCR engagement. CD19 and Btk interactions were not required for initial Btk phosphorylation, but CD19 expression maintained Btk in an activated state following BCR engagement. In primary B cells, CD19 signaling also required downstream Btk function since CD19-induced intracellular Ca2+ ([Ca2+]i) responses were modest in Xid B cells. In addition, CD19 overexpression did not normalize the Xid phenotype and most phenotypic and functional hallmarks of CD19 overexpression were not evident in these mice. However, CD19 and Btk also regulate independent signaling pathways since their combined loss had additive inhibitory effects on BCR-induced [Ca2+]i responses and CD19 deficiency induced a severe immunodeficiency in Xid mice. Thus, CD19 expression amplifies or prolongs Btk-mediated signaling, rather than serving as a required agent for Btk activation. Consistent with this, phosphatidylinositol 3-monophosphate kinase and Akt activation were normal in CD19-/- B cells following IgM engagement, although their kinetics of activation was altered. Thus, these biochemical and compound gene dosage studies indicate that Btk activation and [Ca2+]i responses following BCR engagement are regulated through multiple pathways, including a CD19/Src family kinase-dependent pathway that promotes the longevity of Btk signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Blymphocyte development and responses to Ag are regulated by cell surface signaling molecules including the B cell Ag receptor (BCR).3 BCR engagement activates signaling pathways through distinct non-receptor protein tyrosine kinases (PTKs) including the Src family PTKs (Lyn, Fyn, and Blk), Syk, and the Tec family member Btk. A subset of cellular Lyn constitutively associates with the BCR, but Lyn primarily localizes to the detergent-insoluble cholesterol- and sphingolipid-rich microdomains of the plasma membrane (1). BCR cross-linking may result in its rapid translocation into these lipid rafts, where Ig{alpha}/Ig{beta} are phosphorylated by Lyn (2, 3). Lyn and other Src family PTKs play crucial initiating roles in BCR signaling, which leads to downstream activation of Syk and Bruton’s tyrosine kinase (Btk), and additional downstream pathways that spread and amplify BCR signaling. Impaired Btk function results in human X-linked agammaglobulinemia and murine X-linked immunodeficiency (Xid) (4, 5, 6, 7). Xid represents a point mutation in the pleckstrin homology domain (6, 7) that is crucial for Btk membrane localization and activation (8, 9, 10, 11, 12, 13, 14). Xid and Btk-deficient mice have almost identical phenotypes (15, 16, 17). B cell maturation is altered in Xid mice, resulting in an ~50% reduction of peripheral B cells (18) and the absence of peritoneal B1 cells (19). Btk is limiting for transmission of signals from the BCR so B cells with reduced Btk expression or function are hyporesponsive to IgM and other transmembrane signals and generate decreased humoral responses to certain T cell-independent Ags (20, 21).

Intrinsic and BCR-induced signals are also regulated by cell surface molecules including CD19, an ~95-kDa B lymphocyte-specific member of the Ig superfamily (22). Like Btk, CD19 is expressed during most stages of B cell development until plasma cell differentiation (23). CD19 regulates intrinsic B lymphocyte signaling thresholds and also serves as a costimulatory molecule for amplifying BCR signaling (22). CD19 density on the cell surface is tightly regulated during B cell development (24, 25), with even subtle increases predisposing mice and potentially humans to the development of autoimmunity (26, 27). Changing CD19 expression levels in gene-targeted or transgenic mice results in significant changes in B cell function. B cells from CD19-deficient (CD19-/-) mice are hyporesponsive to transmembrane signals and generate modest immune responses (24, 28, 29, 30, 31, 32). By contrast, CD19 overexpression in transgenic (CD19TG) mice that express mouse CD19 (mCD19) plus human CD19 (hCD19) results in B cells that are hyperresponsive to transmembrane signals, elevated humoral immune responses, and disrupted tolerance with autoantibody production (24, 28, 29, 30, 31, 33, 34). Thus, CD19 functions as a general response regulator or rheostat, which defines intrinsic and BCR-induced signaling thresholds critical for expansion of the peripheral B cell pool.

Recent studies have identified common signaling pathways through which intracellular Btk and cell surface CD19 may be functionally linked to regulate intracellular Ca2+ ([Ca2+]i) responses (reviewed in Refs. 35–37). Btk activation and membrane localization requires Src family PTK and phosphatidylinositol 3-kinase (PI3-kinase) activation (10, 11, 38, 39, 40), with Btk activation contributing to phospholipase C{gamma} activation and [Ca2+]i mobilization (13, 41). A major function for CD19 is the regulation of Src family PTK activity (42, 43, 44). Lyn is the primary PTK to initially phosphorylate CD19 (43), while phosphorylated CD19, in turn, amplifies Src family PTK activity through events termed "processive amplification" (27, 43). CD19 phosphorylation and amplification of Lyn kinase activity facilitates CD19 interactions with Vav and the p85 subunit of PI3-kinase, and initiates downstream events such as the augmentation of [Ca2+]i responses and activation of the CD22/SHP1 regulatory pathway (42, 45, 46, 47, 48, 49, 50). CD19 amplification of Lyn kinase activity is also required for optimal CD22 phosphorylation (45, 51). CD22 phosphorylation induces formation of a CD22-Shc-Grb-2 ternary complex that may down-regulate [Ca2+]i responses through Src homology 2 domain-containing inositol polyphosphate 5-phosphatase (SHIP) recruitment (52). Simultaneous CD19 and BCR engagement enhances BCR-induced [Ca2+]i responses by sequestering the available pool of functional Lyn away from downstream negative regulatory proteins such as CD22 (53, 54). Since both CD19 and Btk participate in these overlapping signaling pathways, functional and physical interactions between CD19 and Btk were assessed in the current study using B cells with altered CD19 expression and/or deficient in Btk function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and lymphoblastoid cell lines

CD19-/- mice (129 x C57BL/6) and human CD19 transgenic (CD19TG) mice (SJL x C57BL/6) were as described elsewhere (28). Xid mice (CBA/CaHN-Btkxid) and wild-type control (CBA/Ca) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). CD19-/- mice carrying the Xid mutation were generated by breeding CD19-/- mice with Xid (CBA/Ca) mice. Heterozygous offspring were backcrossed with Xid (CBA/Ca) mice for at least three more generations before they were used in heterozygous matings (CD19+/- Xid x CD19+/- Xid) to generate homozygous Xid/CD19-/- offspring and Xid/CD19+/+ littermates that were used as controls. Using the same strategy, CD19TG mice that had been backcrossed with B6 mice for at least seven generations were crossed with Xid mice for at least four generations before being used to generate homozygous Xid/CD19TG+/+ offspring and Xid/CD19TG-/- littermate controls. Identical results were obtained with control Xid/CD19+/+ littermates, control Xid/CD19TG-/- littermates, and Xid (CBA/Ca) mice; therefore, the data obtained from these mice were pooled and reported as Xid values. All mice used were 2–3 mo of age and were housed in a specific pathogen-free barrier facility. All studies and procedures were approved by the Animal Care and Use Committee of Duke University Medical Center (Durham, NC).

A20 cells (55) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FCS, L-glutamine, streptomycin, penicillin, and 2-ME. CD19-negative lines were generated by repetitively removing the CD19-positive population using biotinylated anti-CD19 Ab (MB19-1; Ref. 29) and avidin-coated magnetic beads (Dynal Biotech, Lake Success, NY).

Reagents and immunofluorescence analysis

The mAbs used in this study included anti-mCD19 (MB19-1), anti-hCD19 (HB12b; Ref. 56), anti-CD22 (CY34.1, TIB163); biotinylated or FITC-conjugated anti-B220 (RA3-6B2); PE-conjugated anti-CD5 (53-7.3; BD PharMingen, San Diego, CA); and HRP-conjugated anti-phosphotyrosine Ab (4G10; Upstate Biotechnology, Lake Placid, NY and PY99, Santa Cruz Biotechnology, Santa Cruz, CA). Antisera used included F(ab')2 of goat anti-mouse IgM (Cappel, Durham, NC); F(ab')2 of goat anti-mouse IgG (H + L) (Zymed Laboratories, South San Francisco, CA); anti-Btk (Santa Cruz Biotechnology); anti-Btk-phosphoY223 and anti-Btk-phosphoY551 (57); anti-phospho-Akt (Ser473) and anti-Akt antisera (New England Biolabs, Beverly, MA); anti-mCD19 (generously provided by Dr. M. Grove, Duke University); and biotinylated, FITC-conjugated, or alkaline phosphatase-conjugated goat anti-mouse IgM, IgG, IgD, and IgA isotype-specific Abs (Southern Biotechnology Associates, Birmingham, AL). PE-conjugated streptavidin (Fischer Scientific, Pittsburgh, PA) was used to reveal biotin-coupled Ab staining.

Blood leukocytes and single-cell leukocyte suspensions from spleen, bone marrow, peripheral lymph nodes, and the peritoneal cavity were counted using a hemocytometer. Leukocytes (0.5–1 x 106) were stained for two-color immunofluorescence analysis at 4°C using predetermined optimal concentrations of Abs for 20 min as described previously (33). Blood erythrocytes were lysed after staining using FACS Lysing Solution (BD Biosciences, San Jose, CA). Cells with the forward and side light scatter properties of mononuclear cells were analyzed on a FACScan flow cytometer (BD Biosciences). Positive and negative populations of cells were determined using unreactive isotype-matched Abs (BD PharMingen, San Diego, CA) as controls for background staining.

B cell activation, immunoprecipitations, and Western blot analysis

Splenic B cells were purified by removing T cells with anti-Thy1.2 Ab-coated magnetic beads (Dynal Biotech). B cells were stimulated with goat anti-mouse IgG Ab F(ab')2 (10 µg/ml; Zymed Laboratories) or goat anti-mouse IgM Ab F(ab')2 (40 µg/ml; Cappel) and subsequently lysed in buffer containing 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 1 mM sodium orthovanadate, 2 mM EDTA, 50 mM NaF, and protease inhibitors. The lysates were either analyzed by SDS-PAGE or subjected to immunoprecipitation. For Btk immunoprecipitations, the cell lysates were precleared twice by incubating with appropriate control Abs plus protein G-Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ), followed by incubating with protein G beads plus rabbit or goat antiserum for 4 h at 4°C. For CD19 immunoprecipitations, the lysates were precleared with Affigel 10 beads (Bio-Rad, Hercules, CA) conjugated with mouse IgA Ab, then incubated for 4 h with Affigel 10 beads bearing anti-CD19 Ab (MB19-1). Following SDS-PAGE, the proteins were transferred to membranes that were incubated with HRP-conjugated anti-phosphotyrosine Ab or were incubated with specific Abs. These blots were incubated with HRP-conjugated donkey anti-rabbit IgG Abs (Jackson ImmunoResearch Laboratories, West Grove, PA) and developed using an ECL kit (Pierce, Rockford, IL). To verify equivalent amounts of protein in each lane, the blots were stripped and reprobed with Abs against the proteins of interest.

Measurement of [Ca2+]i

Spleen cells were isolated at room temperature, washed, and resuspended at 107/ml in RPMI 1640 medium containing 5% FCS and 10 mM HEPES, and loaded with 1 µM indo-1-AM ester (Molecular Probes, Eugene, OR) at 37°C for 30 min. The cells were washed and stained with FITC-conjugated anti-B220 mAb for 15 min at room temperature, washed, and resuspended at 2 x 106 cells/ml. A20 cells were treated likewise except for B220 staining. For analysis, the ratio of fluorescence (525:405 nm) of B220+ cells was determined using a FACStar flow cytometer (BD Biosciences). Baseline fluorescence ratios were collected for 1 min before treatment with Abs including F(ab')2 anti-mouse IgM (40 µg/ml), F(ab')2 anti-mouse IgG (H + L) (10 µg/ml), and/or anti-mCD19 Ab (MB19-1; 10 or 40 µg/ml for suboptimal or optimal concentrations, respectively) and anti-hCD19 Ab (HB12b; 40 µg/ml). Fluorescence ratios were collected at real time for 7 min following Ab addition. To inhibit PI3-kinase activity, wortmannin (50 nM; Sigma-Aldrich) and LY294002 (15 µM; Sigma-Aldrich) were added 10 min before measuring the fluorescence ratios in some experiments. Results were plotted as the fluorescence ratio at 20-s intervals with the background subtracted. Increased fluorescence ratios indicate increased [Ca2+]i.

In vitro PI3-kinase assays

After purified B cell lysates were normalized to the same protein concentration, they were immunoprecipitated with Abs to the p85 subunit of PI3-kinase and protein A-Sepharose beads for 2 h at 4°C. The beads were washed four times in lysis buffer and twice in reaction buffer (20 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 0.5 mM EGTA) and then incubated with 0.2 mg/ml sonicated phosphatidylinositol (Sigma-Aldrich), 10 µCi of [{gamma}-32P]ATP, and 20 µM ATP in 50 µl of reaction buffer for 15 min at 30°C. The reaction was stopped by adding 80 µl of 1 M HCl, and the phospholipids were extracted with 1/2 chloroform/methanol. The phospholipids were separated by TLC, and 32P incorporation into phosphatidylinositol 3-monophosphate (PI3P) was quantified using a phosphor imager. PI3P was identified using 32P-labeled PI3P as a positive control (generously provided by Dr. M. E. Cardenas, Duke University). Wortmannin (500 nM; Sigma-Aldrich) was used to inhibit PI3-kinase activity.

B cell proliferation

Purified spleen B cells (2 x 105 cells) were cultured in 0.2 ml of culture medium containing F(ab')2 anti-mouse IgM Abs in triplicate wells of 96-well flat-bottom tissue culture plates. Proliferation was assessed by the incorporation of [3H]thymidine (1 µCi/well) added during the last 16 h of culture followed by scintillation counting.

Serum Ig ELISAs

Mouse Ig isotype-specific ELISAs were performed as previously described (28) with affinity-purified mouse IgM, IgG1, IgG2a, IgG2b, and IgG3 (Southern Biotechnology Associates) used to generate standard curves.

Statistical analysis

All data are shown as mean values ± SEM. Comparisons between groups were made using the Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD19 expression influences Btk tyrosine phosphorylation

Since Btk phosphorylation is characteristically difficult to detect in primary B cells, a CD19-deficient B cell line was generated to assess physical and functional CD19-Btk interactions. A CD19-negative cell population was isolated from the A20 B lymphoblastoid cell line (A20-CD19neg) by repetitive depletion of CD19+ cells. A20-CD19neg cells did not express detectable CD19 as assessed by indirect immunofluorescence staining (Fig. 1GoA) and immunoprecipitation analysis (data not shown), and remained CD19neg after extended culture. A20-CD19neg cells expressed 2-fold higher levels of cell surface IgG (188 ± 14% in four experiments), although B220, CD21, CD22, and MHC class II expression levels were not altered (Fig. 1GoA and data not shown). Despite increased IgG expression, tyrosine phosphorylation of cellular proteins was reduced in A20-CD19neg cells following IgG ligation when compared with parental A20 cells (Fig. 1GoB). Thus, A20-CD19neg cells and B cells from CD19-/- mice are similar, since surface Ig levels are increased on B cells from CD19-/- mice (24), while BCR-induced tyrosine phosphorylation of cellular proteins is reduced in B cells from CD19-/- mice (24, 42, 49).



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  		FIGURE 1. Characterization of A20 cells lacking CD19 expression. A, Cell surface molecule expression by parental A20 (thin line) and A20-CD19neg cells (bold line) detected using FITC-conjugated mAbs with flow cytometry analysis. Immunofluorescence staining with an unreactive, isotype-matched, control mAb is also shown (dashed line). Results represent those obtained in four experiments. B, Protein tyrosine phosphorylation following cell surface IgG cross-linking. Parental A20 and A20-CD19neg cells (4 x 105/lane) were incubated with either medium alone (time 0) or with anti-IgG Abs for the indicated times. Cell lysates were subjected to SDS-PAGE analysis, electrophoretic transfer to nitrocellulose membranes, and subsequent antiphosphotyrosine immunoblotting. Molecular mass standards ( x 10-3) are shown on the right. C and D, Btk tyrosine phosphorylation in A20 cells. Parental and A20-CD19neg cells (3 x 107/lane) were incubated with F(ab')2 anti-IgG Abs for the times shown, detergent lysed, and incubated with protein G beads and either anti-Btk Ab or control rabbit IgG (CTL). Immunoprecipitated proteins were subjected to SDS-PAGE and transferred onto membranes for antiphosphotyrosine (C, anti-pTyr) immunoblotting, anti-phospho-Y223Btk (D), or anti-phospho-Y551Btk immunoblotting. Results represent those obtained in three experiments. E, Kinetics of CD19 phosphorylation in A20 cells. Parental A20 cells (1 x 107/lane) were treated as in C with CD19 immunoprecipitated from cell lysates. Immunoprecipitated proteins were divided, subjected to SDS-PAGE, and transferred onto membranes for immunoblotting with either anti-pTyr Ab (top) or rabbit anti-CD19 cytoplasmic domain Ab (bottom). F, CD19 tyrosine phosphorylation in Xid B cells. Purified splenic B cells (4 x 107/sample) were incubated with anti-IgM Ab for 0, 3, or 10 min. Cell lysates were incubated with beads bearing either anti-CD19 or isotype-matched control (CTL) mAbs and processed as described in E. Results represent those obtained in three experiments.

 
Whether CD19 expression affects Btk activation was assessed by immunoprecipitating Btk from parental A20 and A20-CD19neg cells before and after cell surface IgG cross-linking. Although maximal Btk tyrosine phosphorylation was similar in both cell populations after BCR ligation, Btk phosphorylation in A20-CD19neg cells was markedly reduced by 10 min after BCR ligation (Fig. 1GoC). These results were similar to those obtained by others using plasmacytoma cell lines, where BCR-mediated Btk activation was modest and relatively transient in the absence of CD19 expression (37). Nonetheless, the shortened time course for Btk phosphorylation in A20-CD19neg cells was also observed when Btk phosphorylation was assessed using antisera reactive with the Btk autophosphorylation (Btk-Y223) and the Lyn phosphorylation (Btk-Y551) sites (Fig. 1GoD). By contrast, CD19 expression was not necessary for the initial activation of Btk in A20 cells since Btk-Y551 phosphorylation was consistently increased in unstimulated A20-CD19neg cells and BCR-stimulated cells at 1 min when compared with parental cells (Fig. 1GoD). Thus, CD19 expression influenced the sites and duration of Btk phosphorylation in A20 cells.

Btk immunoprecipitated from parental A20 cells coprecipitated an ~100-kDa phosphoprotein that was not coprecipitated from A20-CD19neg cells (Fig. 1GoC). Reprobing of the immunoblots with an anti-CD19 antiserum confirmed that the ~100-kDa protein band contained CD19. However, CD19 was maximally coprecipitated by Btk at time points after maximal Btk phosphorylation (Fig. 1GoC) and at time points after maximal CD19 phosphorylation in A20 cells (Fig. 1GoE). These results demonstrate that CD19 expression prolongs Btk phosphorylation and activation and suggest that CD19/Btk interactions contributes to this.

CD19 regulation of BCR-induced [Ca2+]i responses

Despite increased IgG expression by A20-CD19neg cells, their [Ca2+]i responses following IgG ligation were markedly reduced (Fig. 2GoA). By contrast, B cells from CD19-/- mice generated near-normal [Ca2+]i responses following IgM ligation (Fig. 2GoB), with a delayed peak during the acute phase and a more prolonged late-phase response (24, 42). To determine whether A20-CD19neg cells are similar to B cells from CD19-/- mice, [Ca2+]i responses were assessed following BCR ligation with optimal concentrations of anti-Ig Abs. Total BCR ligation induced significantly higher [Ca2+]i responses than optimal IgM ligation in wild-type B cells (Fig. 2GoB). When CD19-overexpressing B cells were stimulated with optimal concentrations of either anti-IgM or anti-Ig Abs, initial [Ca2+]i responses were higher and faster with diminished late phase responses (Fig. 2GoB) as described for IgM ligation (53). Despite this, [Ca2+]i responses in B cells from CD19-/- mice were significantly reduced following total Ig ligation, with late-phase [Ca2+]i responses similar to those of B cells from wild-type littermates. Thus, altered CD19 expression affects B cell [Ca2+]i responses in characteristic ways with initial BCR-induced [Ca2+]i responses of both A20-CD19neg cells and CD19-/- B cells maximally inhibited when total surface Ig was ligated.



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  		FIGURE 2. Ca2+ responses following BCR ligation. A, [Ca2+]i responses of parental A20 and A20-CD19neg cells following BCR ligation using optimal concentrations of anti-IgG Ab. Cells were loaded with indo-1-AM ester and examined for relative [Ca2+]i levels by flow cytometry with anti-BCR Ab added to the cell mixtures at the indicated times (arrow). B, [Ca2+]i responses of B cells from wild-type, CD19-/-, and CD19TG mice. Spleen B cells stained with FITC-labeled anti-B220 mAb were treated using optimal concentrations of anti-IgM (left) or anti-Ig (right) Ab and examined for relative [Ca2+]i levels after gating on the B220+ population of cells. An increase in [Ca2+]i over time is shown as an increase in the ratio of indo-1 fluorescence. Results represent those obtained in at least three experiments.

 
Remarkably, the amplitudes of IgM- and total Ig-induced [Ca2+]i responses were nearly identical in CD19-/- B cells (Fig. 2GoB). Thereby, BCR engagement activates CD19-independent signaling pathways which generate [Ca2+]i responses of a defined magnitude. When CD19 is expressed, it further amplifies the BCR-induced response, particularly when total surface IgM plus IgD is ligated compared with IgM ligation alone. Thus, BCR-induced [Ca2+]i responses normally represent the sum of CD19-dependent and -independent pathways.

CD19 and Btk can independently influence BCR-induced [Ca2+]i responses

Functional interactions between CD19 and Btk during BCR-induced [Ca2+]i responses were assessed by generating CD19-/- and CD19TG mice with the Xid mutation. Although altered [Ca2+]i responses have been reported for Xid B cells (37, 58), splenic B cells from Xid mice generated near-normal [Ca2+]i responses following IgM ligation (Fig. 3GoA). CD19-/-B cells generated near-normal [Ca2+]i responses following IgM engagement, while CD19TG B cells generated faster initial [Ca2+]i responses with diminished late-phase responses (Figs. 2GoB and 3A). When compared with Xid or CD19-/- B cells, Xid/CD19-/- B cells had markedly reduced [Ca2+]i responses although the late-phase responses were more similar. Xid B cells that overexpressed CD19 generated faster initial [Ca2+]i responses after IgM ligation with diminished late-phase responses (Fig. 3GoA). IgM ligation induced nearly identical [Ca2+]i responses in B cells from mice with C57BL/6, CBA, or (CBA x C57BL/6) genetic backgrounds (data not shown). When either anti-IgM or anti-Ig Abs were used for BCR ligation with Xid B cells, Xid/CD19-/- B cells, or Xid/CD19TG B cells, similar results were obtained in each case (data not shown). That anti-IgM- and anti-Ig-induced [Ca2+]i responses were similar in Xid B cells presumably reflects their predominant cell surface IgM expression with relatively little IgD. Nonetheless, Xid and CD19 deficiency had additive effects, which suggests that Btk and CD19 influence BCR-induced [Ca2+]i responses through independent or partially overlapping pathways.



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  		FIGURE 3. Ca2+ responses following BCR and/or CD19 ligation. A, Splenocytes from wild-type, Xid, CD19-/-, Xid/CD19-/-, CD19TG, and Xid/CD19TG mice; B, wild-type and Xid mice; C, wild-type, CD19TG, and Xid/CD19TG mice. B cells loaded with indo-1 and stained with FITC-labeled anti-B220 mAb were examined for relative [Ca2+]i levels by flow cytometry after gating on the B220+ population of cells as described in Fig. 2Go legend. Abs were added at the indicated times (arrows): A, Optimal concentrations of anti-IgM; B, optimal concentrations of anti-IgM, anti-CD19, or optimal concentrations of anti-IgM with suboptimal concentrations of anti-CD19; or C, optimal concentrations of anti-mCD19 and anti-hCD19 mAbs. Results represent those obtained from at least three experiments.

 
CD19-induced [Ca2+]i responses are Btk dependent

In contrast to BCR-induced [Ca2+]i responses, CD19 engagement induces slower and less dramatic [Ca2+]i responses, while simultaneous BCR and CD19 engagement generates robust [Ca2+]i responses in wild-type B cells (Fig. 3GoB). Surprisingly, CD19 ligation on Xid B cells did not induce a significant [Ca2+]i response (Fig. 3GoB). By contrast, simultaneous BCR and CD19 engagement generated a wild-type [Ca2+]i response in Xid B cells (Fig. 3GoB), confirming that signal transduction pathways activated during simultaneous CD19 and BCR ligation are qualitatively different from CD19 ligation alone (53). Nonetheless, [Ca2+]i responses induced by CD19 ligation alone were predominantly dependent on Btk function.

To further assess the dependence of CD19 signaling on Btk function, B cell [Ca2+]i responses were assessed in CD19TG mice with the Xid mutation. Ligation of mCD19 induced lower [Ca2+]i responses in CD19TG B cells than in wild-type B cells (Fig. 3GoC), presumably due to higher overall CD19 density reducing the pool of Lyn and other downstream effector molecules available to mCD19 (53). Regardless, mCD19-induced [Ca2+]i responses were not significant in Xid/CD19TG B cells (Fig. 3GoC). Ligation of both mCD19 and hCD19 generated more robust [Ca2+]i responses in CD19TG B cells (Fig. 3GoC). Again however, simultaneous mCD19 and hCD19 ligation induced significantly lower [Ca2+]i responses in Xid/CD19TG B cells. Although the CD19TG mice were on a C57BL/6 genetic background and the Xid/CD19TG mice were on a (CBA x B6) background, mCD19 ligation induced identical [Ca2+]i responses in B cells from wild-type C57BL/6 and CBA mice (data not shown). In addition, the Xid mutation did not affect B cell surface expression of mCD19 or hCD19 (data not shown). Therefore, optimal CD19-induced [Ca2+]i mobilization is Btk dependent.

Btk expression does not significantly influence CD19 tyrosine phosphorylation

Whether Btk expression influences CD19 phosphorylation was assessed using B cells purified from Xid mouse spleens. Antiphosphotyrosine immunoblots of CD19 immunoprecipitated from B cells after cell surface IgM cross-linking revealed that CD19 was likely to be phosphorylated normally in Xid B cells (Fig. 1GoF). In two of four experiments, CD19 phosphorylation was slightly lower in Xid B cells than in wild-type B cells. However, these small differences may reflect maturity differences in Xid B cells. Therefore, it is likely that Btk expression does not substantially regulate CD19 phosphorylation after BCR ligation, but that CD19 expression influences downstream Btk activation.

BCR-induced PI3-kinase activation does not require CD19 expression

Since PI3-kinase plays an important role in Btk activation (13, 59) and PI3-kinase binding to CD19 may influence PI3-kinase function, the contribution of PI3-kinase to BCR-induced [Ca2+]i responses was assessed using potent PI3-kinase inhibitors. Wortmannin and LY294002 pretreatment reduced the magnitude of [Ca2+]i responses in both wild-type and CD19-/- B cells following IgM ligation (Fig. 4GoA and data not shown). Interestingly, PI3-kinase inhibitors inhibited CD19-/- B cell [Ca2+]i responses more significantly than wild-type B cells responses. In addition, late-phase [Ca2+]i responses in CD19-/- and wild-type B cells were less affected by PI3-kinase inhibitor treatment than acute [Ca2+]i responses. Thus, PI3-kinase activity contributed to [Ca2+]i responses regardless of CD19 expression, although [Ca2+]i responses were more dependent on PI3-kinase activity when CD19 was not expressed.



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  		FIGURE 4. PI3-kinase activity in CD19-/- B cells. A, [Ca2+]i responses following IgM ligation in splenic B cells from CD19-/- and wild-type littermates in the presence or absence of PI3-kinase inhibitors. Experiments were as described in Fig. 2Go legend except that some samples were pretreated with 50 nM wortmannin for 10 min. Similar results were obtained using LY294002 at 15 µM. Results represent those obtained in at least three experiments. B, PI3-kinase activation following BCR cross-linking in B cells from CD19-/- and wild-type (WT) littermates. Purified splenic B cells (107/lane) were incubated with anti-IgM Abs, lysed, and the lysates were immunoprecipitated with anti-PI3-kinase Abs or normal rabbit serum (CTL). Precipitated proteins were then incubated with phosphatidylinositol and [{gamma}-32P]ATP and separated by TLC. 32P incorporated into PI3P was quantified using a phosphor imager. The top panel represents results from three independent experiments which are described in the bottom graph as relative mean (±SEM) quantities of 32P incorporated into PI3P in all experiments. In each experiment, the kinase activities in control and unstimulated wild-type B cells were defined as 0 and 100%, respectively. C, Akt phosphorylation following BCR cross-linking in B cells from CD19-/- mice (top) and A20-CD19neg cells (bottom). Lysates (2 x 10-6 cells) were subjected to SDS-PAGE and transferred to membranes that were immunoblotted with anti-phospho-Akt Abs. The blots were stripped and reprobed with anti-Akt Abs as controls.

 
To quantify PI3-kinase activation in wild-type and CD19-/- B cells, the functional activity of immunoprecipitated PI3-kinase was assessed using in vitro kinase assays. Surprisingly, baseline PI3-kinase activity was ~50% higher in CD19-/- B cells than in wild-type B cells (p < 0.05; Fig. 4GoB). Following IgM cross-linking, the degree of activation relative to baseline levels of PI3-kinase activity was much lower in CD19-/- B cells than in wild-type B cells (p < 0.05). Regardless, PI3-kinase activity remained higher in CD19-/- B cells compared with wild-type B cells at 1 and 5 min. In identical experiments, differences in PI3-kinase activation between A20-CD19neg cells and parental A20 cells following IgG engagement were not significant (data not shown). Wortmannin treatment eliminated all kinase activity in these assays. Thus, CD19 expression was not required for PI3-kinase activation in B cells following BCR ligation, although the relative increase in PI3-kinase activation following BCR ligation was modest in CD19-/- cells relative to background levels. This distinction may clarify the apparent discrepancy between the current findings and those previously reported by others (49). In that study, all data assessing PI3-kinase activity were reported as fold increases relative to unstimulated B cells rather than differences between cell types relative to starting levels of PI3-kinase activity. Therefore, CD19 expression was not required for PI3-kinase activation before and following BCR ligation.

Since CD19 expression may be required for PI3-kinase membrane localization, PI3-kinase function was assessed by examining phosphorylation of the Akt protein kinase. Akt phosphorylation and activation are dependent on the PI3-kinase product 3'-phosphorylated inositol phosphate and wortmannin treatment blocks Akt activation (60, 61). Detergent lysates of purified B cells and A20 cells were subjected to SDS-PAGE before and after BCR cross-linking, followed by immunoblotting with Ab that binds activated Akt phosphorylated on Ser473. Phosphorylated Akt also undergoes a slight shift in electrophoretic mobility. Basal levels of Akt phosphorylation were generally higher in CD19-/- B cells (Fig. 4GoB). IgM-induced Akt phosphorylation increased faster in CD19-/- B cells, but Akt phosphorylation levels were generally normal relative to wild-type B cells (Fig. 4GoB). Higher constitutive Akt phosphorylation and faster IgG-induced phosphorylation were also observed with A20-CD19neg cells (Fig. 4GoC). Rapid Akt phosphorylation in CD19-/- B cells contrasts with recent observations by others (62). This difference may have resulted from variations in A20-CD19neg lines and IgM vs Ig stimulation of splenic B cells. Nonetheless, CD19 loss did not block activation of the PI3-kinase/Akt pathway following BCR ligation, but influenced the time course of Btk phosphorylation, PI3-kinase activation, and Akt phosphorylation.

B cell development in Xid mice with altered CD19 expression

The in vivo consequences of altered Btk and CD19 function were assessed using CD19-/- and CD19-overexpressing mice with the Xid mutation. These studies were limited to comparisons between littermates from fourth generation backcrosses that were wild type, Xid, Xid/CD19-/-, and Xid/CD19TG because each mouse line originated from different genetic backgrounds. There were no obvious differences among control wild-type littermates from Xid, Xid/CD19-/-, and Xid/CD19TG breedings; therefore, it is unlikely that differences resulted from genetic variation. Altered CD19 expression did not dramatically affect the generation of IgM-B220low pro/pre-B cells, IgM+B220low immature B cells, or IgM+B220high mature B cells in the bone marrow of Xid mice (Fig. 5GoA and Table IGo). Deletion or overexpression of CD19 alone has modest effects on the generation of IgM-B220low pro/pre-B cells or IgM+B220low immature B cells (28). By contrast, the frequency of mature B cells is increased in the bone marrow of CD19-/- mice, yet decreased in CD19-overexpressing mice, presumably due to increased negative selection. Thus, altering CD19 expression did not inhibit or rescue abnormal B cell development in Xid mice.



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  		FIGURE 5. Lymphocytes present in lymphoid tissues of Xid mice with altered CD19 expression. Single-cell suspensions of leukocytes were isolated from wild-type, Xid, Xid/CD19-/-, and Xid/CD19TG mice and examined by two-color immunofluorescence staining with flow cytometry analysis. Quadrant gates indicate negative and positive populations of cells as determined using isotype-matched unreactive control mAbs. Results represent those obtained with four to seven 2-mo-old mice of each genotype.

 

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  		Table I. Frequency, number, and cell surface IgM density of B lymphocytes in Xid mice with altered CD19 expression1

 
Eliminating CD19 expression reduced the number of circulating IgM+B220+ B cells in Xid mice by 4-fold (Fig. 5GoB and Table IGo). Normally, circulating B cell numbers are only modestly decreased, if at all, in CD19-/- mice (28). Therefore, the combined defects in Btk and CD19 expression were at least additive. Similar to blood, there was a severe reduction in the number of splenic and lymph node B cells in Xid/CD19-/- mice (Fig. 5GoC and Table IGo). However, the 85% decrease in spleen B cell numbers in Xid/CD19-/- mice was beyond the 50% decrease that normally results from CD19 deficiency alone. Decreased numbers of B220+IgM+ B cells in the peritoneal cavity, with a 96% decrease in B1 cells, was also a prominent feature of both Xid and Xid/CD19-/- mice (Fig. 5GoD and Table IGo). There was also an ~80% reduction in the number of peritoneal B220+CD5- B cells in Xid/CD19-/- mice compared with Xid mice. Thus, CD19 and Xid functions appeared additive for the production and maintenance of peripheral B cells, which suggests that CD19 and Btk can influence independent signaling pathways.

CD19 overexpression in Xid mice significantly reduced the frequency of circulating B cells (Fig. 5GoB and Table IGo), although the extent of the reduction was less dramatic than the normal >90% reduction that occurs with CD19 overexpression alone (28). Similar to blood, there was a severe reduction in the number of splenic and lymph node B cells in Xid/CD19TG mice (Fig. 5GoC and Table IGo). CD19 overexpression alone normally results in an 80% decrease in splenic B cells, so the 60% decrease in Xid B cell numbers overexpressing CD19 was less profound than anticipated. Nonetheless, decreased numbers of B220+IgM+ B cells in the peritoneal cavity was also a prominent feature of both Xid and Xid/CD19TG mice (Fig. 5GoD and Table IGo). Although CD19 overexpression normally increases B1 lineage cell numbers dramatically, this did not rescue the impaired development or maintenance of B1 cells caused by the Xid mutation. Rather, the loss of Btk function blunted the alterations in B cell numbers normally observed with CD19 overexpression. Thus, Btk appears responsible in part for the transmission of signals generated by CD19 overexpression.

IgM expression in Xid mice with altered CD19 expression

Increased surface IgM expression is a characteristic feature of Xid B cells, presumably due to altered maturation (18). Increased surface IgM expression is a characteristic feature of CD19-/- B cells and decreased surface IgM expression is characteristic of CD19-overexpressing B cells, presumably due to reciprocal alterations in signaling thresholds (24, 28). When compared with Xid mice, Xid/CD19-/- mice had significantly increased surface IgM density on their B220+ B cells in blood, spleen, and lymph nodes (Fig. 5Go and Table IGo). By contrast, Xid/CD19TG B cells expressed surface IgM densities similar to those of Xid B cells rather than the expected decrease in IgM levels (Fig. 5Go and Table IGo). In all cases, cell size differences (light scatter properties) were not detectable for Xid, Xid/CD19-/-, or Xid/CD19TG B cells when compared with comparable wild-type B cells. Therefore, IgM expression by Xid/CD19-/- B cells was synergistically regulated by both CD19 and Btk, while CD19 overexpression did not lower IgM expression levels in the absence of Btk function.

B cell proliferation in Xid mice with altered CD19 expression

B cell responses to anti-IgM Ab stimulation were assessed to clarify how CD19 and Btk differentially regulate B cell proliferation. Xid B cell proliferative responses were significantly lower than those of wild-type B cells, regardless of CD19 expression levels (Xid, 89 ± 4%; Xid/CD19-/-, 92 ± 4%; Xid/CD19TG, 78 ± 6% lower at 10 µg/ml; p < 0.01 in three experiments relative to wild-type B cells, Fig. 6Go). There were no significant differences in proliferation among Xid, Xid/CD19-/-, and Xid/CD19TG B cells. B cells from these three mouse lines also had decreased responses to LPS stimulation and anti-CD38 plus IL-4 stimulation (data not shown). B cells from CD19TG mice are normally hyperresponsive to mitogens, whereas CD19-/- or Xid B cells are hyporesponsive to BCR engagement. Thus, increased or decreased CD19 expression in the absence of normal Btk function did not significantly influence B cell proliferation.



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  		FIGURE 6. B cell proliferation in response to BCR stimulation. Spleen B cells (1 x 105/well) from wild-type, Xid, Xid/CD19-/-, and Xid/CD19TG mice were enriched using mAb-coated magnetic beads to remove T cells. B cells were cultured with the indicated concentrations of anti-IgM F(ab')2 Ab fragments. Proliferation was assessed by the incorporation of labeled thymidine (1 µCi/well) added during the last 16 h of 72-h cultures. Values represent the mean cpm (±SEM) obtained from triplicate cultures. Mean values significantly different from wild-type proliferation levels are indicated; **, p < 0.01. Results represent those obtained in three experiments.

 
Humoral immunity in Xid mice with altered CD19 expression

Serum Ig levels were examined in Xid mice with altered CD19 expression to assess B cell differentiation. Xid/CD19-/- mice had more severe hypogammaglobulinemia than Xid mice (Fig. 7Go), although this could result in part from severely decreased B cell numbers (Table IGo). Xid and Xid/CD19TG mice had similar levels of each Ab isotype as Xid, except IgG1 levels in Xid/CD19TG mice were significantly higher (Fig. 7Go). Normally, CD19-/- mice have reduced serum Ab levels of all isotypes, while CD19TG mice have increased levels of serum IgM and most IgG isotypes despite their reductions in peripheral B cell numbers (28). Thus, CD19 and Btk function synergistically during the generation of immune responses, yet CD19 overexpression did not compensate for Btk deficiency.



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  		FIGURE 7. Serum Ig levels. Values represent mean (±SEM) Ig levels for 5–10 wild-type, Xid, Xid/CD19-/-, and Xid/CD19TG mice as determined by isotype-specific ELISA. Mean values significantly different from wild-type levels are indicated; **, p < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies demonstrate that Btk and CD19 physically interact and share overlapping signaling pathways. Specifically, CD19 coprecipitated with Btk following BCR engagement in A20 B lymphoblastoid cells (Fig. 1GoC). Physical interactions between CD19 and Btk following BCR ligation may be important for optimal BCR signaling and the amplification of other transmembrane signals, but were not required for initial Btk phosphorylation (Fig. 1GoD). Rather, CD19 expression may maintain Btk in an activated state, since BCR-induced Btk phosphorylation was prolonged in A20 cells expressing CD19. In support of this notion, associations between CD19 and Btk were most obvious 10–20 min following BCR ligation, time points subsequent to optimal CD19 and Btk phosphorylation (Fig. 1Go, C–E). Btk does not bind to phosphotyrosine containing peptides representing phosphorylation sites in CD19 (43). Thus, Btk may bind CD19 indirectly through other molecules. Alternatively, the Btk Src homology 3 domain may mediate CD19-Btk interactions as occurs with Itk and CD28 in T cells (63). Prolonged Btk phosphorylation by CD19 expression may be functionally important since Btk mutations in human B cells abrogate sustained [Ca2+]i responses following BCR ligation (59). Although physical associations between CD19 and Btk are a provocative predictor of useful interactions, functional studies also demonstrated that Btk signaling activities were influenced by CD19 expression and engagement.

CD19 and Btk are likely to share overlapping signaling pathways with some component of Btk signaling downstream of CD19 (Fig. 8Go). In support of this, CD19-induced [Ca2+]i responses were essentially absent in Xid B cells (Fig. 3Go, B and C), while IgM-induced [Ca2+]i responses were only modestly affected (Fig. 3GoA). Similarly, CD19 overexpression augmented [Ca2+]i responses in wild-type B cells (Fig. 2GoB), but CD19-induced [Ca2+]i responses were modest in Xid/CD19TG B cells (Fig. 3GoC). Since the cytoplasmic domain of CD19 generates and maintains an autonomous Src family kinase activation loop that establishes basal levels of Lyn activation (42, 43), the Btk-Y551 Lyn phosphorylation site may be a direct and/or downstream target of this CD19/Lyn signaling pathway. Consistent with a requirement for Btk function downstream of CD19 signaling, CD19 tyrosine phosphorylation was not affected by the absence of Btk function (Fig. 1GoF). Furthermore, CD19 overexpression in Xid mice did not normalize the Xid phenotype and most phenotypic hallmarks of CD19 overexpression were not evident in Xid/CD19TG mice ( Figs. 5–7GoGoGo). For example, IgM expression on circulating B cells is significantly reduced in mice that overexpress CD19 (24). However, CD19 overexpression did not affect IgM expression on B cells from Xid mice (Fig. 5GoB and Table IGo). Similarly, CD19 overexpression significantly expands the frequency of peritoneal cavity B1 lineage cells (29), with CD19 and Btk deficiencies dramatically reducing the frequency of peritoneal B1 cell numbers (19, 28). However, CD19 overexpression did not induce the generation of peritoneal B1 cells in Xid mice (Fig. 5Go and Table IGo). Likewise, CD19 overexpression makes B cells hyperresponsive to mitogenic signals, while Xid/CD19TG B cell proliferation was similar to that of Xid B cells (Fig. 6Go). Therefore, Btk function contributes to the hyperresponsive phenotype of CD19TG B cells, with either direct CD19-Btk interactions or downstream Btk activation required for CD19-generated signals that regulate [Ca2+]i responses and B cell function.



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  		FIGURE 8. Potential models for how CD19 and Btk influence downstream [Ca2+]i responses. A, BCR ligation results in Lyn and Syk activation. Activated Lyn phosphorylates CD19, and, in turn, CD19 amplifies Lyn kinase activity that also leads to CD22 phosphorylation. B, BCR ligation in the absence of CD19 expression reduces Lyn amplification and CD22 phosphorylation. C, CD19 cross-linking activates Lyn, but not the Syk pathway. D, Simultaneous BCR and CD19 ligation results in enhanced activation of multiple pathways for PI3-kinase/Btk activation, but also extinguishes CD22 phosphorylation.

 
Although CD19 and Btk physically interact and may share a common signaling pathway, CD19-independent signaling pathways also influenced Btk function (Fig. 8GoB). For example, CD19 expression was not required for initial Btk activation in the A20 B cell line (Fig. 1Go, C and D). Furthermore, the loss of both CD19 and Btk significantly dampened IgM-induced [Ca2+]i responses in primary B cells (Fig. 3GoA), although IgM-induced [Ca2+]i responses were only modestly affected by the absence of Btk function (Fig. 3GoA) or CD19 expression (Figs. 2GoB and 3A). The concept of additive CD19 and Btk functions is also supported by the phenotype of Xid mice that lack CD19, where a combined CD19/Btk deficiency resulted in a more severe phenotype ( Figs. 5–7GoGoGo and Table IGo). For example, a combined CD19/Xid deficiency augmented IgM expression on circulating B cells (Fig. 5GoB) that was 4-fold beyond the effects observed with either deficiency alone. Cell surface IgM expression is normally augmented 3-fold on circulating CD19-/- B cells that mature normally (24). Increased IgM expression is unlikely to solely reflect the maturation state of B cells in CD19/Xid-deficient mice since CD19neg A20 cells had 2-fold higher levels of cell surface IgG when compared with parental CD19+ A20 cells (Fig. 1GoA). Thus, CD19 and Btk are likely to cooperatively regulate signaling thresholds that influence constitutive cell surface BCR density. Also in support of CD19-independent Btk signaling pathways, Btk/Lyn-deficient mice have a more severe immunodeficiency than Btk-deficient mice (64), and Xid/Lyn-deficient mice have a more severe immunodeficiency than Xid mice (65).

BCR ligation regulates [Ca2+]i responses through CD19-dependent and -independent pathways (Fig. 8Go). The magnitude of the CD19-dependent signaling pathway depends on whether IgM or total cell surface Ig was cross-linked (Fig. 2GoB), as reported previously (37). The CD19-independent signaling pathway appears to generate a [Ca2+]i response of defined magnitude (Fig. 2GoB) that may primarily rely on a Syk-dependent signal transduction pathway since Syk phosphorylation and kinase activity are intact in CD19-/- B cells following BCR ligation, CD19 ligation does not induce detectable Syk activation in primary B cells (42), and BCR-associated Src family PTKs initiate a Syk autophosphorylation activation loop that amplifies Syk activity (66). PI3-kinase activation is downstream of Syk activation following BCR signaling, with phosphatidylinositol-3,4,5-trisphosphate production by PI3-kinase decreased by 50% in the absence of Syk function (67). Thereby, Btk function is partly downstream of Syk and PI3-kinase activation since Btk activation is dependent on membrane recruitment via phosphatidylinositol-3,4,5-trisphosphate (10, 11, 13). Although CD19 and PI3-kinase interact after BCR ligation (43, 48), baseline PI3-kinase activity was elevated in B cells from CD19-/- mice with modest increases in enzymatic activity following IgM ligation (Fig. 4GoB). Nonetheless, total PI3-kinase activity induced by BCR engagement was not decreased in B cells from CD19-/- mice (Fig. 4GoA) or A20-CD19neg cells (data not shown). In addition, IgM-induced [Ca2+]i responses in CD19-/- B cells were more susceptible to wortmannin inhibition of PI3-kinase activity than wild-type B cells (Fig. 4GoA). These results are consistent with the recent demonstration that PI3-kinase activation is downstream of CD19/Lyn, but requires BCAP adapter protein interactions rather than CD19/PI3-kinase interactions (68). In addition, Akt phosphorylation in B cells from CD19-/- mice and A20-CD19neg cells mirrored the changes observed in PI3-kinase activity (Fig. 4GoC). Akt phosphorylation was accelerated in B cells from CD19-/- mice and A20-CD19neg cells, and Akt phosphorylation was not sustained in B cells from CD19-/- mice following IgM ligation (Fig. 4GoC). Likewise, baseline Btk-Y551 phosphorylation was elevated in the absence of CD19 expression and declined faster following BCR ligation in the absence of CD19 expression (Fig. 1GoD). Thus, Btk and PI3-kinase activation following BCR engagement may occur through a Syk/PI3-kinase-dependent pathway and a CD19/Src family kinase-dependent pathway, with the sum of these signaling pathways having specific quantitative and qualitative effects on [Ca2+]i responses and downstream signaling events.

Although CD19, PI3-kinase, and Btk are each important for the induction and maintenance of optimal [Ca2+]i responses induced by BCR engagement, other effector molecules and signaling pathways are likely to contribute as well. As an example, simultaneous BCR/CD19 ligation was not significantly affected by the Xid mutation while CD19-induced [Ca2+]i responses were significantly affected (Fig. 3GoC). This may be explained in part by the recent finding that amplified [Ca2+]i responses induced by simultaneous CD19 and BCR ligation may result from Lyn sequestration by CD19, which extinguishes phosphorylation of CD22 and perhaps other negative regulatory molecules (45, 53). Thereby, CD19 may also influence Btk function by regulating CD22 phosphorylation (Fig. 8Go), which induces the formation of a CD22-Grb-2-Shc-SHIP quaternary complex that may facilitate SHIP phosphorylation (52) and regulate Btk’s association with the membrane (13). Thus, functional interactions between the CD19 and Btk signaling pathways are likely to be dynamic and dependent on the state of B cell activation and the activating stimuli.

In summary, these biochemical and compound gene dosage studies demonstrate that Btk and CD19 physically interact and share overlapping signaling pathways with Btk downstream of CD19 signaling. In addition, CD19 and Btk can be regulated independently. These findings explain some of the similarities and differences observed between CD19-deficient, Xid, and Btk-deficient mice. Mice deficient in Btk (or Xid mice), PI3-kinase {alpha}, BLNK, and phospholipase C{gamma}2 exhibit remarkably similar phenotypes, suggesting that these molecules are linearly associated in a "B cell signalosome" (69). Each mouse demonstrates impaired B cell maturation, decreased serum IgM and IgG3 levels, normal T cell-dependent but modest T cell-independent Ab responses, as well as decreased numbers of peritoneal B1 cells. Since CD19-/- mice have normal B cell maturation, decreased serum IgM, IgG1, and IgG2 levels, modest T cell-dependent but near-normal T-independent Ab responses, their phenotype is distinct (24, 28, 29, 30, 31, 32). Thus, CD19 ligation is likely to influence the function of this downstream "signalosome," but CD19 is not a linear component of this signaling pathway (Fig. 8Go). Thus, while CD19 and Btk are physically and functionally linked, both molecules also play broader roles in B cell signaling and [Ca2+]i homeostasis.


   Acknowledgments
 
We thank Dr. M. E. Cardenas for assistance with the PI3-kinase assays and Dr. M. Grove for providing reagents.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants CA81776 and CA54464. A.B.S. is a Special Fellow of the Leukemia Society of America and O.N.W. is an Investigator of the Howard Hughes Medical Institute. Back

2 Address correspondence and reprint requests to Dr. Thomas F. Tedder, Department of Immunology, Box 3010, Duke University Medical Center, Durham, NC 27710. E-mail address: thomas.tedder{at}duke.edu Back

3 Abbreviations used in this paper: BCR, B cell Ag receptor; Btk, Bruton’s tyrosine kinase; [Ca2+]i, intracellular Ca2+; TG, transgenic; h, human; m, mouse; PI3-kinase, phosphatidylinositol 3-kinase; PI3P, phosphatidylinositol 3-monophosphate; PTK, protein tyrosine kinase; SHIP, Src homology 2 domain-containing inositol polyphosphate 5-phosphatase; Xid, X-linked immunodeficiency. Back

Received for publication December 7, 2001. Accepted for publication March 21, 2002.


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 Introduction
 Materials and Methods
 Results
 Discussion
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