3966.full

IMMUNOBIOLOGY

Wiskott-Aldrich syndrome protein (WASP) and N-WASP are critical forperipheral B-cell development and functionLisa S. Westerberg,1-4 Carin Dahlberg,4 Marisa Baptista,4 Christopher J. Moran,1-3 Cynthia Detre,5 Marton Keszei,5Michelle A. Eston,1-3 Frederick W. Alt,6,7 Cox Terhorst,5 Luigi D. Notarangelo,8 and Scott B. Snapper1-3,9

1Gastrointestinal Unit and 2Center for the Study of Inflammatory Bowel Diseases, Massachusetts General Hospital, Boston, MA; 3Department of Medicine,Harvard Medical School, Boston, MA; 4Translational Immunology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden; 5Division ofImmunology, Beth Israel Deaconess Medical Center, Boston, MA; 6Department of Genetics, 7Immune Disease Institute, Howard Hughes Medical Institute, and8Divison of Immunology and The Manton Center for Orphan Disease Research, Childrens Hospital Boston, Boston, MA; and 9Gastroenterology Division,Childrens Hospital, Harvard Medical School, Boston, MA

The Wiskott-Aldrich syndrome protein(WASP) is a key cytoskeletal regulator ofhematopoietic cells. Although WASP-knockout (WKO) mice have aberrant B-cell cytoskeletal responses, B-cell devel-opment is relatively normal. Wehypothesized that N-WASP, a ubiqui-tously expressed homolog of WASP, mayserve some redundant functions withWASP in B cells. In the present study, wegenerated mice lacking WASP and N-WASP in B cells (conditional double

knockout [cDKO] B cells) and show thatcDKO mice had decreased numbers offollicular and marginal zone B cells in thespleen. Receptor-induced activation ofcDKO B cells led to normal proliferationbut a marked reduction of spreading com-pared with wild-type and WKO B cells.Whereas WKO B cells showed decreasedmigration in vitro and homing in vivocompared with wild-type cells, cDKOB cells showed an even more pronounceddecrease in the migratory response

in vivo. After injection of 2,4,6-trinitrophenol(TNP)Ficoll, cDKO B cells had reducedantigen uptake in the splenic marginalzone. Despite high basal serum IgM, cDKOmice mounted a reduced immune re-sponse to the T cellindependent antigenTNP-Ficoll and to the T celldependentantigen TNPkeyhole limpet hemocyanin.Our results reveal that the combined activ-ity of WASP and N-WASP is required forperipheral B-cell development and func-tion. (Blood. 2012;119(17):3966-3974)

IntroductionB cells are generated via sequential differentiation steps in the BMand enter the circulation as immature, surface IgM-expressingcells.1 Immature B cells migrate into the spleen, where theydifferentiate into mature, naive B cells through highly regulateddevelopmental steps. Naive, mature B cells recirculate through thebloodstream and enter into peripheral lymph nodes, peritoneal orpleural cavities, gut-associated lymphatic tissue, and the spleen,where they differentiate into effector cells in response to specificantigenic challenge. In the spleen, B cells can undergo an importantcell-fate decision to become either a follicular (FO) or a marginalzone (MZ) B cell.1 FO B cells reside inside B-cell follicles, wherethey can undergo affinity maturation and class-switch recombina-tion in response to antigenic challenge.2 MZ B cells reside in thesplenic MZ, a location that provides a first line of defense againstblood-borne pathogens. Peripheral B-cell development, activation,and function require both migration and adhesive properties. FOB cells depend on signaling by the chemokine receptor CXCR5 tolocalize to the follicles, whereas MZ B cells are sensitive tosphingosine-1-phosphate (S1P), which is highly concentrated inblood.1 Adhesion by MZ B cells to ICAM-1 and 41 integrin iscritical for MZ B-cell retention in the MZ, an area that is exposed tothe sheer stress of blood flow.1

The Wiskott-Aldrich syndrome protein (WASP) coordinatescell-surface signaling to changes in the actin cytoskeleton and is akey organizer of migration and adhesion in hematopoietic cells.3,4

In recent years, it has become clear that WASP deficiency affectsspecific aspects of B-cell biology. Although WASP seems dispens-able for B-cell development in the BM, WASP serves a critical rolein peripheral B-cell homeostasis and lack of WASP leads to aspecific reduction of MZ precursor cells and MZ B cells.5-8WASP-deficient MZ B cells fail to respond to S1P and showaberrant integrin clustering downstream of BCR engagementduring formation of the B-cell immunologic synapse.5,8 Two recentpapers show that cell-intrinsic loss of WASP in B cells causebreakdown of B-cell tolerance in the setting of normal T-cellfunction.9,10

WASP belongs to the family of proteins that includes N-WASPand several WAVE molecules.11 WASP is expressed exclusively inleukocytes. N-WASP is the closest homolog and shares 50%homology with WASP; it is ubiquitously expressed and is criticalfor development because N-WASP deficiency is embryonicallylethal.12 Conditional deletion of N-WASP in keratinocytes hasrevealed that N-WASP deficiency leads to epidermal hyperprolifera-tion and progressive loss of hair follicle cycling.13,14 AlthoughWASP plays a key role in the function of most leukocytes, thefunctional contribution of N-WASP in these cell types is less clear.Compared with WASP deficiency alone, combined deletion ofWASP and N-WASP in T cells leads to a profound block inthymopoiesis, resulting in marked reduction of CD4 and CD8T cells in the periphery and a more pronounced defect in T-cell

Submitted September 20, 2010; accepted February 14, 2012. Prepublishedonline as Blood First Edition paper, March 12, 2012; DOI 10.1182/blood-2010-09-308197.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked advertisement in accordance with 18 USC section 1734.

2012 by The American Society of Hematology

3966 BLOOD, 26 APRIL 2012 VOLUME 119, NUMBER 17

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migration.15 N-WASP deletion alone had no apparent effect onT-cell function. The role of N-WASP in the development andfunction of other hematopoietic cells, including B cells, remainsunknown.

In the present study, we sought to explore the unique andredundant activity of WASP and N-WASP in B cells, and found thatthe combined activity of WASP and N-WASP is required forperipheral B-cell development and for the capacity of B cells totake up and respond to antigens.

MethodsAnimals

Mice were housed at Bostons Childrens Hospital and at MassachusettsGeneral Hospital under specific pathogen-free conditions. Animal experi-ments were carried out after approval and in accordance with guidelinesfrom the Subcommittee on Research Animal Care of Childrens Hospitaland Massachusetts General Hospital. Wild-type (WT), WASP-knockout(WKO), conditionally targeted N-WASPknockout (cNWKO), and WASPand N-WASP conditional double-knockout (cDKO) mice were littermatesfrom breedings of WT 129Sv mice, WKO mice on a129Sv background,conditional N-WASP KO mice on a 129Sv background, and CD19-Cremice on a C57Bl/6 background.

Proliferation, spreading, chemotaxis, and in vivo homing

The proliferative response was assessed in vitro as described previouslyusing [3H]thymidine incorporation.15,16 B cells were purified with theCellSep B-cell enrichment kit (StemCell Technologies) and cultured withAbs for IgM and CD40 (eBiosciences), lipopolysaccharide (Sigma-Aldrich), and IL-4 (PeproTech). For in vivo proliferation/expansion, micewere fed with bromodeoxyuridine (BrdU; Sigma-Aldrich) for 6 days, andBrdU cells were identified using a BrdU-labeling kit (BD Biosciences).For B-cell spreading, B cells were cultured for 48 hours in lipopolysaccha-ride and IL-4 and incubated on anti-CD44 Abcoated slides that had beenprecoated with poly-L-lysine for 8 hours. Cells were fixed and stained withAlexa Fluor 488phalloidin. Spread B cells were defined as having at leastone protrusion longer than one cell diameter in length compared withnonspread cells. In vitro migration of B cells to CXCL12 was assessed asdescribed previously.7,15 In vivo homing of B cells was performed asdescribed previously.17 Single-cell suspensions of spleen B cells wereprepared from WT, WKO, and cDKO mice. Cells were labeled with eitherCFSE or tetramethylrhodamine isothiocyanate (both from Invitrogen) andinjected IV into WT recipient mice at a 1:1 ratio. After 12-15 hours, spleen,peripheral, and mesenteric lymph nodes, Peyer patches, BM, and bloodlymph nodes were analyzed by flow cytometry. The relative frequency ofthe 2 donor-cell populations was determined for each individual organ, anda homing index was calculated as described previously.17 Similar resultswere obtained when WT, WKO, and cDKO cells were stained with thealternative labeling agent.

Flow cytometry and immunohistochemistry

For flow cytometry, single-cell suspensions were labeled with fluorescentlyconjugated antimouse Abs including B220, CD5, CD11b, CD19, CD21,CD23, CD43, CD93, IgD, and IgM (all eBiosciences) and Fas, GL7,LFA-1, and 2,4,6-trinitrophenol (TNP, all BD Biosciences). Data wereacquired on a FACSCalibur flow cytometer (BD Biosciences) and analyzedusing FlowJo Version 8.2 software for Mac (TreeStar). For immunohisto-chemistry, sections were fixed in ice-cold acetone and labeled withfluorescently conjugated antimouse Abs including MOMA, MARCO,SIGN-R1 (Serotec) CD1d, B220, and TNP (all BD Biosciences) and peanutagglutinin (Vector Laboratories). All slides were viewed at room tempera-ture with an Olympus Provis AX70 research system microscope using anUplanFl lens at 100 and Mowiol medium (Calbiochem). Images wereacquired using a U-PHOTO Universal Photo System camera (Olympus)

model U-CMAD-2 and were processed with MagnaFire 2.1c (Optronics)and Adobe Photoshop CS Version 8.0. Germinal center (GC) areas weremeasured on images of random sections using ImageJ 1.45 software forMac and were calculated as a percentage of total spleen area in a particularimage. A mean value of measurements from 3 images of each spleen wasthen determined.

Immunizations

For TI-2 antigen responses, mice were injected intravenously with TNP-Ficoll (Biosearch Technologies) and uptake of TNP-Ficoll in the MZ and byMZ B cells was examined 30 minutes and 3 hours after injection. ForT celldependent antigen response, mice were immunized by IP injection ofTNPkeyhole limpet hemocyanin (TNP-KLH) in alum. Three weeks later,mice were boosted with a second injection of TNP-KLH. GC cells and GCareas were quantified by flow cytometry and immunohistochemistry. Todetermine anti-TNP Ab titers in response to TNP-Ficoll and TNP-KLHimmunization, anti-TNP IgM, IgG3, and IgG1 were measured by ELISA.The samples were run in triplicate and corrected for background binding.

Statistics

Data are expressed as means SD where indicated. Statistical significancebetween groups was assessed by 2-tailed Student t test and ANOVA.Differences were considered significant when P .05.

ResultsSpecific deletion of WASP and N-WASP in B cells results indecreased number of peripheral B cells

WKO mice have normal B-cell development except for a markeddecrease of MZ B cells.5,7,8,16 We have demonstrated previouslythat both WASP and N-WASP are critical for T-cell development.15We hypothesized that N-WASP may also have overlapping func-tion with WASP in B-cell development and in the present studysought to address the unique and redundant function of N-WASPduring B-cell lymphopoiesis. To circumvent the embryonic lethal-ity associated with N-WASP deficiency in the mouse germline,12,18we inactivated N-WASP specifically in B cells using the Cre-loxPsystem. We bred cNWKO mice generated previously15 with WKOmice and transgenic mice expressing Cre recombinase under theB cellspecific CD19 promoter (CD19-Cre) to generate cDKOmice. The use of the CD19-Cretransgenic mouse results indeletion of loxP-flanked genes in BM-derived pre-B cells.19 PCRand Western blot analyses showed nearly complete excision ofN-WASP exon 2 and reduced N-WASP protein expression, respec-tively, in splenic B cells expressing the Cre protein (Figure 1A-C).B-cell development in the BM was similar in WT, WKO, andcDKO mice (supplemental Figure 1, available on the Blood Website; see the Supplemental Materials link at the top of the onlinearticle). Peripheral B cells were present at normal numbers in WT,cNWKO, and WKO mice, whereas the absolute B-cell number incDKO mice was reduced (Figure 2A). We next analyzed theabsolute and relative number of cellular subsets in peripheral B-celldevelopment (Figure 2B-D). WT, cNWKO, and WKO mice hadnormal numbers of transitional type 1 (T1) cells and FO B cells(Figure 2C-D). As shown previously, transitional type 2 (T2)MZPand MZ B cells were decreased in WKO mice (Figure 2C-D).5,8cDKO mice had a normal number of T1 B cells, a decreasednumber of FO and T2-MZP B cells, and a markedly decreasednumber of MZ B cells (Figure 2C-D). Whereas there was nodifference in the number of T2-MZP B cells in cNWKO mice,these mice did have a decreased number of MZ B cells (Figure 2C).To address the reason that cNWKO, WKO, and cDKO mice had

WASP AND N-WASP ARE CRITICAL FOR B LYMPHOCYTES 3967BLOOD, 26 APRIL 2012 VOLUME 119, NUMBER 17


reduced numbers of MZ B cells, we examined the expression ofLFA-1, an adhesion molecule critical for retention of MZ B cells inthe MZ. As expected, we detected increased expression of LFA-1as WT B cells progressed from T1 to MZ B cells (supplementalFigure 2A). The expression of LFA-1 was modestly decreased inT2-MZP and MZ B cells from cNWKO, WKO, and cDKO micecompared with WT mice (supplemental Figure 2B). To delineate ifthe decreased number of precursor cells may explain the reducednumber of FO B cells in cDKO mice, we examined FO precursorcells: T2 and transitional type 3 (T3) cells. Whereas WKO mice haddecreased numbers of T3 cells, cDKO mice had decreased numbersof both T2 and T3 subsets, indicating a decreased number of FOprecursor cells (supplemental Figure 3). We next examined theB-cell compartment in the peritoneum. Whereas WT, cNWKO,WKO, and cDKO mice had similar numbers and frequencies of

B1a cells, cDKO mice had a decreased number of B1b cells (Figure2F-G). Both WKO and cDKO mice had decreased numbers ofperitoneal B2 cells, which are an intermediate between splenic B2and peritoneal B1 cells (Figure 2F-G).

WASP- and N-WASPdeficient B cells alter the MZ architecture

The splenic MZ is the site where blood flows into the spleen.MZ-resident cells include MZ B cells and MZ macrophages thatexpress scavenger receptors for rapid uptake of blood-bornesubstances and apoptotic cells. Another subset of macrophages,metallophilic macrophages, delineates the border between the outerMZ and the inner B-cell follicle. WKO mice have a decreasednumber of both MZ B cells and MZ macrophages and fairly normalnumbers of metallophilic macrophages.6,8,20 To address whether thecombined activity of WASP and N-WASP in B cells is importantfor MZ architecture, we examined spleen sections of WT, cNWKO,WKO, and cDKO mice to identify MZ B cells, MZ macrophages,and metallophilic macrophages. MZ B cells (CD1d) were presentin the MZ of WT and cNWKO mice and not detectable in WKOand cDKO mice (Figure 3A). Metallophilic macrophages (MOMA)were present at normal numbers in WT, cNWKO, and WKO mice,whereas cDKO mice showed a marked reduction in metallophilicmacrophages (Figure 3A). To identify MZ macrophages, we usedstaining for the scavenger receptors SIGN-R1 and MARCO. Therim of MZ macrophages surrounding the B-cell follicle was clearlyidentified in WT and cNWKO mice, whereas WKO and cDKOmice had a decreased number of MZ macrophages (Figure 3B-D).

WASP and N-WASP regulate B-cell spreading and migration

To evaluate whether the reduced number of peripheral B cellsmight result from decreased survival or proliferation, we firstinvestigated whether WASP and N-WASP are required for receptor-mediated B-cell proliferation and found that WT, WKO, cNWKO,and cDKO B cells showed similar proliferative responses (Figure4A). To address how B-cell turnover/proliferation in vivo may beaffected by WASP and N-WASP double deficiency, we fed micewith BrdU for 6 days and analyzed BrdU cells in different B-cellcompartments. We detected no differences in expansion of T1, FO,and T2-MZP B cells comparing WT, cNWKO, WKO, and cDKOmice (supplemental Figure 4). A higher proliferation of MZ B cellswas detected in WKO and cDKO mice, suggesting increasedhomeostatic expansion of MZ B cells (supplemental Figure 4). Wenext examined cell death in cultured B cells and determined thatthe frequency of necrotic (7-amino-actinomycin D positive andannexin V positive) and apoptotic (7-amino-actinomycin D nega-tive and annexin V positive) cells was similar in B cells from allmice (supplemental Figure 5). Because WASP family membersplay a critical role in cytoskeletal reorganization and in traffickingof immune cells, the receptor-mediated cytoskeletal responses ofB cells was evaluated. To assess the spreading response, activatedB cells were incubated on surfaces coated with Abs to CD44. WTand cNWKO B cells showed a high percentage of spread cells,defined as having at least 1 long protrusion, whereas WKO B cellshad a reduced number of spread cells,21 and cDKO B cells showedan even more pronounced defect in the formation of long protru-sions (Figure 4B-C). An in vitro chemotaxis assay was used todetermine whether WASP and N-WASP double deficiency influ-enced the migration of B cells to the chemokine CXCL12, which iscritical for the homing of mature B cells into lymphoid organs.Migration to CXCL12 was reduced in both WKO and cDKOB cells compared with WT and cNWKO B cells (Figure 4D).

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Figure 1. Generation of mice devoid of WASP and N-WASP in B cells.(A) Schematic of the PCR strategy used to monitor conditional targeting of N-WASP.Displayed is a portion of the WT N-WASP allele, the conditionally targeted allele withexon 2 flanked by loxP sites (L2L), and the conditionally targeted allele afterCre-mediated excision in B cells (L). LoxP sites are denoted by black arrowheads.Labeled arrows denote primers used in the PCR strategy to simultaneously detectL2L and L using primers a, b, and c. (B) PCR analysis of N-WASP deletion in splenicB cells from WT, WKO, cNWKO, and cDKO mice. Note that the WKO mouse in thisexperiment has the N-WASP L2L allele but lacks expression of CD19-Cre for deletionof N-WASP L2L, and therefore expresses N-WASP. (C) N-WASP protein detection byWestern blotting in splenic B cells from WT, WKO, cNWKO, and cDKO mice using anAb for N-WASP (top panel). IgH was used as loading control (bottom panel). Theweak expression of N-WASP seen in cells from cNWKO and cDKO mice may reflectthe incomplete deletion of N-WASP by CD19-Cre or the contribution of otherhematopoietic cells left after B-cell purification (90%-95% B-cell purity). The asteriskdenotes a band present in cNWKO B cells that may represent WASP, because theantiN-WASP Ab shows some cross-reactivity with WASP.

3968 WESTERBERG et al BLOOD, 26 APRIL 2012 VOLUME 119, NUMBER 17


Although the analysis of cNWKO mice showed a reduction of MZB cells (Figure 2C-D), in functional assays such as migration andspreading, cNWKO B cells responded similarly to WT B cells andwe therefore did not analyze the cNWKO mice further. To evaluatehow reduced spreading and migratory capacity of cDKO B cellswould influence B-cell trafficking into lymphoid organs, weperformed an in vivo homing assay. WT and KO (WKO orcDKO) B cells were labeled with tetramethylrhodamine isothio-cyanate (red fluorescence) or CFSE (green fluorescence) andmixed at a ratio of 1:1 immediately before IV injection into aWT recipient mouse. To monitor the homing of labeled cells,

recipient mice were killed 12 hours after the adoptive B-celltransfer and the percentage of labeled cells was analyzed usingflow cytometry (Figure 5A). Compared with WT cells, bothWKO and cDKO B cells showed reduced homing into second-ary lymphoid organs including the spleen and the peripheral andmesenteric lymph nodes (Figure 5B). Homing defects weremore pronounced in cDKO B cells compared with WKO B cellsin Peyer patches (Figure 5B). To address competitive homingbetween WKO and cDKO B cells directly, a 1:1 ratio cell ratiowas injected into WT recipient mice and homing into lymphoidorgans was assessed. We found no difference in the homing

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Figure 2. Specific deletion of WASP and N-WASP inB cells results in a decreased number of peripheralB cells. (A) Total absolute number of spleen B cells inWT, WKO, cNWKO, and cDKO mice. (B) Schematicdiagram depicting B-cell development in the spleen(left) and flow cytometric analysis to define T1, FO,T2-MZP, and MZ B cells (right) in absolute number(C) and relative number (D) of cells. (E) Flow cytometryof peritoneum cells to define B1a, B1b, and B2 cells inabsolute (F) and relative (G) numbers. Each grouprepresents averages SD from 14 (WT, WKO, andcDKO) and 10 (cNWKO) mice for panels A throughD and 6 mice per group in panels E through G. *P .05compared with WT.



capacity of WKO and cDKO B cells (Figure 5C). The pro-nounced defect of homing into Peyer patches by cDKO B cellsmay result from the increase in flow rates (ie, shear stress)unique to this lymphoid compartment.

cDKO mice show a reduced immune response to TNP-Ficoll

Positioning of MZ B cells in the MZ is critical for the uptake ofblood-borne antigens and for antigen delivery to follicular dendriticcells.22 We reasoned that the pronounced decrease in MZ B cellsand defects in B-cell adhesion and migration in cDKO mice wouldreduce the uptake and delivery of blood-borne antigen to the B-cellfollicle. WT, WKO, and cDKO mice were immunized withTNP-Ficoll, a type 2 T-independent antigen, and antigen uptakewas examined on splenic sections by immunohistology. In WTmice, as has been shown previously, TNP-Ficoll was detected inthe MZ 30 minutes after injection and was localized exclusively tothe B-cell follicles 3 hours after injection (Figure 6A).22 In WKOmice, less TNP-Ficoll was detected in the MZ at 30 minutes and inthe follicle at 3 hours (Figure 6A).8 In cDKO mice, only scatteredTNP-Ficoll was detected in the MZ at 30 minutes, and TNP-Ficollwas undetectable in the follicle at 3 hours (Figure 6A). Tocomplement these studies, we next examined specific antigenuptake by MZ B cells using flow cytometry. Compared with WTMZ B cells, we found that WKO MZ B cells had reduced uptake ofTNP-Ficoll, and this uptake defect was even more pronounced in

cDKO MZ B cells (Figure 6B). To evaluate the specific Abresponse to TNP-Ficoll, we measured TNP-specific serum titers byELISA. Unchallenged WT mice had low TNP-reactive IgM Absthat increased at day 7 after immunization with TNP-Ficoll (Figure6C left panel). As shown previously, unchallenged WKO mice hadincreased serum titers of TNP-reactive IgM Abs and a decreasedspecific immune response to TNP-Ficoll at day 7 compared withWT mice (Figure 6C left panel).8 Unchallenged cDKO mice hadmarkedly elevated, TNP-reactive IgM Abs and, after immuniza-tion, there was no increased specific response to TNP-Ficoll(Figure 6C left panel). To determine whether cDKO B cells couldundergo class-switch recombination after antigenic challenge, weexamined TNP-specific IgG3 Abs after TNP-Ficoll immunization.WT mice responded to TNP-Ficoll with increased TNP-specificIgG3 Abs at day 7, whereas WKO and cDKO mice showed asignificantly reduced response (Figure 6C right panel).

WASP and N-WASP are required for the B-cell immuneresponse to TNP-KLH

To assess the responsiveness of cDKO mice to a T-dependentantigen, WT and mutant mice were immunized with TNP-KLH inalum. TNP-specific serum Ab titers were measured by ELISA atdays 7, 14, and 21 in the primary immune response and after asecond administration of TNP-KLH to assess the secondaryresponse at day 28. Unchallenged WT mice had low TNP-reactive

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Figure 3. WASP and N-WASP-deficient B cells alterthe MZ architecture. Immunohistochemistry of spleensections from WT, WKO, and cDKO mice. (A) FO B cellswere visualized with B220, MZ B cells with CD1d, andmetallophilic macrophages with MOMA-1 Ab staining. MZmacrophages were visualized with SIGNR1 (B) andMARCO (C) Ab staining. (D) Quantification of MARCOcells from spleen sections shown in panel C. Each grouprepresents quantification from 3 mice and 3 sections fromeach mouse. Note the marked reduction of FO B cells,MZ B cells, metallophilic macrophages, and MZ macro-phages in cDKO mice. Original magnification was 10.This experiment is representative of analysis of at least3 WT, WKO, and cDKO mice.



IgM Abs and showed increased TNP-specific Abs at day 7 afterimmunization with TNP-KLH (Figure 7A). WKO mice had adelayed response with increased TNP-specific IgM Abs presentfirst at day 14 (Figure 7A). In contrast, unchallenged cDKO micehad markedly elevated, TNP-reactive IgM Abs and lacked aspecific immune response to TNP-KLH at day 7-21 (Figure 7A).WT, WKO, and cDKO mice had similar specific IgM Ab titers inthe secondary response at day 28 (Figure 7A). To determinewhether cDKO B cells could undergo class-switch recombinationto a T-dependent antigen, we next examined TNP-specific IgG1Abs after TNP-KLH immunization. WT and WKO mice respondedto TNP-KLH with increased TNP-specific IgG1 at days 14 and 21,whereas cDKO mice showed a significantly reduced response(Figure 7B). At day 28, WT, WKO, and cDKO mice showedsimilar titers of TNP-specific IgG1 (Figure 7B). To determinewhether the high TNP-specific IgG1 titers in cDKO mice at day 28represented a delayed primary immune response or a normalsecondary response, we investigated whether GCs had developednormally by immunohistochemistry and flow cytometry. Well-organized GCs were frequently detected in WT and WKO spleens,

whereas cDKO mice showed reduced GC areas with looselyorganized peanut agglutininpositive GC cells (Figure 7C). Toassess the frequency of GC B cells directly, we examined B cells inthe spleen after TNP-KLH immunization at day 28. cDKO miceshowed reduced frequency of B cells compared with WT and WKOmice (Figure 7D left panel). Moreover, the frequency and absolutenumber of GC B cells (FasGL7B220DAPI) was significantlydecreased in cDKO mice (Figure 7D right panel). Considering thatcDKO mice had a decreased GC area and reduced GC B cells in thesecondary response at day 28, we reasoned that the high TNP-specific IgG1 Ab titers we detected at day 28 in cDKO mice mayreflect a delayed primary response to TNP-KLH. These resultssuggest that cDKO mice have a reduced capacity to form a specificprimary and secondary immune response with GC formation.

Discussion

FO and MZ B cells are B-cell subsets with unique functions. FOB cells have a defined life span measured in weeks and have theability to undergo affinity maturation in response to antigenicchallenge to form long-lived, Ab-secreting plasma cells andmemory cells. The newly formed MZ B cells migrate into the MZ,

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Figure 4. WASP and N-WASP are dispensable for B-cell proliferation, butregulate B-cell spreading and migration. (A) Proliferation. Splenic B cells werestimulated for 48 hours with the indicated stimulus, followed by a 16-hour pulse with[3H]thymidine to determine the proliferative response. Bars represent mean values ofcpm ([3H]thymidine) SD of triplicate wells from 1 of at least 3 independentexperiments. (B) Spreading. Spreading of activated B cells was assessed onanti-CD44 Ab-coated surfaces. White arrowhead depicts the formation of longprotrusions of spread B cells. (C) Graphs showing the average of relative numbers( SD) of spread B cells in triplicate representative of 3 experiments. (D) Migration.Splenic B or T cells were allowed to migrate to CXCL12 for 3 hours using an in vitrochemotaxis chamber. Migrating cells were collected and enumerated by flowcytometry with reference beads. The percentage of migrating cells is shown as meanvalues SD of triplicate wells and data are representative of at least 3 experiments.*P .05 compared with WT.

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Figure 5. WASP and N-WASP regulate in vivo homing of B cells. (A) SplenicB cells from WT, WKO, and cDKO mice were labeled with tetramethylrhodamineisothiocyanate (WT, red) or CFSE (WKO or cDKO, green) and mixed at a 1:1 ratiobefore IV injection to WT mice. (B) Competitive homing of WT and WKO or cDKOB cells. Spleen, peripheral and mesenteric lymph nodes, Peyer patches, BM, andblood of recipient mice were harvested after 12-15 hours, and the percentage of WKOand cDKO B-cell homing relative to WT cells was determined. (C) Competitivehoming of WKO (tetramethylrhodamine isothiocyanatelabeled) and cDKO (CFSE-labeled) B cells. Shown are averages SD of combined data from 2 experimentsincluding 4 mice per group. Dashed line represents the input percentage. *P .05compared with WKO.



where they are retained and acquire self-renewing capacity with anunlimited life span. The cell-fate decision controlling MZ B-celldifferentiation has been well characterized in mutant mousemodels. There are 4 categories of proteins that govern MZ B-celldevelopment: (1) proteins involved in BCR signaling strength,(2) proteins involved in BAFFR and NF-B signaling, (3) Notchfamily proteins, and (4) proteins regulating integrin and chemokineactivation.1 We and others have shown previously that WASPregulation of integrin and chemokine signaling is critical for MZB-cell development.5,8 The specific signaling pathways leading tothe formation of FO B cells are poorly defined. In the present study,we have addressed how cytoskeletal regulation by WASP andN-WASP regulate peripheral B-cell development. By specificallydeleting these proteins in B cells, we show that the combinedactivity of WASP and N-WASP is required for the development andfunction of both MZ and FO B cells (Figure 7E).

Our results reveal a complex regulation of B-cell developmentand function by WASP and N-WASP. cDKO mice had nosignificant change in frequency of pro-B, pre-B, or immatureB cells, suggesting that WASP and N-WASP are not required forthe generation and expansion of early B-lineage progenitors,although we did not determine N-WASP quantity in cDKOpro-B cells directly because of their limited number. Likewise,cDKO mice had a normal number of T1 B cells in the spleen,suggesting that circulating T1 cells can enter into peripheral

lymphoid organs in cDKO mice. In contrast, our results demon-strate profound abnormalities at later developmental stages. cDKOmice had diminished numbers of splenic FO B cells and, comparedwith WKO mice, the decrease in MZ precursor cells and MZB cells was exacerbated in cDKO mice. Our data highlight theimportance of cytoskeletal regulation in peripheral B-cell develop-ment, and are consistent with recent data from studies of micelacking upstream signaling molecules of WASP family members.Mice that lack the small GTPases Rac1 and Rac2 and mice lackingthe guanine exchange factors Vav1-3 undergo normal B-celldevelopment in the BM while having a marked reduction of bothFO and MZ B cells.23,24 A recent study demonstrated thatRac1/Rac2/ transitional B cells fail to exit the red pulp toenter the white pulp of the spleen.25 This defect is partlyexplained by the decreased migratory response to chemokinesrequired for entry into the white pulp cords, where the cellsreceive survival signals from BAFF and BCR signaling anddevelop into FO and MZ B cells.25,26 The phenotype of Rac1/Rac2/ B cells is strikingly similar to that of the cDKO B cellsstudied herein, including decreased migratory and spreadingresponses. However, one important difference between Rac1/Rac2/ and cDKO B cells is that the former have aberrant BCRand BAFFR signaling and therefore do not receive propersurvival signals. In contrast, those cDKO B cells that enter intothe white pulp are likely to receive BCR-dependent survivalsignals, because in the present study, cDKO B cells proliferatednormally in response to BCR stimulation and showed noevidence of increased cell death after receptor stimulation. Wepropose that cDKO B cells have a reduced capacity to developinto FO and MZ B cells because of the markedly decreasedmigratory and spreading response. One unifying implication isthat deletion of both WASP and N-WASP alters intrasplenicmigration, preventing correct homing of T1 cells to the anatomi-cal location for the development of MZ and FO B cells.

The MZ is critical for clearance of blood-borne pathogens.1 Thedevelopment of MZ B cells depends on signals from the highlyphagocytic MZ macrophages and on B cellintrinsic signalinginvolving WASP8,27,28 and N-WASP (this study). In addition toreduced MZ macrophages, the cDKO mice also had reducedmetallophilic macrophages. This differs from WKO mice, whichhad a normal number of metallophilic macrophages. This suggeststhat MZ B cells regulate the development and/or retention of thesemacrophages and that only when MZ B cells are much reduced innumbers (as in the cDKO mice) is the MZ rim of metallophilicmacrophages disrupted. The metallophilic macrophages delineatethe border between the MZ and the inner B-cell follicles and serve arole in antigen transport, at least in lymph nodes.29 It is possible thata breach in the MZ leads to altered uptake of blood-borne antigens.Artificial disruption of the MZ by deletion of the MZ andmetallophilic macrophages using diphtheria toxin leads to reducedclearance of apoptotic cells and may be associated with thedevelopment of autoantibodies.30 We addressed the possibility thatcDKO mice would have a breach in the MZ by examining theuptake of blood-borne TNP-Ficoll, and found that TNP-Ficoll wasvirtually absent from spleens of cDKO mice. cDKO mice failed tomount a specific IgM response to TNP-Ficoll because the TNP-reactive IgM serum titer in nonchallenged mice was markedlyelevated. Increased serum titers of such natural IgM have beenassociated with low-affinity and potentially autoreactive IgMresponses.31 In response to T celldependent antigenic challenge(ie, with TNP-KLH), cDKO mice had a reduced primary Ab

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Figure 6. cDKO mice show a reduced immune response to TNP-Ficoll. WT,WKO, and cDKO mice were injected IV with 2.5 g of TNP-Ficoll. (A) Uptake ofTNP-Ficoll in the spleen 30 minutes and 3 hours after injection. Spleen sections werelabeled to detect TNP-Ficoll and MOMA metallophilic macrophages to define theMZ. Note the marked reduction of TNP in the MZ at 30 minutes (top panel) and in thefollicle at 3 hours (bottom panel) in cDKO mice compared with WT mice. Originalmagnification was 10. (B) MZ B cells were labeled with anti-TNP Abs and analyzedby flow cytometry. (C) Anti-TNP IgM and IgG3 Ab titers were determined at day 7 afterimmunization by ELISA. Test samples were corrected for background binding. Eachgroup represents 6 WT, 3 WKO, and 4 cDKO mice. Black bar represents the meanvalue for the group. Non-B indicates lymphocytes negative for CD21 and IgM; andRP, red pulp. *P .05 compared with WT.



response and a diminished capacity to form a well-defined GC inthe spleen after secondary challenge. The GC is the site of B-cellaffinity maturation that relies critically on the migratory andadhesive responses of B cells.32 B-cell localization to the GC darkand light zones is regulated by the chemokines CXCL12 andCXCL13, whereas the orphan G proteincoupled receptor EBI2 iscritical for the retention of B cells in the outer follicle.33,34 Althoughbeyond the scope of this study, it is possible that cDKO B cellshave altered affinity maturation caused by decreased response tocues directing migration and adhesion within the GC reaction.

In conclusion, by studying cytoskeletal regulation in B cells, inthe present study, we have identified WASP and N-WASP as keyproteins in the transition from T1 cells to FO and MZ B cells. Theseobservations provide novel insights into the critical regulation ofthe cell cytoskeleton for peripheral B-cell development and function.

AcknowledgmentsThis work was supported by a postdoctoral fellowship from theSwedish Society for Medical Research and research grants fromthe Swedish Research Council to L.S.W. and by the NationalInstitutes of Health (grants HL-59561 to S.B.S. and L.D.N.,2P30DK034854-26 and AI-50950 to S.B.S., DK-43351 to S.B.S.and C.T., and AI-076210 to C.T. and L.D.N.).

AuthorshipContribution: L.S.W., L.D.N., and S.B.S. designed the research;L.S.W., C. Dahlberg, M.B., C.J.M., C. Detre, M.K., and M.A.E.

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Figure 7. WASP and N-WASP are required for the B-cell immuneresponse to TNP-KLH. WT, WKO, and cDKO mice were immunized by IPinjection of TNP-KLH in alum and boosted 3 weeks later with another doseof TNP-KLH. Anti-TNP IgM (A) and anti-TNP IgG1 (B) Ab titers weredetermined by ELISA. Test samples were corrected for backgroundbinding. (C) Spleen sections from immunized mice at day 28 were labeledto detect GCs (peanut agglutinin positive) and MOMA metallophilicmacrophages to define the MZ. GC areas were quantified and indicated inarbitrary units. Original magnification was 10. (D) Quantification of GCB cells (B220DAPI-GL7Fas) from immunized mice at day 28 by flowcytometry. Note the marked reduction of GC B cells in cDKO micecompared with WT mice. Each group represents 8 mice. Black barrepresents the mean value for the group. *P .05 compared with WT.(E) Schematic model of how WASP and N-WASP activity regulateperipheral B-cell homeostasis.



performed the research; F.W.A., C.T., and L.D.N. contributed newreagents or analytical tools; L.S.W., C. Dahlberg, M.B., C.J.M.,C. Detre, M.K., and S.B.S. analyzed the data; and L.S.W. andS.B.S. wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Scott B. Snapper, MD, PhD, Harvard MedicalSchool, Gastroenterology Division, Childrens Hospital Boston,300 Longwood Ave, Boston, MA 02115; e-mail: [email protected]; or Lisa S. Westerberg, PhD, KarolinskaInstitutet, Department of Medicine, Translational Immunology UnitL2:04, 171 76 Stockholm, Sweden; e-mail: [email protected].

References1. Pillai S, Cariappa A. The follicular versus mar-

ginal zone B lymphocyte cell fate decision. NatRev Immunol. 2009;9(11):767-777.

2. Victora GD, Nussenzweig MC. Germinal centers.Annu Rev Immunol. 2012;30:429-457

3. Thrasher AJ, Burns SO. WASP: a key immuno-logical multitasker. Nat Rev Immunol. 2010;10(3):182-192.

4. Bosticardo M, Marangoni F, Aiuti A, Villa A,Grazia Roncarolo M. Recent advances in under-standing the pathophysiology of Wiskott-Aldrichsyndrome. Blood. 2009;113(25):6288-6295.

5. Meyer-Bahlburg A, Becker-Herman S,Humblet-Baron S, et al. Wiskott-Aldrich syn-drome protein deficiency in B cells results inimpaired peripheral homeostasis. Blood. 2008;112(10):4158-4169.

6. Vermi W, Blanzuoli L, Kraus MD, et al. The spleenin the Wiskott-Aldrich syndrome: histopathologicabnormalities of the white pulp correlate with theclinical phenotype of the disease. Am J SurgPathol. 1999;23(2):182-191.

7. Westerberg L, Larsson M, Hardy SJ, Fernandez C,Thrasher AJ, Severinson E. Wiskott-Aldrich syn-drome protein deficiency leads to reduced B-cell ad-hesion, migration, and homing, and a delayed hu-moral immune response. Blood. 2005;105(3):1144-1152.

8. Westerberg LS, de la Fuente MA, Wermeling F,et al. WASP confers selective advantage for spe-cific hematopoietic cell populations and serves aunique role in marginal zone B-cell homeostasisand function. Blood. 2008;112(10):4139-4147.

9. Recher M, Burns SO, de la Fuente MA, et al.B cell-intrinsic deficiency of the Wiskott-Aldrichsyndrome protein causes severe abnormalities ofthe peripheral B-cell compartment in mice. Blood.2012;119(12):2819-2828.

10. Becker-Herman S, Meyer-Bahlburg A, Schwartz MA,et al. WASp-deficient B cells play a critical, cell-intrinsic role in triggering autoimmunity. J ExpMed. 2011;208(10):2033-2042.

11. Pollitt AY, Insall RH. WASP and SCAR/WAVE pro-teins: the drivers of actin assembly. J Cell Sci.2009;122(pt 15):2575-2578.

12. Snapper SB, Takeshima F, Anton I, et al.N-WASP deficiency reveals distinct pathways forcell surface projections and microbial actin-basedmotility. Nat Cell Biol. 2001;3(10):897-904.

13. Lefever T, Pedersen E, Basse A, et al. N-WASP isa novel regulator of hair-follicle cycling that con-trols antiproliferative TGF{beta} pathways. J CellSci. 2010;123(pt 1):128-140.

14. Lyubimova A, Garber JJ, Upadhyay G, et al. Neu-ral Wiskott-Aldrich syndrome protein modulatesWnt signaling and is required for hair follicle cy-cling in mice. J Clin Invest. 2010;120(2):446-456.

15. Cotta-de-Almeida V, Westerberg L, Maillard MH,et al. Wiskott Aldrich syndrome protein (WASP)and N-WASP are critical for T cell development.Proc Natl Acad Sci U S A. 2007;104(39):15424-15429.

16. Snapper SB, Rosen FS, Mizoguchi E, et al.Wiskott-Aldrich syndrome protein-deficient micereveal a role for WASP in T but not B cell activa-tion. Immunity. 1998;9(1):81-91.

17. Weninger W, Crowley MA, Manjunath N,von Andrian UH. Migratory properties of naive,effector, and memory CD8() T cells. J ExpMed. 2001;194(7):953-966.

18. Lommel S, Benesch S, Rottner K, Franz T,Wehland J, Kuhn R. Actin pedestal formation byenteropathogenic Escherichia coli and intracellu-lar motility of Shigella flexneri are abolished inN-WASP-defective cells. EMBO Rep. 2001;2(9):850-857.

19. Rickert RC, Roes J, Rajewsky K. B lymphocyte-specific, Cre-mediated mutagenesis in mice.Nucleic Acids Res. 1997;25(6):1317-1318.

20. Blundell MP, Bouma G, Calle Y, Jones GE,Kinnon C, Thrasher AJ. Improvement of migratorydefects in a murine model of Wiskott-Aldrich syn-drome gene therapy. Mol Ther. 2008;16(5):836-844.

21. Westerberg L, Greicius G, Snapper SB,Aspenstrom P, Severinson E. Cdc42, Rac1, andthe Wiskott-Aldrich syndrome protein are in-volved in the cytoskeletal regulation of B lympho-cytes. Blood. 2001;98(4):1086-1094.

22. Cinamon G, Zachariah MA, Lam OM, Foss FW Jr,Cyster JG. Follicular shuttling of marginal zoneB cells facilitates antigen transport. Nat Immunol.2008;9(1):54-62.

23. Walmsley MJ, Ooi SK, Reynolds LF, et al. Criticalroles for Rac1 and Rac2 GTPases in B cell devel-opment and signaling. Science. 2003;302(5644):459-462.

24. Fujikawa K, Miletic AV, Alt FW, et al. Vav1/2/3-nullmice define an essential role for Vav family pro-teins in lymphocyte development and activationbut a differential requirement in MAPK signalingin T and B cells. J Exp Med. 2003;198(10):1595-1608.

25. Henderson RB, Grys K, Vehlow A, et al. A novelRac-dependent checkpoint in B cell developmentcontrols entry into the splenic white pulp and cellsurvival. J Exp Med. 2010;207(4):837-853.

26. Su TT, Guo B, Wei B, Braun J, Rawlings DJ. Sig-naling in transitional type 2 B cells is critical forperipheral B-cell development. Immunol Rev.2004;197:161-178.

27. Wermeling F, Chen Y, Pikkarainen T, et al. ClassA scavenger receptors regulate tolerance againstapoptotic cells, and autoantibodies against thesereceptors are predictive of systemic lupus. J ExpMed. 2007;204(10):2259-2265.

28. Karlsson MC, Guinamard R, Bolland S, Sankala M,Steinman RM, Ravetch JV. Macrophages control theretention and trafficking of B lymphocytes in thesplenic marginal zone. J Exp Med. 2003;198(2):333-340.

29. Phan TG, Green JA, Gray EE, Xu Y, Cyster JG.Immune complex relay by subcapsular sinusmacrophages and noncognate B cells drives anti-body affinity maturation. Nat Immunol. 2009;10(7):786-793.

30. Miyake Y, Asano K, Kaise H, Uemura M,Nakayama M, Tanaka M. Critical role of macro-phages in the marginal zone in the suppression ofimmune responses to apoptotic cell-associatedantigens. J Clin Invest. 2007;117(8):2268-2278.

31. Zhang M, Carroll MC. Natural IgM-mediated in-nate autoimmunity: a new target for early inter-vention of ischemia-reperfusion injury. ExpertOpin Biol Ther. 2007;7(10):1575-1582.

32. Allen CD, Okada T, Cyster JG. Germinal-centerorganization and cellular dynamics. Immunity.2007;27(2):190-202.

33. Allen CD, Ansel KM, Low C, et al. Germinal cen-ter dark and light zone organization is mediatedby CXCR4 and CXCR5. Nat Immunol. 2004;5(9):943-952.

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online March 12, 2012 originally publisheddoi:10.1182/blood-2010-09-308197

2012 119: 3966-3974

SnapperKeszei, Michelle A. Eston, Frederick W. Alt, Cox Terhorst, Luigi D. Notarangelo and Scott B. Lisa S. Westerberg, Carin Dahlberg, Marisa Baptista, Christopher J. Moran, Cynthia Detre, Marton

peripheral B-cell development and functionWiskott-Aldrich syndrome protein (WASP) and N-WASP are critical for

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mz b cells

wko b cells

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immature b cells

naive b cells

marginal zone b cells

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