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B CellsControlling Actin Cytoskeleton Dynamics inAntigen Processing and Presentation by Btk Regulates B Cell Receptor-Mediated

Shruti Sharma, Gregory Orlowski and Wenxia Song

http://www.jimmunol.org/content/182/1/329doi: 10.4049/jimmunol.182.1.329

2009; 182:329-339; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2009 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Btk Regulates B Cell Receptor-Mediated Antigen Processingand Presentation by Controlling Actin Cytoskeleton Dynamicsin B Cells1

Shruti Sharma, Gregory Orlowski, and Wenxia Song2

The high efficiency of Ag processing and presentation by B cells requires Ag-induced BCR signaling and actin cytoskeletonreorganization, although the underlying mechanism for such requirements remains elusive. In this study, we identify Bruton’styrosine kinase (Btk) as a linker connecting BCR signaling to actin dynamics and the Ag transport pathway. Using xid mice anda Btk inhibitor, we show that BCR engagement increases actin polymerization and Wiskott-Aldrich syndrome protein activationin a Btk-dependent manner. Concurrently, we observe Btk-dependent increases in the levels of phosphatidylinositide-4,5-bisphos-phate and phosphorylated Vav upon BCR engagement. The rate of BCR internalization, its movement to late endosomes, andefficiency of BCR-mediated Ag processing and presentation are significantly reduced in both xid and Btk inhibitor-treated B cells.Thus, Btk regulates actin dynamics and Ag transport by activating Wiskott-Aldrich syndrome protein via Vav and phosphati-dylinositides. This represents a novel mechanism by which BCR-mediated signaling regulates BCR-mediated Ag processing andpresentation. The Journal of Immunology, 2009, 182: 329–339.

A ntigen encounter initiates two critical cellular processesin B cells: signal transduction and Ag processing andpresentation. Multivalent Ags result in the translocation

of the BCR into lipid rafts in the vicinity of Src kinases, inducingsignaling cascades (1) and subsequent activation of transcriptionfactors (2). The BCR internalizes and transports Ags to the endo-somal compartments, where Ags are fragmented and loaded ontoMHC class II, generating ligands for T cells. Together, BCR-ini-tiated signaling and T cell help acquired through Ag presentationprovide the two crucial signals required for B cell activation andsubsequent T cell-dependent Ab responses.

The BCR can thus serve as both signal transducer and Ag trans-porter. By increasing the kinetics and specificity of Ag capture,uptake, and transport, the BCR increases the efficiency of Ag pro-cessing and presentation by B cells (3, 4), which enables B cells topresent even sparsely occurring Ags. Key signaling intermediates,such as the tyrosine kinase Syk and the adaptor protein BLNK (5,6) are involved in the timely transport of BCR-Ag complexes fromthe cell surface to Ag-processing compartments. BCR signalingblockade by the tyrosine kinase inhibitor genistein or PP2 (7, 8), orloss-of-function mutants for Lyn or Syk (6, 9), has been shown toimpede Ag uptake. Moreover, tyrosine phosphorylation of clathrinin lipid rafts upon BCR cross-linking (XL)3 is required for BCR

internalization (10), revealing the entwined nature of signaling andAg-transport pathways of the BCR.

The binding of the BCR to Ags not only induces the reorgani-zation of the actin cytoskeleton but also triggers its associationwith the BCR and signaling molecules, including Lyn, Syk, andGTP-binding proteins (11–13). Tyrosine kinase inhibitors blockBCR-induced actin polymerization (14), suggesting that actin re-modeling is downstream of BCR proximal signaling. Disruptingthe actin cytoskeleton does not inhibit BCR-induced tyrosine phos-phorylation or the translocation of the BCR into lipid rafts (15);however, it blocks BCR internalization by inhibiting the pinchingoff of clathrin-coated vesicles from the plasma membrane (PM)(16). An actin-dependent, but clathrin-independent, internalizationpathway for the BCR has recently been observed (17), underscor-ing the importance of actin in BCR internalization. These studieslead to the hypothesis that a dynamic actin cytoskeleton is a de-termining factor for the correct intracellular routing of BCR-boundAgs and that there is a regulatory relationship between the actincytoskeleton and BCR signaling and Ag-transport pathways.

The mechanistic links between the interrelated pathways ofBCR signaling, Ag transport, and the actin cytoskeleton have notbeen well studied. Wiskott-Aldrich syndrome protein (WASP) ispotentially one such link. WASP is a hematopoietic cell-specificactin regulator that links upstream signals to actin polymerizationand branching by stabilizing Arp2/3 complexes (18). WASP con-tains multiple interacting domains, including WASP homology-1(WH1), basic (B), GTPase-binding (GBD), proline-rich domains,and C-terminal verprolin homology, cofilin homology, and acidic(VCA) domains (19). The interaction of GTP-Cdc42 with theGBD, phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) withthe B domain, and phosphorylation at tyrosine 256 and 291 andserine 242 and 483/484 of WASP regulate its activity (20–23). Agbinding to the BCR has been shown to induce the phosphorylationof Rho family GTPase guanidine nucleotide exchange factor

Department of Cell Biology and Molecular Genetics, University of Maryland, CollegePark, MD 20742

Received for publication June 2, 2008. Accepted for publication October 28, 2008.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work is supported by a National Institutes of Health Grant AI059617 (to W.S.).2 Address correspondence and reprint requests to Dr. Wenxia Song, Department ofCell Biology and Molecular Genetics, University of Maryland, College Park, MD20742. E-mail address: wenxsong@umd.edu3 Abbreviations used in this paper: XL, cross-linking; AF, Alexa Fluor; B, basic; Btk,Bruton’s tyrosine kinase; CTX-B, cholera toxin subunit B; GBD, GTPase-bindingdomain; GEF, guanidine nucleotide exchange factor; HEL, hen egg lysozyme; PH,pleckstrin homology; PM, plasma membrane; PRD, proline-rich domain; PtdIns-4,5-P2, phosphatidylinositol-4,5-bisphosphate; Tf, transferrin; VCA domains, verprolinhomology, cofilin homology and acidic; WASP, Wiskott-Aldrich syndrome protein;

oWASP, WASP with open and active conformation; pWASP, phosphorylated WASP;MFI, mean fluorescence intensity; wt, wild type; MZ, marginal zone.

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

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(GEF) Vav (24), the activation of Rho family GTPases (25), andmodulation of phosphatidylinositide metabolism (26). Althoughall of these signaling activities potentially regulate WASP, the ex-act mechanism that links BCR signaling to WASP activation re-mains to be defined.

Bruton’s tyrosine kinase (Btk) belongs to the Tec tyrosinekinase family. The significance of Btk in B cell function wasrevealed by the discovery that Btk mutations cause inheritedimmunodeficiencies in both humans (XLA for X-linked agam-maglobulinemia) and mice (xid) (27). The xid mice have a pointmutation at arginine 28 to cysteine (R28C) in the pleckstrin ho-mology (PH) domain of Btk and show B cell developmental de-fects, with 50% reduction in mature B cells, a virtual absence ofthe B1-subset of cells, and a pronounced decrease in serum levelsof IgM and IgG3 (28). In addition to its kinase domain, Btk hasPH, Tec homology, SH3, and SH2 domains (27). Both the kinaseand PH domains are indispensable for Btk activity. Upon BCRactivation, Btk is recruited to the PM by its PH domain bindingPtdIns-3,4,5-P3 (29). At the PM, Btk is phosphorylated by Srckinases at tyrosine 551 in the kinase activation loop and then au-tophosphorylates tyrosine 223 in its SH3 domain (30, 31). Phos-phorylated Btk activates PLC�2 by binding to BLNK and, thus,modulates Ca2� influx (32). Additionally, it facilitates phosphati-dylinositide metabolism by its interaction with phosphatidylinosi-tol 4-phosphate 5-kinase (PIP5K) (33). The xid mutation preventsBtk from binding to PtdIns-3,4,5-P3 and recruiting to the PM, con-sequently inhibiting its activation (34, 35). Recent studies on Tcells from mice deficient in Tec kinases Itk and/or Rlk show im-paired actin polarization and defects in the recruitment of GTP-Cdc42 and its GEF Vav1 to the TCR at the immune synapse (36,37). These indicate a role for Tec kinases in linking upstreamsignaling to actin dynamics.

In this study, we explore the role of Btk, a Tec kinase expressedin B cells, in linking BCR signaling to the actin cytoskeleton andBCR-mediated Ag-processing pathways. We report that the BCR-triggered signaling regulates the dynamics of the actin cytoskele-ton through WASP in a Btk-dependent manner. Btk function isrequired for BCR-induced WASP and Vav activation, increasedcellular PtdIns-4,5-P2 levels, actin rearrangement, and ultimatelyfor BCR-mediated Ag processing and presentation.

Materials and MethodsMice and cells

Splenic B cells were isolated from wild-type (wt) (CBA/CaJ) and xid(CBA/CaHN-Btkxid/J) mice (Jackson Laboratories). Mononuclear cellswere obtained by Ficoll density-gradient centrifugation (Sigma-Aldrich). Tcells were deleted by anti-Thy1.2 mAb (BD Biosciences) and guinea pigcomplement (Rockland Immunochemicals). All procedures involving micewere approved by the Animal Care and Use Committee of University ofMaryland. B cell lymphoma A20 IIA1.6 cells (H-2d, IgG2a�, Fc�IIBR�)were cultured in DMEM supplemented with 10% FBS.

Flow cytometric analysis

B cells were stimulated with 20 �g/ml F(ab�)2-goat-anti-mouse IgG�M[F(ab�)2-anti-Ig] (Jackson ImmunoResearch Laboratories) at 37°C for in-dicated times. To determine the effect of the Btk inhibitor, B cells werepreincubated with LFM A-13 (100 �M; Calbiochem) at 37°C for 1 h, andthe inhibitor was included in the cell medium during B cell stimulation andchase. A nonactive derivative of LFM A-13, LFM A-11 (100 �M), wasused as a control (data not shown). Cells were fixed with 4% paraformal-dehyde, washed, permeabilized with 0.05% saponin, and stained with Al-exa Fluor (AF) 488-phalloidin (Invitrogen) or AF488-anti-phosphorylatedVav Y174 (pVav) (Santa Cruz Biotechnology). The cells were analyzedusing a FACSCalibur (BD Biosciences) flow cytometer. The data are rep-resented as mean fluorescence intensity (MFI). To compare the levels ofphosphorylated WASP (pWASP) in different B cell subpopulations,splenic B cells from wt and xid mice were incubated with AF488-anti-

mouse IgM at 4°C to label the surface BCR. To activate the BCR, the cellswere warmed up to 37°C for 5 min in the presence of AF488-anti-mouseIgM. The cells were then washed, fixed, and incubated with PE-Cy5-anti-mouse B220 and PE-anti-mouse IgD. After fixation and permeabilization,the cells were incubated with anti-mouse pWASP (S483/S484) Ab. Thecells were analyzed using CyAn (Dako) flow cytometer. Three subsets ofsplenic B cells were gated, including B220�IgMlowIgDhigh (mature andfollicular), B220�IgMhighIgDhigh (T2), and B220�IgMhighIgDlow (T1/mar-ginal zone (MZ)) B cells. The pWASP staining levels of different B cellsubsets were determined.

Immunofluorescence microscopy analysis

To analyze BCR internalization, B cells were incubated with 5 �g/ml Cy3-Fab-rabbit anti-mouse IgM (Cy3-Fab-anti-�; Jackson ImmunoResearchLaboratories) at 4°C for 30 min in the presence of 10 �g/ml F(ab�)2-anti-Igto label and cross-link the surface BCR. Cells were chased at 37°C forvarying lengths of time and incubated with AF488-cholera toxin subunit B(CTX-B; Invitrogen) at 4°C to label the PM.

To analyze the movement of the BCR from early to late endosomes, Bcells were incubated with Cy3-Fab-anti-� in the presence or absence ofF(ab�)2-anti-Ig at 18°C for 30 min to allow for BCR internalization. Thecells were warmed up to 37°C for varying lengths of time. To mark earlyendosomes, AF488-holo-transferrin (Tf) (10 �g/ml; Invitrogen) was in-cluded in the incubation medium at both 18 and 37°C. After fixation andpermeabilization, the cells were incubated with anti-CD32/CD16 mAb (BDBiosciences) to block Fc�R, anti-LAMP-1 mAb (1D4B; ATCC) to marklate endosomes, and AF488-phalloidin to label F-actin. To analyze thecellular distribution of oWASP and pWASP, pVav, and PtdIns-4,5-P2,splenic B cells were pulsed with Cy3-Fab-anti-� at 37°C for 10 min andchased in the presence or absence of F(ab�)2-anti-Ig for varying lengths oftime at 37°C. The cells were fixed, permeabilized, preincubated with anti-CD32/CD16 mAb, and incubated with Ab specific for oWASP (UpstateBiotechnology), pWASP S483/S484 (Bethyl Laboratories), pVav, orPtdIns-4,5-P2 (Invitrogen), followed by their corresponding secondaryAbs. Cells were analyzed under a laser-scanning confocal microscope(Zeiss LSM 510) or a Deltavision deconvolution microscope. Pearson’scorrelation coefficients of differently stained proteins, which measure theoverlap of pixels of two staining, were determined using the LSM 510software. Pearson’s linear correlation coefficient (r) is defined as followsbased on the principles described by Manders et al. (38):

rp ����Ri � Ravg��Gi � Gavg��

���Ri � Ravg�2��Gi � Gavg�

2

with Ri and Gi the pixel values in two different channels and Ravg and Gavg

the averages of all the Ri and Gi. The correlation coefficient here indicatesthe strength and direction of a linear relationship between the cellular lo-cations of two proteins. The r values range between �1 and �1 with �1being perfect colocalization, �1 being perfect exclusion, and 0 represent-ing no significant correlation or random distribution.

Analysis of actin nucleation sites

The actin nucleation sites were labeled as previously described (39).Briefly, B cells were serum starved for 1 h and then incubated with Cy3-Fab-anti-� and F(ab�)2-anti-Ig at 37°C for indicated times. In the lastminute of incubation, cells were treated with 0.45 �M AF488-G-actin (Cy-toskeleton) in the presence of 0.025% saponin and then immediately fixed.The cells were analyzed using a confocal fluorescence microscope andquantified using the LSM510 software.

Immunoblotting

B cells untreated or treated with LFM A-13 (100 �M) were activated withF(ab�)2-anti-Ig for indicated times and lysed. Cell lysates were analyzedwith SDS-PAGE and Western blot, and probed for pWASP S483/484 andpVav Y174, respectively. The blots were stripped and reprobed with anti-mouse �-tubulin Ab (Sigma-Aldrich) for establishing loading controls. Theblots were quantified by densitometry. The levels of pWASP and pVavwere normalized against tubulin and expressed as fold increases over un-stimulated levels.

Analysis of BCR internalization by flow cytometry

B cells were incubated with 10 �g/ml biotin-F(ab�)2-anti-mouse IgM at4°C and chased for 0, 5, 10, and 30 min at 37°C. Biotin-F(ab�)2-anti-�gMleft on the cell surface after the chase was stained with PE-streptavidin andquantified using a flow cytometer. The data were expressed as percentagesof the cell surface-associated biotin-F(ab�)2-anti-IgM Ab at time 0. To

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distinguish mature and immature/transitional B cells, splenic B cells werecolabeled with FITC-anti-mouse AA4.1 and PE-Cy5-anti-B220 Abs (BDBiosciences). BCR internalization in mature and immature/transitionalsplenic B cells was compared by gating on B220�AA4.1�(mature),B220�AA4.1� (immature/transitional) subsets, respectively.

Ag-presentation assay

Splenic B cells from wt and xid mice and wt splenic B cells that wereserum-starved and pretreated with or without LFM A-13 (100 �g/ml) wereincubated with hen egg lysozyme (HEL) alone or with the following Absin sequence at 4°C to target HEL to the BCR: anti-CD32/CD16 mAb toblock Fc�Rs, rabbit anti-mouse IgM (5 �g/ml) to bind the BCR, goat

anti-rabbit IgG (5 �g/ml) to link rabbit anti-HEL and rabbit anti-mouseIgM Abs, rabbit anti-HEL Ab (5 �g/ml), and finally HEL (0.5 or 1 �g/ml)as the Ag. Cells were warmed to 37°C with the Ag-Ab complex for 15 min,washed, and incubated at 37°C for 14 h. HEL-I-Ak complexes on the cellsurface were detected by AF488-C4H3 mAb and quantified by flow cy-tometry. To test the ability of B cells to present Ag to T cells, splenic Bcells were incubated with HEL (1 �g/ml) with or without the Ab complexfor 24 h. After washing, the B cells (1 � 106) were cocultured overnightwith KZH T cells (1 � 106) (a gift from Dr. Nilabh Shastri, Department ofMolecular and Cell Biology, University of California, Berkeley, CA) thatare specific for HEL46–61:I-Ak and express a lacZ reporter gene under thecontrol of the IL-2 promoter (40). The lacZ activity was assayed using

FIGURE 1. BCR activation induces the reorganization of the actin cytoskeleton and this actin remodeling is dependent on Btk. A, Wt splenic and A20B cells that were treated or left untreated with Btk inhibitor LFM-A13 (100 �g/ml) and untreated xid splenic B cells were stimulated with F(ab�)2-anti-mouse IgG�M (F(ab�)2-anti-Ig, 10 �g/ml) for 0, 2, 5, and 10 min. The cells were fixed, and F-actin was stained with AF488-phalloidin. The cells wereanalyzed using flow cytometry. Shown are the average fluorescence intensities (SD) of phalloidin staining at the indicated times from three independentexperiments. B–D, Splenic B cells from wt and xid mice were incubated with Cy3-Fab-anti-mouse �-chain (Fab-anti-�) and stimulated with F(ab�)2-anti-Igfor 1, 2, and 5 min at 37°C. In the last minute of the stimulation, cells were incubated with AF488-G-actin in the presence of detergent to label newlypolymerizing F-actin. The cells were immediately fixed and analyzed using a confocal fluorescence microscope. Shown are representative images from threeindependent experiments (B). Bar, 5 �m. Images were quantitatively analyzed to determine the fluorescence intensity of cell-associated AF488-G-actin (C)and the correlation coefficients between the labeled BCR and AF488-G-actin (D). Shown are mean values (SD) from three independent experiments whereover 300 cells were individually analyzed using Zeiss LMS 510 software (�, p � 0.01).

FIGURE 2. BCR stimulation induces Btk-dependentWASP activation. A, The surface BCR on wt splenic Bcells was labeled with Cy3-Fab-anti-� and either leftunstimulated (-XL) or stimulated with F(ab�)2-anti-Ig at37°C for indicated times. The cells were fixed, perme-abilized, and stained with an Ab specific for oWASP.Cells were analyzed using the Deltavision deconvolu-tion microscope. Shown are representative images fromthree independent experiments. Bar, 3 �m. B, The sur-face BCR of splenic B cells from wt (a–h) and xid (i–p)mice were labeled and stimulated as described in A. Af-ter fixation and permeabilization, cells were labeledwith AF488-phalloidin and an Ab specific for oWASP,and analyzed using a confocal fluorescence microscope.Bar, 3 �m. C, The colocalization coefficients betweenoWASP and BCR (a), BCR and F-actin (b), andoWASP and F-actin (c) for wt and xid B cells werequantified using the LSM 510 software. Shown are theaverage values (SD) from three independent experi-ments of �300 cells (�, p � 0.01).

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chlorophenol red �-galactopyranoside (41). The reaction product wasquantified by its absorbance at 595 nm, with 655 nm as the referencewavelength.

ResultsBCR activation induces Btk-dependent actin rearrangement

To investigate the relationship between BCR signaling pathwaysand the actin cytoskeleton, we determined changes in the overalllevels of cellular F-actin and actin polymerization in response toBCR XL. Upon Ag binding, the levels of total F-actin, quantifiedby phalloidin staining and flow cytometry, increased reproduciblyby 2 min followed by a decrease at later time points in both splenicand A20 B cells (Fig. 1A). This result suggests biphasic actin re-organization with polymerization and depolymerization dominatedphases. To follow actin polymerization, AF488-G-actin was intro-duced to cells in the presence of detergent in the last minute ofstimulation. The incorporation of AF488-G-actin into polymeriz-ing ends of actin filaments marks de novo actin nucleation sites.Similar to the cellular levels of F-actin, G-actin incorporation in-creased significantly 1–2 min after stimulation, after which thelevels of G-actin incorporation plateaued (Fig. 1, Ba–d and C). Tostudy the subcellular location of this incorporation relative to theBCR, the correlation indices of individual BCR and G-actin pixelswere calculated. XL the BCR significantly increased the correla-tion between staining for actin nucleation sites and the BCR (Fig.1D). Thus, BCR activation increases actin polymerization in thevicinity of the Ag-bound BCR.

To test whether Btk plays a role in linking BCR signal trans-duction to the actin cytoskeleton, we used xid mice and a Btkinhibitor, LFM A-13 (42). LFM A-13, a membrane permeable in-hibitor, inhibits the kinase activity of Btk. The inhibitor allows forinhibition of Btk activity independent of developmental defectsseen in xid B cells (28). LFM A-13 and Btk xid mutation showedsimilar inhibitory effects on both BCR-triggered gross and Btktyrosine phosphorylation (data not shown). The effect of the Btkmutation and inhibitor on BCR-induced actin rearrangement wasdetermined. LFM A-13 inhibited BCR-induced increases in F-ac-tin levels in both splenic and A20 B cells, while the xid mutationonly slightly reduced F-actin levels (Fig. 1A). The xid mutation notonly blocked BCR-induced increases in actin polymerization butalso drastically reduced the basal level of actin polymerization(Fig. 1, Be–h and C) and the colocalization of the BCR with actinnucleation sites (Fig. 1D). Thus, Btk is required for both consti-tutive actin polymerization and BCR-induced actin reorganizationin B cells.

Ag engagement of the BCR induces Btk-dependent activation ofWASP

To understand the mechanism for BCR-induced actin reorganiza-tion, we examined the cellular behavior of WASP, an actin nucle-ation promoting factor. The activation of WASP was followed bychanges in its conformation and phosphorylation using Abs spe-cific for WASP in its open, active conformation (oWASP) orWASP phosphorylated at S483/S484 (pWASP), respectively. Im-munofluorescence microscopic studies found that upon antigenicstimulation, oWASP was relocated from the cytoplasm to cell sur-face under BCR caps (Fig. 2, Aa–f). At 10 min after the stimula-tion, oWASP was recruited to BCR�-vesicles at the perinuclearlocation (Fig. 2, Ag–i). By 30 min, while some of oWASP re-mained with BCR�-vesicles, the rest appeared to move into thenuclei (Fig. 2, Aj–l). Consistent with these results, BCR activationsignificantly increased the correlation coefficient between thestaining of oWASP and the BCR by 5 min and it remained highuntil at least 30 min (Fig. 2Ca). Although the BCR colocalized

with F-actin in both stimulated and unstimulated cells, BCR XLfurther increased this colocalization (Fig. 2, Ba–h and Cb). Fur-thermore, BCR XL increased the correlation between the stainingof oWASP and F-actin from negative to positive values (Fig. 2Cc).

FIGURE 3. BCR activation increases the phosphorylation of WASP andcolocalization of pWASP with the BCR in a Btk-dependent manner. A,Splenic B cells from wt and xid mice were stained with Cy3-Fab-anti-� for theBCR and stimulated with F(ab�)2-anti-Ig at 37°C for indicated times. The cellswere fixed, permeabilized, and stained with an Ab specific for WASP phos-phorylated at S483/S484 (pWASP). The cells were analyzed using a confocalfluorescence microscope. Shown are representative images of three indepen-dent experiments. Bar, 3 �m. B, Shown are the means (SD) of pWASPfluorescence intensity of 300 cells from three independent experiments (�,p � 0.005). C and D, Cells showing membrane redistribution of pWASP werevisually determined and quantified. The data were plotted as percentages oftotal cells in images (C). The correlation coefficients between the BCR andpWASP in wt and xid B cells were determined using the LSM 510 software(D). Shown are the average results of three independent experiments where300 cells were analyzed (�, p � 0.005). E–H, A20 B cells that were treatedwith or without LFM A-13 (E and G) and splenic B cells from wt and xid mice(F and H) were stimulated with F(ab�)2-anti-Ig for indicated times. The cellswere lysed, and the cell lysates were analyzed using SDS-PAGE and Westernblot, and probed for pWASP S483/S484. The blots were stripped and reprobedfor tubulin as loading controls. The blots were analyzed by densitometry.pWASP levels were normalized against tubulin levels, and the data were plot-ted as fold increases over unstimulated levels (G and H). Shown are represen-tative blots and plots of three independent experiments (�, p � 0.05).

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The phosphorylation of WASP was analyzed by both immuno-fluorescence microscopy and Western blot. Immunofluorescencemicroscopic studies showed a significant increase in pWASP stain-ing over time in the splenic B cells upon BCR XL (Fig. 3, Aa, Ad,Ag, Aj, and B). The average level of pWASP staining peaked at 2min (Fig. 3B). Meanwhile, pWASP was relocated from the cyto-plasm to the PM where it colocalized with the surface BCR (Fig.3, Aa–i, C, and D). At 5 min, �50% of cells showed predominantcell surface staining of pWASP in contrast to �5% of unstimu-lated cells (Fig. 3C). By 10 min, the number of cells showingsignificant surface staining of pWASP were reduced to �25%(Fig. 3C). Correlation analyses showed continued increase in thecolocalization between pWASP and the BCR with time (Fig. 3D).pWASP colocalized with BCRs at the cell surface at early timepoints (Fig. 3, Ad–i, C, and D) and with internalized BCRs at latertime points (Fig. 3, Aj–l and D). Quantitative analysis of pWASPby Western blot showed that XL the BCR significantly increasedthe level of pWASP in both A20 (Fig. 3, E and G) and splenic Bcells (Fig. 3, F and H), which peaked at 2 min after stimulation.Thus, BCR activation induces phosphorylation and conformationalchanges in WASP and the recruitment of WASP to the cell surfaceand BCR�-vesicles.

The induction of WASP activation by BCR XL suggests thatBCR-derived signals regulate the actin cytoskeleton throughWASP. The lack of BCR-triggered actin reorganization in the Btk-deficient models implicates Btk in regulating WASP functions. Wethus measured the effect of the xid mutation and Btk inhibitor onthe cellular distribution and levels of oWASP and pWASP. Incomparison with wt splenic B cells, the staining level of oWASPin xid splenic B cells was much lower (Fig. 2B), suggesting adefect in WASP activation in xid B cells. Although BCR activationdid cause an initial increase in the colocalization between the BCRand oWASP in xid B cells, the correlation was significantly lowerthan in wt B cells (Fig. 2Ca). In contrast to wt splenic B cells,where the colocalization of the BCR and oWASP was sustained,this colocalization declined in xid B cells over time (Fig. 2Ca).Although the xid mutation had less of an inhibitory effect on thecorrelation of the BCR with F-actin (Fig. 2Cb), it significantlydecreased the correlation of oWASP with F-actin (Fig. 2Cc). BCRactivation failed to significantly increase the staining level ofpWASP (Fig. 3, Am–x and B) or induce comparable redistributionof the pWASP to the cell surface in xid B cells (Fig. 3, Am–x andC), similar to the results with oWASP. In xid B cells, the BCR andpWASP showed a negative correlation (Fig. 3D), indicating thatthe BCR and pWASP do not colocalize, rather they exclude fromeach other. Western blot analysis further confirmed that LFM A-13(Fig. 3, E and G) and the xid mutation (Fig. 3, F and H) both blockBCR-induced phosphorylation of WASP. Thus inhibition of Btkblocks BCR-induced activation and recruitment of WASP to theBCR, indicating a role for Btk in regulating WASP activity.

To test whether the inhibition of WASP activation in xidmice is an indirect effect of B cell developmental defects causedby the Btk mutation, we compared the levels of pWASP be-tween different subpopulations of splenic B cells by flow cy-tometry. We found that BCR XL increased pWASP levels in allthe splenic B cell subpopulations from wt mice, including Ig-DhighIgMlow mature follicular B cells, IgDhighIgMhigh T2 Bcells, and IgDlowIgMhigh T1 and MZ B cells, but failed to in-crease the pWASP levels in all the splenic B cell subpopula-tions from xid mice (Fig. 4). This indicates that it is the xidmutation, but not delayed B cell development, which inhibitsBCR-induced WASP activation.

BCR-induced biogenesis of PtdIns-4,5-P2 depends on Btk

To examine the mechanism for Btk-mediated activation of WASP,we determined the effect of Btk deficiency on the cellular level anddistribution of PtdIns-4,5-P2, a coactivator of WASP, using im-munofluorescence microscopy and flow cytometry. Significant in-creases in PtdIns-4,5-P2 staining levels were observed in both ac-tivated splenic (Fig. 5, Aa–i) and A20 B cells (Fig. 5C) comparedto unstimulated cells (-XL). Additionally, PtdIns-4,5-P2 appearedto be recruited to the cell periphery and BCR�-vesicles after acti-vation for 10 min (Fig. 5, Ag–i). The correlation analysis showed

FIGURE 4. The Btk xid mutation inhibits BCR-induced WASP phos-phorylation in all subsets of splenic B cells. Splenic B cells from wt and xidmice were incubated with AF488-anti-mouse IgM at 4°C. To activate theBCR, cells were warmed up to 37°C for 5 min in the presence of AF488-anti-mouse IgM. The cells were then washed, fixed, and stained with PE-Cy5-anti-mouse B220 and PE-anti-mouse IgD. After fixation and perme-abilization, cells were incubated with anti-mouse pWASP (S483/S484) Ab.The cells were analyzed using flow cytometry. Three subsets of splenic Bcells were gated, including B220�IgMlowIgDhigh mature follicular (FO),B220�IgMhighIgDhigh transitional T2, and B220�IgMhighIgDlow T1 andMZ B cells (A). Shown are representative histograms of pWASP levels ineach B cell subset from wt and xid spleens, with (�XL) and without (-XL)BCR XL, from three independent experiments (B).

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an increased colocalization between BCR and PtdIns-4,5-P2 stain-ing (Fig. 5B). This BCR-induced increase in the levels of PtdIns-4,5-P2 and its redistribution were severely dampened in xid splenicB cells (Fig. 5, Aj–r and B). LFM A-13 treatment not only blockedBCR-triggered increases in PtdIns-4,5-P2 but also dramatically re-duced constitutive levels of PtdIns-4,5-P2 in unstimulated A20 B cells(Fig. 5C). Thus, cellular biogenesis of PtdIns-4,5-P2 in response toBCR stimulation is dependent on the unimpaired activity of Btk.

BCR-triggered Vav activation requires Btk

In addition to PtdIns-4,5-P2 binding to the B domain, the coordi-nated binding of GTP-Cdc42 to the GBD domain of WASP isrequired for WASP activation (23). Vav serves as a GEF for Cdc42(43). To test whether Btk-dependent WASP activation is mediatedthrough Vav, we determined the effect of the xid mutation andLFM A-13 on Vav activation. Vav activation was followed by itsrecruitment to the cell surface and its phosphorylation in responseto BCR stimulation (44) using an Ab specific for Vav phosphor-ylated at Y174 (pVav) by immunofluorescence microscopy, flowcytometry, and Western blot. In wt splenic B cells, BCR XL no-ticeably increased the staining levels of pVav, compared with un-stimulated B cells (-XL) (Fig. 6, Aa–i). This BCR-triggered in-crease in pVav staining was drastically reduced in the xid B cells(Fig. 6, Aj–r). pVav accumulated primarily at the cell surfacewhere it heavily colocalized with the BCR at early times after

activation (�2 min) (Fig. 6, Ad–f). At later times (10 min), pVavcolocalized not only with the surface BCR but also the internalizedBCR at the perinuclear location (Fig. 6, Ag–i). Correlation analysisshowed an increase in the colocalization of pVav and the BCRupon BCR XL (data not shown). Both flow cytometry (Fig. 6B)and Western blot (Fig. 6, C–F) analyses confirmed that BCR XLincreased pVav levels in wt splenic and A20 B cells, which werereduced by the xid mutation and LFM A-13 treatment. Thus, BCR-triggered Vav phosphorylation and colocalization of pVav with theBCR are dependent on the activity of Btk.

Btk inhibitor and deficiency inhibit BCR-mediated Aginternalization and transport to Ag-processing compartments

BCR-mediated Ag uptake and transport is dependent upon theintegrity of signaling pathways and the actin cytoskeleton.Btk’s roles in modulating both signaling and the actin cytoskel-eton implicate its role in BCR-mediated Ag processing. To testthis hypothesis, we determined the effects of Btk deficiency onthe internalization and movement of the BCR from the cellsurface to Ag-processing compartments. The surface-labeledBCR was chased for 30 min at 37°C. The cell surface wasidentified with CTX-B which binds GM1, early endosomes withholo-Tf, and late endosomes/lysosomes by LAMP-1. After 30min of Ab XL, the BCR moved from the PM to a perinuclearlocation (Fig. 7, Aa and Ac), where most of BCRs colocalizedwith LAMP-1 (Fig. 7, Bg–i) while a small portion was foundwith Tf (Fig. 7, Ba– c). In contrast, after the same treatment, thesurface labeled BCR in splenic xid B cells remained colocalizedwith CTX-B at the PM (Fig. 7Ad) and showed little to no in-ternalization and colocalization with Tf (Fig. 7, Bd–f) orLAMP-1 (Fig. 7, Bj–l). The correlation between the BCR andLAMP-1 staining increased with time in wt splenic B cells, butthis increase was abrogated in xid splenic B cells (Fig. 7C). Wenext determined the effect of Btk deficiency on the kinetics ofBCR internalization, which was followed by decreases in thelevels of surface-labeled BCR after the chase using flow cy-tometry. We found that the rate of BCR internalization wasdramatically reduced in the xid B cells, in comparison with thewt B cells (Fig. 7D). Similarly, LFM A-13 significantly reducedBCR internalization rates in both splenic and A20 B cells (Fig.7D). Furthermore, by gating on B220�AA4.1� immature/tran-sitional B cells and B220�AA4.1� mature B cells, we foundthat both mature and immature B cells from the spleen of xidmice showed reduced rates of BCR internalization comparedwith respective wt B cell subsets (Fig. 8), indicating that thedecrease in the rate of BCR internalization is not the result ofB cell developmental defects in xid mice. These results dem-onstrate a requirement for Btk-mediated signals in efficientBCR internalization and transport of Ag to Ag-processingcompartments.

Btk-deficiency and Btk inhibition reduce BCR-mediated Agpresentation

The inhibitory effect on BCR internalization and transport tolate endosomes suggests that Btk deficiency interferes withBCR-mediated Ag processing. To test this, we compared theefficiencies of BCR-mediated Ag presentation by wt and xid Bcells and by wt B cells treated with or without Btk inhibitorLFM A-13. A model Ag, HEL, was targeted to the BCR forBCR-mediated Ag processing and presentation using an Abcomplex. The surface levels of HEL46 – 61-loaded MHC class III-Ak (HEL46 – 61:I-Ak) was determined by mAb C4H3 (45) andflow cytometry. To test the sensitivity and efficiency of Ag pro-cessing and presentation, splenic B cells were pulsed with HEL

FIGURE 5. BCR activation induces Btk-dependent increase in PdtIns-4,5-P2 levels. Splenic B cells from wt (Aa-i) and xid (Aj-r) mice were incu-bated with Cy3-Fab-anti-Ig to label the BCR and activated with F(ab�)2-anti-Igfor indicated times at 37°C. The cells were fixed, permeabilized, and stainedwith an anti-PtdIns-4,5-P2 mAb followed by a Cy2-conjugated secondary Ab.The cells were analyzed by a confocal fluorescence microscope. Shown arerepresentative images from three independent experiments (A). Bar, 3 �m. Thecolocalization between the BCR and PtdIns-4,5-P2 (PIP2) staining in wt andxid B cells was quantified as correlation coefficients using the LSM 510 soft-ware (B). Shown are the average results (SE) of two independent experi-ments where 200 cells were analyzed. PtdIns-4,5-P2 (PIP2) levels in un-treated and LFM A-13-treated A20 cells were analyzed by flow cytometry (C).Shown are the MFI (SD) of PtdIns-4,5-P2 that were plotted against time(�, p � 0.01).

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(0.5 or 1 �g/ml) either alone (for pinocytosis-mediated Ag pro-cessing) or in presence of the Ab complex (for BCR-mediatedAg processing) for 15 min, washed, and incubated at 37°C for14 h. The wt B cells pulsed with HEL plus the Ab complexdisplayed much higher levels of surface HEL46 – 61:I-Ak thanthose pulsed with HEL alone (Fig. 9A). This indicates thatBCR-mediated Ag processing and presentation has a higher ef-ficiency than nonspecific pinocytosis. The wt and xid B cellspulsed with HEL alone showed similar surface levels ofHEL46 – 61:I-Ak (Fig. 9A, dotted line), suggesting that Btk defi-ciency does not significantly affect pinocytic rates of Ag pro-cessing and presentation. Although both wt and xid B cellsshow similar levels of MHC class II I-Ak on their surfaces (datanot shown), the surface levels of HEL46 – 61:I-Ak on xid B cellspulsed with HEL-Ab complex were much lower than those onwt B cells, even though they were slightly higher than those ofxid B cells pulsed with HEL alone (Fig. 9, A and C). Althoughboth wt and xid B cells increased surface HEL46 – 61: I-Ak levelsas the Ag concentration increased, the increase shown by xid Bcells was much smaller than that by wt B cells (Fig. 9C). Sim-ilarly, the Btk inhibitor, LFM A-13, significantly decreased the

surface levels of HEL46 – 61:I-Ak in treated B cells compared tountreated B cells (Fig. 9, B and D). To further compare theabilities of wt and xid B cells to present Ag and activate T cells,splenic B cells were first incubated with HEL or HEL-Ab com-plex for 24 h, washed, and then incubated with KZH T cells.The KZH T cells, specific for the same peptide-MHC class IIcomplex recognized by the C4H3 mAb, express a lacZ reportergene under the control of the IL-2 promoter (40). T cell acti-vation was monitored by the expression of the reporter lacZ.Similar to the surface HEL46 – 61: I-Ak levels, wt and xid B cellspulsed with HEL alone activated the KZH T cells to similarextents (Fig. 9E). However, when HEL was targeted to the BCRby the Ab complex, wt B cells stimulated the KZH T cells to amuch higher level than xid B cells (Fig. 9E). These data dem-onstrate a role for Btk in regulating BCR-mediated Ag process-ing and presentation.

DiscussionThe binding of Ags to the BCR induces a series of cellular eventsthat are essential for B cell activation, including signaling cas-cades, actin reorganization, and Ag internalization for processing

FIGURE 6. BCR activation induces Btk-dependentphosphorylation of Vav and recruitment of phosphory-lated Vav to the BCR. A, The surface BCR of splenic Bcells from wt (a–i) and xid mice (j–r) were labeled withCy3-Fab-anti-� and activated with F(ab�)2-anti-Ig forvarying lengths of time. The cells were fixed, perme-abilized, and stained with an Ab specific for phosphor-ylated Vav at Y174 (pVav). Images were acquired usinga confocal fluorescence microscope. Shown are repre-sentative images from three independent experiments.Bar, 3 �m. B, The wt splenic B cells that were treated oruntreated with LFM A-13 and xid splenic B cells wereactivated with F(ab�)2-anti-Ig for varying lengths oftime. The cells were fixed, permeabilized, and stainedwith an Ab specific for pVav Y174. The MFI of pVavwas quantified using flow cytometry. Shown is a repre-sentative plot of pVav MFI vs the time from three in-dependent experiments. C–F, A20 B cells that weretreated with LFM A-13 or left untreated (C and D) aswell as splenic wt and xid B cells (E and F) were stim-ulated with F(ab�)2-anti-Ig for indicated times and lysed.The lysates were analyzed by SDS-PAGE and Westernblot, and probed for pVav Y174. The blots werestripped and reprobed for tubulin as loading controls.The blots were analyzed using densitometry. pVavlevels were normalized against tubulin levels, pre-sented as fold increases over unstimulated B cells,and plotted as a function of time. Shown are repre-sentative blots and average pVav levels from threeindependent experiments.

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and presentation. Although there is a regulatory relationship be-tween these cellular events, no distinct link between these cellularpathways has been defined. In this study, we identify Btk as alinker that transduces signals from the BCR into actin reorganiza-tion by controlling the activity of WASP, Vav, and PtdIns-4,5-P2

biogenesis. Significantly, Btk-dependent actin cytoskeleton re-modeling is required for the high efficiency of BCR-mediated Aguptake, processing, and presentation.

BCR activation is known to trigger changes in the actin cy-toskeleton (12, 46). We further characterize it as a biphasic processwith a rapid but transient increase in cellular F-actin in the first fewminutes post BCR XL, followed by a decline during the next fewminutes. Moreover, XL of the BCR triggers site-directed actin po-lymerization near the BCR. Localized actin polymerization shownhere provides an explanation for the dependency of BCR internal-ization on the actin cytoskeleton we reported previously (16). BCRactivation induces actin assembly at BCR internalization sites andBCR�-vesicles, where this polymerization may provide the driv-

ing force for fission of clathrin-coated vesicles and inward move-ment of BCR�-vesicles. BCR-triggered depolymerization of F-actin, in contrast, may loosen the “fence” formed by the corticalactin cytoskeleton, allowing for inward movement of BCR-contain-ing vesicles. Thus, these biphasic actin dynamics may be essential forrestructuring the actin cytoskeleton in response to BCR activation.

The abrogation of BCR-triggered actin cytoskeleton rearrange-ment in the presence of tyrosine kinase inhibitors and Syk defi-ciency (6, 7, 9) indicates a regulatory role for BCR-mediated sig-naling in actin dynamics. Using two model systems, xid mice andBtk inhibitor LFM A-13, we demonstrate that BCR-dependent ac-tin polymerization and even the constitutive level of actin poly-merization is dependent on the functionality of Btk. This studyreveals for the first time that Btk is the major signaling componentthat links BCR signals to the actin cytoskeleton in B cells.

Although the Btk xid mutation and LFM A-13 exhibit similarinhibitory effects on Btk activity and actin dynamics in B cells,each of them could influence actin dynamics through a mechanismdifferent from Btk, as the xid mutation causes B cell developmen-tal delays (47) and LFM A-13 can inhibit Jak2 (48). Our findingthat both mature and immature/transitional subsets of splenic Bcells increase the level of pWASP to similar degrees and exhibitsimilar BCR internalization rates demonstrates that the observedinhibitory effect of the xid mutation is not caused by B cell de-velopmental defects. Because BCR activation does not induce Jak2

FIGURE 7. Btk inhibitor and xid mutation inhibit BCR internalizationand intracellular movement to late endosomes. A–C, Splenic B cells fromwt and xid mice were incubated with Cy3-Fab-anti-� at 4°C to label thesurface BCR and treated with or without F(ab�)2-anti-Ig for 30 min at 37°C.Then cells were incubated with AF488-CTX-B at 4°C to demarcate the cellsurface (A). The cells were fixed, permeabilized, and stained for LAMP-1using a mAb (ID4B) for marking late endosomes (Bg–l). To mark earlyendosomes, splenic B cells from wt and xid mice were labeled with Cy3-Fab-anti-� in the presence of F(ab�)2-anti-Ig at 18°C for 30 min and chasedat 37°C for 30 min in the presence of AF488-holo-Tf (Ba–f). The cells wereanalyzed using a confocal fluorescence microscope. Representative imagesfrom three independent experiments are shown. Bar, 3 �m. C, The corre-lation coefficients between BCR and LAMP-1 staining were determinedfrom images of �300 cells from three independent experiments using theZeiss LSM 510 software (�, p � 0.05). D, Splenic B cells from wt and xidmice and LFM A-13-treated wt and A20 B cells were incubated with bi-otinylated F(ab�)2-anti-Ig at 4°C to label the surface BCR. After washing,cells were incubated at 37°C for indicated times. Biotin-F(ab�)2-anti-Igremaining on the cell surface after the chase was detected with PE-strepta-vidin and quantified using flow cytometry. Shown are the average percent-ages (SD) of biotin-F(ab�)2-anti-Ig remaining on the cell surface fromthree independent experiments (�, p � 0.05).

FIGURE 8. The Btk xid mutation decreases the rate of BCR internal-ization in both mature and transitional splenic B cells. Splenic B cells fromwt and xid mice were labeled with PE-Cy5 anti-mouse B220, FITC-anti-mouse AA4.1 (CD93), and biotin-F(ab�)2-anti-mouse IgM at 4°C andchased at 37°C for varying lengths of time. BCR internalization was ana-lyzed as described in Fig. 7. BCR internalization in mature and immatureB cell subsets (B) was measured by gating for B220�AA4.1� (mature) andB220�AA4.1� (immature/transitional B cells) (A). Shown are data fromtwo independent experiments.

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phosphorylation and BCR-induced STAT activation is indepen-dent of Jaks (49), the effect of LFM A-13 on actin polymerizationis unlikely due to its inhibition of Jak2.

The results presented here demonstrate that BCR-triggered, Btk-dependent actin cytoskeleton rearrangement in B cells is mediatedthrough WASP. Binding of Ag to the BCR increases the levels ofoWASP, triggers its phosphorylation at S483/484, and recruits ac-tivated WASP to the PM and the BCR. The concordance of Ag-bound BCR, actin nucleation sites, and active WASP strongly sug-gests that WASP mediates actin polymerization and branching atBCR internalization sites. A fraction of the active WASP wasfound to maintain its colocalization with the BCR, even after it hadbeen internalized, suggesting a role for WASP beyond internaliza-tion, possibly in driving the inward movement and/or membranefusion of BCR�-vesicles to late endosomes. The relationship ofdefects in BCR-triggered WASP activation and actin reorganiza-tion in xid and Btk inhibitor-treated B cells reinforces that Btk canfunnel BCR signaling cues to the cytoskeleton through WASP.

The activation mechanism for WASP has been extensively stud-ied and a general model for its activation has emerged. In theabsence of stimuli, WASP is present in an autoinhibited state me-diated by the interaction of its GBD with VCA region. This auto-inhibition is released when its GBD, B, and proline-rich domainscoordinately bind to GTP-Cdc42, PtdIns-4,5-P2, and a SH3 do-main-containing protein, respectively, freeing the VCA region forbinding Arp2/3 (19). The phosphorylation of WASP at Y291 andS483/484 (22) increases the actin polymerization activity ofWASP, by stabilizing its open, active conformation. In B cells,BCR activation has been shown to induce transient tyrosine phos-phorylation of WASP (50), and an interaction of WASP with Btkin vitro has been reported (50–52). Our results show that BCRactivation induces the cell surface recruitment and phosphorylationof Cdc42 GEF Vav and increases the cellular biogenesis of PtdIns-4,5 P2 and the surface recruitment and serine phosphorylation ofWASP, all of which occur in a Btk-dependent manner. These re-

sults suggest that Btk activates WASP by several related mecha-nisms, by regulating the activity of Vav, the generation of PtdIns-4,5-P2, and the phosphorylation of WASP. The direct interactionof WASP with Btk reported previously (50–52) provides anothermechanism for Btk to regulate the phosphorylation and subcellularlocation of WASP, whereby Btk recruits WASP to the PM whereWASP interacts with PtdIns-4,5-P2 and is phosphorylated bykinases.

Reports on the involvement of Tec kinases in regulating Vavactivity in T cells (37, 53) and our study support a general functionof Tec kinases in regulating WASP activity by controlling theactivity of Vav. How Btk activates Vav in B cells remains to beelucidated. Possible mechanisms include direct phosphorylation ofVav by Btk or Btk-mediated recruitment of Vav to BCR signalingmicrodomains, where it is phosphorylated by Src or Syk. Theadaptor function of Btk brings to the cell surface PIP5K, the pri-mary PtdIns-4,5-P2-generating lipid kinase (33). In this study, weshow that Btk is able to regulate the local metabolism of PtdIns-4,5-P2 that in turn is a substrate or coactivator for downstreameffectors of Btk such as PLC�2 and WASP (23, 32). Btk-depen-dent PtdIns-4,5-P2 generation activates WASP in cooperation withGTP-Cdc42, the product of Btk-dependent Vav activation.

Btk’s capacity in BCR signaling has been well characterized(54–57). The results of this study show for the first time that Btkis part of a regulatory mechanism for efficient Ag uptake and trans-port that leads to Ag processing and presentation. This appears tocontradict previous findings that xid and Btk�/� mice have pro-found defects in response to T-independent Ags but not in theirT-dependent responses to protein Ags. The high efficiency of Bcells to process and present Ag relies on the abilities of the BCRto capture Ags with high specificity and affinity, to rapidly inter-nalize them, and specifically target them to the Ag-processingcompartment. This allows B cells to present Ags even when ex-posed to low levels of Ags for short periods of time. The disruption

FIGURE 9. The Ag-presentation efficiency is reduced in both Btk-deficient B cells and Btk inhibitor-treated B cells. A and B, Splenic B cells from xidand wt mice (A) or splenic B cells from wt mice that were serum starved and treated with or without LFM A-13 (B) were pulsed with HEL (1 �g/ml) aloneor with the Ab complex that targets HEL to the BCR at 37°C for 15 min. After washing, cells were incubated at 37°C for 14 h. MHC class II I-Ak loadedwith HEL peptides (HEL46–61: I-Ak) on the cell surface was detected using a mAb (C4H3) and quantified using flow cytometry. Shown are representativehistograms of three independent experiments. C and D, Shown are the ratios of MFI of HEL46–61:I-Ak on the surface of B cells that were pulsed with theHEL-Ab complex, which targets HEL to the BCR, vs those B cells that were pulsed with HEL alone, where HEL was internalized through pinocytosis.Shown are average values (SD) of three independent experiments (�, p � 0.01). E, Splenic B cells (1 � 106) from wt and xid mice were either incubatedwith HEL (1 �g/ml) alone or with the Ab complex for 24 h. After washing, the B cells (1�106) were cocultured overnight with KZH T cells (1 � 106).The activity of LacZ that is under the control of IL-2 promoter in the T cells was measured using a colorimetric LacZ substrate. Shown is the OD of theLacZ enzymatic product over time and representative data of three independent experiments with triplicates (�, p � 0.005).

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of Btk function, either by the R28C mutation or the inhibitor, re-duces the rates of BCR internalization and its movement to theAg-processing compartment. This consequently decreases theamount of Ag available to the processing and presentation ma-chinery. It is important to note that the processing and presentationof Ags endocytosed by pinocytosis is not significantly affected bythe Btk xid mutation. This implies that Btk’s role in this process isinstigated by the BCR binding to cognate Ags. Since the process-ing and presentation of Ag internalized through nonspecific pino-cytosis was not affected by the Btk xid mutation, T-dependent Abresponse will not be completely blocked or significantly affected,especially when Ag is abundant as in the case of animal immuni-zation models. The specific effect of the Btk xid mutation and Btkinhibitor on BCR-induced Ag presentation would reduce the sen-sitivity and efficiency of B cells to process and present Ag, espe-cially when Ag is not in abundance like at the beginning of aninfection. This notion is supported by an early report that the ac-tivation of B cells from xid mice is sensitive to the concentrationof T-dependent Ag (58). The defects of BCR-mediated Ag pro-cessing and BCR-triggered actin cytoskeleton rearrangement in thexid B cells suggest that Btk connects BCR signaling activity to itsAg transport and processing functions by mobilizing the actincytoskeleton.

A working model for the interactions among BCR signaling, theactin cytoskeleton, and BCR Ag-processing pathway, thus,emerges. The binding of the BCR by multivalent Ags triggers theformation of surface signaling microdomains. The production ofPtdIns-3,4,5-P3 by PI3K recruits Btk to the signaling microdo-mains where Btk is activated by phosphorylation. Subsequently,Btk recruits WASP to the signaling microdomains and activates itby inducing its phosphorylation, activating Vav and consequentlyCdc42, and increasing local PtdIn-4,5-P2 levels. Activated WASPpromotes actin assembly and branching in the vicinity of the BCR.This provides the driving force for the formation of BCR�

clathrin-coated vesicles and the inward movement and fusion ofthese Ag-containing vesicles with the Ag-processing compart-ment. This cross-talk mechanism between signaling, actin cy-toskeleton, and membrane transport machineries might be im-portant for all lymphocytes to transduce antigenic signals intocellular responses. The functional and physical interaction be-tween Btk and WASP may not be the only link between BCRsignaling and the actin cytoskeleton. B cells from WASPknockout mice only show mild defects in general (59), suggest-ing the presence of additional links. Other members of theWASP family proteins, N-WASP and WAVE (60), potentiallycompensate for WASP downstream of BCR signaling. BCR sig-naling could also regulate the actin cytoskeleton through otheractin regulators that do not belong to the WASP family, such asthe regulation of HS1, an actin-binding protein and a homo-logue of cortactin expressed in lymphocytes, by Syk kinases(61) and actin-binding protein 1 by BCR signaling (62). More-over, redundant functions provided by other Tec kinases cannotbe eliminated, as the recently created Btk/Tec double knockoutmice (63) display defects that are more severe than Btk knock-out alone. Future studies will examine additional molecularlinks and interaction mechanisms between the actin cytoskele-ton and BCR-mediated signaling and Ag-1processing pathways.

AcknowledgmentsWe thank Drs. Kenneth Frauwirth, Lian-Yong Gao, and Silvia Bolland forcritical reading of the manuscript and Amy Beaven for technical assistanceon confocal fluorescence microscope.

DisclosuresThe authors have no financial conflict of interest.

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