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IMMUNOBIOLOGY

Wiskott-Aldrich syndrome protein (WASP) and N-WASP are critical for

peripheral B-cell development and function
Lisa S. Westerberg,1-4 Carin Dahlberg,4 Marisa Baptista,4 Christopher J. Moran,1-3 Cynthia Detre,5 Marton Keszei,5
Michelle 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 of
Immunology, Beth Israel Deaconess Medical Center, Boston, MA; 6Department of Genetics, 7Immune Disease Institute, Howard Hughes Medical Institute, and
8Divison 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 of
hematopoietic cells. Although WASPknockout (WKO) mice have aberrant Bcell cytoskeletal responses, B-cell development is relatively normal. We
hypothesized that N-WASP, a ubiquitously expressed homolog of WASP, may
serve some redundant functions with
WASP in B cells. In the present study, we
generated mice lacking WASP and NWASP in B cells (conditional double

knockout [cDKO] B cells) and show that

cDKO mice had decreased numbers of
follicular and marginal zone B cells in the
spleen. Receptor-induced activation of
cDKO B cells led to normal proliferation
but a marked reduction of spreading compared with wild-type and WKO B cells.
Whereas WKO B cells showed decreased
migration in vitro and homing in vivo
compared with wild-type cells, cDKO
B cells showed an even more pronounced
decrease in the migratory response

in vivo. After injection of 2,4,6-trinitrophenol

(TNP)Ficoll, cDKO B cells had reduced
antigen uptake in the splenic marginal
zone. Despite high basal serum IgM, cDKO
mice mounted a reduced immune response to the T cellindependent antigen
TNP-Ficoll and to the T celldependent
antigen TNPkeyhole limpet hemocyanin.
Our results reveal that the combined activity of WASP and N-WASP is required for
peripheral B-cell development and function. (Blood. 2012;119(17):3966-3974)

Introduction
B cells are generated via sequential differentiation steps in the BM
and enter the circulation as immature, surface IgM-expressing
cells.1 Immature B cells migrate into the spleen, where they
differentiate into mature, naive B cells through highly regulated
developmental steps. Naive, mature B cells recirculate through the
bloodstream and enter into peripheral lymph nodes, peritoneal or
pleural cavities, gut-associated lymphatic tissue, and the spleen,
where they differentiate into effector cells in response to specific
antigenic challenge. In the spleen, B cells can undergo an important
cell-fate decision to become either a follicular (FO) or a marginal
zone (MZ) B cell.1 FO B cells reside inside B-cell follicles, where
they can undergo affinity maturation and class-switch recombination in response to antigenic challenge.2 MZ B cells reside in the
splenic MZ, a location that provides a first line of defense against
blood-borne pathogens. Peripheral B-cell development, activation,
and function require both migration and adhesive properties. FO
B cells depend on signaling by the chemokine receptor CXCR5 to
localize to the follicles, whereas MZ B cells are sensitive to
sphingosine-1-phosphate (S1P), which is highly concentrated in
blood.1 Adhesion by MZ B cells to ICAM-1 and 41 integrin is
critical for MZ B-cell retention in the MZ, an area that is exposed to
the sheer stress of blood flow.1
The Wiskott-Aldrich syndrome protein (WASP) coordinates
cell-surface signaling to changes in the actin cytoskeleton and is a
key organizer of migration and adhesion in hematopoietic cells.3,4

In recent years, it has become clear that WASP deficiency affects

specific aspects of B-cell biology. Although WASP seems dispensable for B-cell development in the BM, WASP serves a critical role
in peripheral B-cell homeostasis and lack of WASP leads to a
specific reduction of MZ precursor cells and MZ B cells.5-8
WASP-deficient MZ B cells fail to respond to S1P and show
aberrant integrin clustering downstream of BCR engagement
during formation of the B-cell immunologic synapse.5,8 Two recent
papers show that cell-intrinsic loss of WASP in B cells cause
breakdown of B-cell tolerance in the setting of normal T-cell
function.9,10
WASP belongs to the family of proteins that includes N-WASP
and several WAVE molecules.11 WASP is expressed exclusively in
leukocytes. N-WASP is the closest homolog and shares 50%
homology with WASP; it is ubiquitously expressed and is critical
for development because N-WASP deficiency is embryonically
lethal.12 Conditional deletion of N-WASP in keratinocytes has
revealed that N-WASP deficiency leads to epidermal hyperproliferation and progressive loss of hair follicle cycling.13,14 Although
WASP plays a key role in the function of most leukocytes, the
functional contribution of N-WASP in these cell types is less clear.
Compared with WASP deficiency alone, combined deletion of
WASP and N-WASP in T cells leads to a profound block in
thymopoiesis, resulting in marked reduction of CD4 and CD8
T cells in the periphery and a more pronounced defect in T-cell

Submitted September 20, 2010; accepted February 14, 2012. Prepublished

online as Blood First Edition paper, March 12, 2012; DOI 10.1182/blood-201009-308197.

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

The online version of this article contains a data supplement.

2012 by The American Society of Hematology

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BLOOD, 26 APRIL 2012 VOLUME 119, NUMBER 17

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BLOOD, 26 APRIL 2012 VOLUME 119, NUMBER 17

migration.15 N-WASP deletion alone had no apparent effect on

T-cell function. The role of N-WASP in the development and
function of other hematopoietic cells, including B cells, remains
unknown.
In the present study, we sought to explore the unique and
redundant activity of WASP and N-WASP in B cells, and found that
the combined activity of WASP and N-WASP is required for
peripheral B-cell development and for the capacity of B cells to
take up and respond to antigens.

Methods
Animals
Mice were housed at Bostons Childrens Hospital and at Massachusetts
General Hospital under specific pathogen-free conditions. Animal experiments were carried out after approval and in accordance with guidelines
from the Subcommittee on Research Animal Care of Childrens Hospital
and Massachusetts General Hospital. Wild-type (WT), WASP-knockout
(WKO), conditionally targeted N-WASPknockout (cNWKO), and WASP
and N-WASP conditional double-knockout (cDKO) mice were littermates
from breedings of WT 129Sv mice, WKO mice on a129Sv background,
conditional N-WASP KO mice on a 129Sv background, and CD19-Cre
mice on a C57Bl/6 background.
Proliferation, spreading, chemotaxis, and in vivo homing
The proliferative response was assessed in vitro as described previously
using [3H]thymidine incorporation.15,16 B cells were purified with the
CellSep B-cell enrichment kit (StemCell Technologies) and cultured with
Abs for IgM and CD40 (eBiosciences), lipopolysaccharide (SigmaAldrich), and IL-4 (PeproTech). For in vivo proliferation/expansion, mice
were fed with bromodeoxyuridine (BrdU; Sigma-Aldrich) for 6 days, and
BrdU cells were identified using a BrdU-labeling kit (BD Biosciences).
For B-cell spreading, B cells were cultured for 48 hours in lipopolysaccharide and IL-4 and incubated on anti-CD44 Abcoated slides that had been
precoated with poly-L-lysine for 8 hours. Cells were fixed and stained with
Alexa Fluor 488phalloidin. Spread B cells were defined as having at least
one protrusion longer than one cell diameter in length compared with
nonspread cells. In vitro migration of B cells to CXCL12 was assessed as
described previously.7,15 In vivo homing of B cells was performed as
described previously.17 Single-cell suspensions of spleen B cells were
prepared from WT, WKO, and cDKO mice. Cells were labeled with either
CFSE or tetramethylrhodamine isothiocyanate (both from Invitrogen) and
injected IV into WT recipient mice at a 1:1 ratio. After 12-15 hours, spleen,
peripheral, and mesenteric lymph nodes, Peyer patches, BM, and blood
lymph nodes were analyzed by flow cytometry. The relative frequency of
the 2 donor-cell populations was determined for each individual organ, and
a homing index was calculated as described previously.17 Similar results
were obtained when WT, WKO, and cDKO cells were stained with the
alternative labeling agent.
Flow cytometry and immunohistochemistry
For flow cytometry, single-cell suspensions were labeled with fluorescently
conjugated 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 were
acquired on a FACSCalibur flow cytometer (BD Biosciences) and analyzed
using FlowJo Version 8.2 software for Mac (TreeStar). For immunohistochemistry, sections were fixed in ice-cold acetone and labeled with
fluorescently conjugated antimouse Abs including MOMA, MARCO,
SIGN-R1 (Serotec) CD1d, B220, and TNP (all BD Biosciences) and peanut
agglutinin (Vector Laboratories). All slides were viewed at room temperature with an Olympus Provis AX70 research system microscope using an
UplanFl lens at 100 and Mowiol medium (Calbiochem). Images were
acquired using a U-PHOTO Universal Photo System camera (Olympus)

WASP AND N-WASP ARE CRITICAL FOR B LYMPHOCYTES

3967

model U-CMAD-2 and were processed with MagnaFire 2.1c (Optronics)

and Adobe Photoshop CS Version 8.0. Germinal center (GC) areas were
measured on images of random sections using ImageJ 1.45 software for
Mac and were calculated as a percentage of total spleen area in a particular
image. A mean value of measurements from 3 images of each spleen was
then determined.
Immunizations
For TI-2 antigen responses, mice were injected intravenously with TNPFicoll (Biosearch Technologies) and uptake of TNP-Ficoll in the MZ and by
MZ B cells was examined 30 minutes and 3 hours after injection. For
T celldependent antigen response, mice were immunized by IP injection of
TNPkeyhole limpet hemocyanin (TNP-KLH) in alum. Three weeks later,
mice were boosted with a second injection of TNP-KLH. GC cells and GC
areas were quantified by flow cytometry and immunohistochemistry. To
determine anti-TNP Ab titers in response to TNP-Ficoll and TNP-KLH
immunization, 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 significance
between groups was assessed by 2-tailed Student t test and ANOVA.
Differences were considered significant when P .05.

Results
Specific deletion of WASP and N-WASP in B cells results in
decreased number of peripheral B cells

WKO mice have normal B-cell development except for a marked

decrease of MZ B cells.5,7,8,16 We have demonstrated previously
that both WASP and N-WASP are critical for T-cell development.15
We hypothesized that N-WASP may also have overlapping function with WASP in B-cell development and in the present study
sought to address the unique and redundant function of N-WASP
during B-cell lymphopoiesis. To circumvent the embryonic lethality associated with N-WASP deficiency in the mouse germline,12,18
we inactivated N-WASP specifically in B cells using the Cre-loxP
system. We bred cNWKO mice generated previously15 with WKO
mice and transgenic mice expressing Cre recombinase under the
B cellspecific CD19 promoter (CD19-Cre) to generate cDKO
mice. The use of the CD19-Cretransgenic mouse results in
deletion of loxP-flanked genes in BM-derived pre-B cells.19 PCR
and Western blot analyses showed nearly complete excision of
N-WASP exon 2 and reduced N-WASP protein expression, respectively, in splenic B cells expressing the Cre protein (Figure 1A-C).
B-cell development in the BM was similar in WT, WKO, and
cDKO mice (supplemental Figure 1, available on the Blood Web
site; see the Supplemental Materials link at the top of the online
article). Peripheral B cells were present at normal numbers in WT,
cNWKO, and WKO mice, whereas the absolute B-cell number in
cDKO mice was reduced (Figure 2A). We next analyzed the
absolute and relative number of cellular subsets in peripheral B-cell
development (Figure 2B-D). WT, cNWKO, and WKO mice had
normal numbers of transitional type 1 (T1) cells and FO B cells
(Figure 2C-D). As shown previously, transitional type 2 (T2)MZP
and MZ B cells were decreased in WKO mice (Figure 2C-D).5,8
cDKO mice had a normal number of T1 B cells, a decreased
number of FO and T2-MZP B cells, and a markedly decreased
number of MZ B cells (Figure 2C-D). Whereas there was no
difference 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

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3968

BLOOD, 26 APRIL 2012 VOLUME 119, NUMBER 17

WESTERBERG et al

N-WASP PCR strategy

exon3

exon2

exon1

loxP-exon2-loxP (L2L)

Cre

exon1

loxP (L)

exon3
c

N-WASP PCR
L2L
L
primers: a b c
N-WASP

WT

L2L

WASP

CD19-Cre

+
+

WT

WKO

cNWKO

cDKO

WT

N-WASP Western
N-WASP

*
IgH

WT

WKO

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

wildtype N-WASP (WT)

exon3

exon2

exon1

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

cNWKO

cDKO

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 with
exon 2 flanked by loxP sites (L2L), and the conditionally targeted allele after
Cre-mediated excision in B cells (L). LoxP sites are denoted by black arrowheads.
Labeled arrows denote primers used in the PCR strategy to simultaneously detect
L2L and L using primers a, b, and c. (B) PCR analysis of N-WASP deletion in splenic
B cells from WT, WKO, cNWKO, and cDKO mice. Note that the WKO mouse in this
experiment has the N-WASP L2L allele but lacks expression of CD19-Cre for deletion
of N-WASP L2L, and therefore expresses N-WASP. (C) N-WASP protein detection by
Western blotting in splenic B cells from WT, WKO, cNWKO, and cDKO mice using an
Ab for N-WASP (top panel). IgH was used as loading control (bottom panel). The
weak expression of N-WASP seen in cells from cNWKO and cDKO mice may reflect
the incomplete deletion of N-WASP by CD19-Cre or the contribution of other
hematopoietic cells left after B-cell purification (90%-95% B-cell purity). The asterisk
denotes a band present in cNWKO B cells that may represent WASP, because the
antiN-WASP Ab shows some cross-reactivity with WASP.

reduced numbers of MZ B cells, we examined the expression of

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

The splenic MZ is the site where blood flows into the spleen.
MZ-resident cells include MZ B cells and MZ macrophages that
express scavenger receptors for rapid uptake of blood-borne
substances and apoptotic cells. Another subset of macrophages,
metallophilic macrophages, delineates the border between the outer
MZ and the inner B-cell follicle. WKO mice have a decreased
number of both MZ B cells and MZ macrophages and fairly normal
numbers of metallophilic macrophages.6,8,20 To address whether the
combined activity of WASP and N-WASP in B cells is important
for 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 present
in the MZ of WT and cNWKO mice and not detectable in WKO
and 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 metallophilic
macrophages (Figure 3A). To identify MZ macrophages, we used
staining for the scavenger receptors SIGN-R1 and MARCO. The
rim of MZ macrophages surrounding the B-cell follicle was clearly
identified in WT and cNWKO mice, whereas WKO and cDKO
mice 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 cells

might result from decreased survival or proliferation, we first
investigated whether WASP and N-WASP are required for receptormediated B-cell proliferation and found that WT, WKO, cNWKO,
and cDKO B cells showed similar proliferative responses (Figure
4A). To address how B-cell turnover/proliferation in vivo may be
affected by WASP and N-WASP double deficiency, we fed mice
with BrdU for 6 days and analyzed BrdU cells in different B-cell
compartments. We detected no differences in expansion of T1, FO,
and T2-MZP B cells comparing WT, cNWKO, WKO, and cDKO
mice (supplemental Figure 4). A higher proliferation of MZ B cells
was detected in WKO and cDKO mice, suggesting increased
homeostatic expansion of MZ B cells (supplemental Figure 4). We
next examined cell death in cultured B cells and determined that
the frequency of necrotic (7-amino-actinomycin D positive and
annexin V positive) and apoptotic (7-amino-actinomycin D negative and annexin V positive) cells was similar in B cells from all
mice (supplemental Figure 5). Because WASP family members
play a critical role in cytoskeletal reorganization and in trafficking
of immune cells, the receptor-mediated cytoskeletal responses of
B cells was evaluated. To assess the spreading response, activated
B cells were incubated on surfaces coated with Abs to CD44. WT
and cNWKO B cells showed a high percentage of spread cells,
defined as having at least 1 long protrusion, whereas WKO B cells
had a reduced number of spread cells,21 and cDKO B cells showed
an even more pronounced defect in the formation of long protrusions (Figure 4B-C). An in vitro chemotaxis assay was used to
determine whether WASP and N-WASP double deficiency influenced the migration of B cells to the chemokine CXCL12, which is
critical for the homing of mature B cells into lymphoid organs.
Migration to CXCL12 was reduced in both WKO and cDKO
B cells compared with WT and cNWKO B cells (Figure 4D).

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BLOOD, 26 APRIL 2012 VOLUME 119, NUMBER 17

B cells

20
Cells (10^6)

FO

Marginal
T2zone
T2
MZP

10

T1

MZ

FO

MZ
CD23

Follicle

T1

FSC

T O O O
W WK WK DK
c
cN

CD21
IgM

T1

20

FO

10
1

100

20

90

15

80

10

70

60

Cells (10^6)

B cells

CD3

Cells (%)

0.2

T2-MZP

* *

1.0

0.4

0.5

0.2

*
* *
T O O O
W WK WK DK
c
cN

15

T O O O
W WK WK DK
c
cN

B1b

MZ

10
5

2
0

MZ

0.0

0.6

1.0

* *
T O O O
W WK WK DK
c
cN

B2

0.8

80

0.6

B1a

30
20

B1b

* *
T O O O
W WK WK DK
c
cN

60

B2

40
20

10

O O O
T
W WK WK DK
c
N
c

0.2

O O O
T
W WK WK DK
c
N
c

40
20

0.4
0

O O O
T
W WK WK DK
c
N
c

60

Although the analysis of cNWKO mice showed a reduction of MZ

B cells (Figure 2C-D), in functional assays such as migration and
spreading, cNWKO B cells responded similarly to WT B cells and
we therefore did not analyze the cNWKO mice further. To evaluate
how reduced spreading and migratory capacity of cDKO B cells
would influence B-cell trafficking into lymphoid organs, we
performed an in vivo homing assay. WT and KO (WKO or
cDKO) B cells were labeled with tetramethylrhodamine isothiocyanate (red fluorescence) or CFSE (green fluorescence) and
mixed at a ratio of 1:1 immediately before IV injection into a
WT recipient mouse. To monitor the homing of labeled cells,

* *

0.4

T O O O
W WK WK DK
c
cN

B1a

CD11b

0.2

1.5

B1a
B2 B1b

CD5

0.6

0.4

O O O
T
W WK WK DK
c
cN

CD19

0.8

0.6

FO

50

O O O
T
W WK WK DK
c
cN

1.0

0.8

O O O
T
W WK WK DK
c
cN

T1

25

T2-MZP

0.0

O O O
T
W WK WK DK
c
cN

Cells (%)

1.0

15

3969

T2-MZP

15

Cells (10^6)

Figure 2. Specific deletion of WASP and N-WASP in

B cells results in a decreased number of peripheral
B cells. (A) Total absolute number of spleen B cells in
WT, WKO, cNWKO, and cDKO mice. (B) Schematic
diagram 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 cytometry
of peritoneum cells to define B1a, B1b, and B2 cells in
absolute (F) and relative (G) numbers. Each group
represents averages SD from 14 (WT, WKO, and
cDKO) and 10 (cNWKO) mice for panels A through
D and 6 mice per group in panels E through G. *P .05
compared with WT.

WASP AND N-WASP ARE CRITICAL FOR B LYMPHOCYTES

* *

O O O
T
W WK WK DK
c
N
c

T O O O
W WK WK DK
c
cN

recipient mice were killed 12 hours after the adoptive B-cell

transfer and the percentage of labeled cells was analyzed using
flow cytometry (Figure 5A). Compared with WT cells, both
WKO and cDKO B cells showed reduced homing into secondary lymphoid organs including the spleen and the peripheral and
mesenteric lymph nodes (Figure 5B). Homing defects were
more pronounced in cDKO B cells compared with WKO B cells
in Peyer patches (Figure 5B). To address competitive homing
between WKO and cDKO B cells directly, a 1:1 ratio cell ratio
was injected into WT recipient mice and homing into lymphoid
organs was assessed. We found no difference in the homing

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BLOOD, 26 APRIL 2012 VOLUME 119, NUMBER 17

WESTERBERG et al

WT

cNWKO

WKO

B220 MOMA CD1d

B220 SIGNR1

cDKO

Figure 3. WASP and N-WASP-deficient B cells alter

the MZ architecture. Immunohistochemistry of spleen
sections from WT, WKO, and cDKO mice. (A) FO B cells
were visualized with B220, MZ B cells with CD1d, and
metallophilic macrophages with MOMA-1 Ab staining. MZ
macrophages were visualized with SIGNR1 (B) and
MARCO (C) Ab staining. (D) Quantification of MARCO
cells from spleen sections shown in panel C. Each group
represents quantification from 3 mice and 3 sections from
each mouse. Note the marked reduction of FO B cells,
MZ B cells, metallophilic macrophages, and MZ macrophages in cDKO mice. Original magnification was 10.
This experiment is representative of analysis of at least
3 WT, WKO, and cDKO mice.

B220 MARCO

D
Cells (%)

400

MARCO+ cells

300

* *

200
100
0
T
W

KO

cN

KO DKO
W
c

capacity of WKO and cDKO B cells (Figure 5C). The pronounced defect of homing into Peyer patches by cDKO B cells
may 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 of

blood-borne antigens and for antigen delivery to follicular dendritic
cells.22 We reasoned that the pronounced decrease in MZ B cells
and defects in B-cell adhesion and migration in cDKO mice would
reduce the uptake and delivery of blood-borne antigen to the B-cell
follicle. WT, WKO, and cDKO mice were immunized with
TNP-Ficoll, a type 2 T-independent antigen, and antigen uptake
was examined on splenic sections by immunohistology. In WT
mice, as has been shown previously, TNP-Ficoll was detected in
the MZ 30 minutes after injection and was localized exclusively to
the B-cell follicles 3 hours after injection (Figure 6A).22 In WKO
mice, less TNP-Ficoll was detected in the MZ at 30 minutes and in
the follicle at 3 hours (Figure 6A).8 In cDKO mice, only scattered
TNP-Ficoll was detected in the MZ at 30 minutes, and TNP-Ficoll
was undetectable in the follicle at 3 hours (Figure 6A). To
complement these studies, we next examined specific antigen
uptake by MZ B cells using flow cytometry. Compared with WT
MZ B cells, we found that WKO MZ B cells had reduced uptake of
TNP-Ficoll, and this uptake defect was even more pronounced in

cDKO MZ B cells (Figure 6B). To evaluate the specific Ab

response to TNP-Ficoll, we measured TNP-specific serum titers by
ELISA. Unchallenged WT mice had low TNP-reactive IgM Abs
that increased at day 7 after immunization with TNP-Ficoll (Figure
6C left panel). As shown previously, unchallenged WKO mice had
increased serum titers of TNP-reactive IgM Abs and a decreased
specific immune response to TNP-Ficoll at day 7 compared with
WT mice (Figure 6C left panel).8 Unchallenged cDKO mice had
markedly elevated, TNP-reactive IgM Abs and, after immunization, there was no increased specific response to TNP-Ficoll
(Figure 6C left panel). To determine whether cDKO B cells could
undergo class-switch recombination after antigenic challenge, we
examined TNP-specific IgG3 Abs after TNP-Ficoll immunization.
WT mice responded to TNP-Ficoll with increased TNP-specific
IgG3 Abs at day 7, whereas WKO and cDKO mice showed a
significantly reduced response (Figure 6C right panel).
WASP and N-WASP are required for the B-cell immune
response to TNP-KLH

To assess the responsiveness of cDKO mice to a T-dependent

antigen, WT and mutant mice were immunized with TNP-KLH in
alum. TNP-specific serum Ab titers were measured by ELISA at
days 7, 14, and 21 in the primary immune response and after a
second administration of TNP-KLH to assess the secondary
response at day 28. Unchallenged WT mice had low TNP-reactive

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3H-Thymidine (cpm 10^3)

BLOOD, 26 APRIL 2012 VOLUME 119, NUMBER 17

WASP AND N-WASP ARE CRITICAL FOR B LYMPHOCYTES

whereas cDKO mice showed reduced GC areas with loosely

organized peanut agglutininpositive GC cells (Figure 7C). To
assess the frequency of GC B cells directly, we examined B cells in
the spleen after TNP-KLH immunization at day 28. cDKO mice
showed reduced frequency of B cells compared with WT and WKO
mice (Figure 7D left panel). Moreover, the frequency and absolute
number of GC B cells (FasGL7B220DAPI) was significantly
decreased in cDKO mice (Figure 7D right panel). Considering that
cDKO mice had a decreased GC area and reduced GC B cells in the
secondary response at day 28, we reasoned that the high TNPspecific IgG1 Ab titers we detected at day 28 in cDKO mice may
reflect a delayed primary response to TNP-KLH. These results
suggest that cDKO mice have a reduced capacity to form a specific
primary and secondary immune response with GC formation.

Proliferation

180

WT
cNWKO
WKO
cDKO

140
100
60
20
0

IgM IgM+IL4 LPS CD40+IL4

unstim

WT

cNWKO

WKO

cDKO

3971

Discussion

50
40
30
20

10
0
CD44

*
-

Migration
30

WT
cNWKO
WKO
cDKO

25
20
15
10

5
0
CXCL12

*
-

Figure 4. WASP and N-WASP are dispensable for B-cell proliferation, but
regulate B-cell spreading and migration. (A) Proliferation. Splenic B cells were
stimulated 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 of
cpm ([3H]thymidine) SD of triplicate wells from 1 of at least 3 independent
experiments. (B) Spreading. Spreading of activated B cells was assessed on
anti-CD44 Ab-coated surfaces. White arrowhead depicts the formation of long
protrusions 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 vitro
chemotaxis chamber. Migrating cells were collected and enumerated by flow
cytometry with reference beads. The percentage of migrating cells is shown as mean
values SD of triplicate wells and data are representative of at least 3 experiments.
*P .05 compared with WT.

IgM Abs and showed increased TNP-specific Abs at day 7 after

immunization with TNP-KLH (Figure 7A). WKO mice had a
delayed response with increased TNP-specific IgM Abs present
first at day 14 (Figure 7A). In contrast, unchallenged cDKO mice
had markedly elevated, TNP-reactive IgM Abs and lacked a
specific immune response to TNP-KLH at day 7-21 (Figure 7A).
WT, WKO, and cDKO mice had similar specific IgM Ab titers in
the secondary response at day 28 (Figure 7A). To determine
whether cDKO B cells could undergo class-switch recombination
to a T-dependent antigen, we next examined TNP-specific IgG1
Abs after TNP-KLH immunization. WT and WKO mice responded
to 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 showed
similar titers of TNP-specific IgG1 (Figure 7B). To determine
whether the high TNP-specific IgG1 titers in cDKO mice at day 28
represented a delayed primary immune response or a normal
secondary response, we investigated whether GCs had developed
normally by immunohistochemistry and flow cytometry. Wellorganized GCs were frequently detected in WT and WKO spleens,

B220 positive

red
WT
green
KO

B220

Homing WT vs WKO or cDKO B cells

WKO

cDKO

WT

1
Migration Index

WT
cNWKO
WKO
cDKO

0,8
0,6
0,4

0,2
0

SPL

PLN

PP

MLN

BM

BLO

Homing WKO vs cDKO B cells

1,8
Migration Index

% Spread cells with

long protrusion

Spreading

60

% Migrating cells

FO and MZ B cells are B-cell subsets with unique functions. FO

B cells have a defined life span measured in weeks and have the
ability to undergo affinity maturation in response to antigenic
challenge to form long-lived, Ab-secreting plasma cells and
memory cells. The newly formed MZ B cells migrate into the MZ,

cDKO

1,4
WKO

1
0,6
0,2
0

SPL

PLN

PP

MLN

BM

BLO

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

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3972

BLOOD, 26 APRIL 2012 VOLUME 119, NUMBER 17

WESTERBERG et al

WT

WKO

cDKO

RP

30 min

FO
FO
FO

RP
RP

MOMA TNP-Ficoll
RP

RP

3 hrs
RP

FO

FO

FO

MOMA TNP-Ficoll
MZ B cells: 30 min
nonB
WT

MZ B cells: 3 hrs
TNP-Ficoll (MFI)

WKO
cDKO

180
100
60
20
0

TNP-Ficoll

OD (405 nm)

1.5

TNP-Ficoll: IgM

1.0

0.5
0

1.5
OD (405 nm)

MZ B cells
WT
WKO
cDKO

140

30 min

3 hrs

TNP-Ficoll: IgG3
WT
WKO
cDKO

1.0
0.5

* *

0
pi

d7
TNP-Ficoll

pi

d7
TNP-Ficoll

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 of
TNP-Ficoll in the spleen 30 minutes and 3 hours after injection. Spleen sections were
labeled to detect TNP-Ficoll and MOMA metallophilic macrophages to define the
MZ. Note the marked reduction of TNP in the MZ at 30 minutes (top panel) and in the
follicle at 3 hours (bottom panel) in cDKO mice compared with WT mice. Original
magnification was 10. (B) MZ B cells were labeled with anti-TNP Abs and analyzed
by flow cytometry. (C) Anti-TNP IgM and IgG3 Ab titers were determined at day 7 after
immunization by ELISA. Test samples were corrected for background binding. Each
group represents 6 WT, 3 WKO, and 4 cDKO mice. Black bar represents the mean
value for the group. Non-B indicates lymphocytes negative for CD21 and IgM; and
RP, red pulp. *P .05 compared with WT.

where they are retained and acquire self-renewing capacity with an

unlimited life span. The cell-fate decision controlling MZ B-cell
differentiation has been well characterized in mutant mouse
models. There are 4 categories of proteins that govern MZ B-cell
development: (1) proteins involved in BCR signaling strength,
(2) proteins involved in BAFFR and NF-B signaling, (3) Notch
family proteins, and (4) proteins regulating integrin and chemokine
activation.1 We and others have shown previously that WASP
regulation of integrin and chemokine signaling is critical for MZ
B-cell development.5,8 The specific signaling pathways leading to
the formation of FO B cells are poorly defined. In the present study,
we have addressed how cytoskeletal regulation by WASP and
N-WASP regulate peripheral B-cell development. By specifically
deleting these proteins in B cells, we show that the combined
activity of WASP and N-WASP is required for the development and
function of both MZ and FO B cells (Figure 7E).
Our results reveal a complex regulation of B-cell development
and function by WASP and N-WASP. cDKO mice had no
significant change in frequency of pro-B, pre-B, or immature
B cells, suggesting that WASP and N-WASP are not required for
the generation and expansion of early B-lineage progenitors,
although we did not determine N-WASP quantity in cDKO
pro-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 demonstrate profound abnormalities at later developmental stages. cDKO
mice had diminished numbers of splenic FO B cells and, compared
with WKO mice, the decrease in MZ precursor cells and MZ
B cells was exacerbated in cDKO mice. Our data highlight the
importance of cytoskeletal regulation in peripheral B-cell development, and are consistent with recent data from studies of mice
lacking upstream signaling molecules of WASP family members.
Mice that lack the small GTPases Rac1 and Rac2 and mice lacking
the guanine exchange factors Vav1-3 undergo normal B-cell
development in the BM while having a marked reduction of both
FO and MZ B cells.23,24 A recent study demonstrated that
Rac1/Rac2/ transitional B cells fail to exit the red pulp to
enter the white pulp of the spleen.25 This defect is partly
explained by the decreased migratory response to chemokines
required for entry into the white pulp cords, where the cells
receive survival signals from BAFF and BCR signaling and
develop into FO and MZ B cells.25,26 The phenotype of Rac1/
Rac2/ B cells is strikingly similar to that of the cDKO B cells
studied herein, including decreased migratory and spreading
responses. However, one important difference between Rac1/
Rac2/ and cDKO B cells is that the former have aberrant BCR
and BAFFR signaling and therefore do not receive proper
survival signals. In contrast, those cDKO B cells that enter into
the white pulp are likely to receive BCR-dependent survival
signals, because in the present study, cDKO B cells proliferated
normally in response to BCR stimulation and showed no
evidence of increased cell death after receptor stimulation. We
propose that cDKO B cells have a reduced capacity to develop
into FO and MZ B cells because of the markedly decreased
migratory and spreading response. One unifying implication is
that deletion of both WASP and N-WASP alters intrasplenic
migration, preventing correct homing of T1 cells to the anatomical location for the development of MZ and FO B cells.
The MZ is critical for clearance of blood-borne pathogens.1 The
development of MZ B cells depends on signals from the highly
phagocytic MZ macrophages and on B cellintrinsic signaling
involving WASP8,27,28 and N-WASP (this study). In addition to
reduced MZ macrophages, the cDKO mice also had reduced
metallophilic macrophages. This differs from WKO mice, which
had a normal number of metallophilic macrophages. This suggests
that MZ B cells regulate the development and/or retention of these
macrophages and that only when MZ B cells are much reduced in
numbers (as in the cDKO mice) is the MZ rim of metallophilic
macrophages disrupted. The metallophilic macrophages delineate
the border between the MZ and the inner B-cell follicles and serve a
role in antigen transport, at least in lymph nodes.29 It is possible that
a breach in the MZ leads to altered uptake of blood-borne antigens.
Artificial disruption of the MZ by deletion of the MZ and
metallophilic macrophages using diphtheria toxin leads to reduced
clearance of apoptotic cells and may be associated with the
development of autoantibodies.30 We addressed the possibility that
cDKO mice would have a breach in the MZ by examining the
uptake of blood-borne TNP-Ficoll, and found that TNP-Ficoll was
virtually absent from spleens of cDKO mice. cDKO mice failed to
mount a specific IgM response to TNP-Ficoll because the TNPreactive IgM serum titer in nonchallenged mice was markedly
elevated. Increased serum titers of such natural IgM have been
associated with low-affinity and potentially autoreactive IgM
responses.31 In response to T celldependent antigenic challenge
(ie, with TNP-KLH), cDKO mice had a reduced primary Ab

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BLOOD, 26 APRIL 2012 VOLUME 119, NUMBER 17

A
OD (405 nm)

3973

TNP-KLH: IgM

0,8

WT
WKO
cDKO

0,6
0,4

0,2
0
pi

d 14

d7

d 21

d 28

1st

2nd

TNP-KLH: IgG1

1,6
OD (405 nm)

Figure 7. WASP and N-WASP are required for the B-cell immune
response to TNP-KLH. WT, WKO, and cDKO mice were immunized by IP
injection of TNP-KLH in alum and boosted 3 weeks later with another dose
of TNP-KLH. Anti-TNP IgM (A) and anti-TNP IgG1 (B) Ab titers were
determined by ELISA. Test samples were corrected for background
binding. (C) Spleen sections from immunized mice at day 28 were labeled
to detect GCs (peanut agglutinin positive) and MOMA metallophilic
macrophages to define the MZ. GC areas were quantified and indicated in
arbitrary units. Original magnification was 10. (D) Quantification of GC
B cells (B220DAPI-GL7Fas) from immunized mice at day 28 by flow
cytometry. Note the marked reduction of GC B cells in cDKO mice
compared with WT mice. Each group represents 8 mice. Black bar
represents the mean value for the group. *P .05 compared with WT.
(E) Schematic model of how WASP and N-WASP activity regulate
peripheral B-cell homeostasis.

WASP AND N-WASP ARE CRITICAL FOR B LYMPHOCYTES

WT
WKO
cDKO

1,2

0,8

0,4
0
pi

d 14

d7

d 21

1st

d 28
2nd

WKO

cDKO

PNA MOMA

B220+DAPI-

B220+DAPI-

GC
GC

6
4
Fas

2
0
40

Cells (%)

120

WT
WKO
cDKO

80
40

GC: Fas+GL7+
0,5

GL7

0,4
0,3
0,2

WT
WKO
cDKO

0,1
0,0

B220+DAPI-

GC: Fas+GL7+

30

Cells (%)

Cells (10^6)

10

160

Cells (10^6)

GC area
Arbitrary Units

WT

20
10

3
2
1

Marginal
zone
Follicle

T1

T2 MZP
FOB

MZB

WASP
N-WASP

response and a diminished capacity to form a well-defined GC in

the spleen after secondary challenge. The GC is the site of B-cell
affinity maturation that relies critically on the migratory and
adhesive responses of B cells.32 B-cell localization to the GC dark
and light zones is regulated by the chemokines CXCL12 and
CXCL13, whereas the orphan G proteincoupled receptor EBI2 is
critical for the retention of B cells in the outer follicle.33,34 Although
beyond the scope of this study, it is possible that cDKO B cells
have altered affinity maturation caused by decreased response to
cues directing migration and adhesion within the GC reaction.
In conclusion, by studying cytoskeletal regulation in B cells, in
the present study, we have identified WASP and N-WASP as key
proteins in the transition from T1 cells to FO and MZ B cells. These
observations provide novel insights into the critical regulation of
the cell cytoskeleton for peripheral B-cell development and function.

Acknowledgments
This work was supported by a postdoctoral fellowship from the
Swedish Society for Medical Research and research grants from
the Swedish Research Council to L.S.W. and by the National
Institutes 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.).

Authorship
Contribution: 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.

From www.bloodjournal.org by guest on March 28, 2015. For personal use only.
3974

BLOOD, 26 APRIL 2012 VOLUME 119, NUMBER 17

WESTERBERG et al

performed the research; F.W.A., C.T., and L.D.N. contributed new

reagents 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. and
S.B.S. wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Scott B. Snapper, MD, PhD, Harvard Medical

School, Gastroenterology Division, Childrens Hospital Boston,
300 Longwood Ave, Boston, MA 02115; e-mail: scott.snapper@
childrens.harvard.edu; or Lisa S. Westerberg, PhD, Karolinska
Institutet, Department of Medicine, Translational Immunology Unit
L2:04, 171 76 Stockholm, Sweden; e-mail: lisa.westerberg@ki.se.

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2012 119: 3966-3974

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

Wiskott-Aldrich syndrome protein (WASP) and N-WASP are critical for

peripheral B-cell development and function
Lisa S. Westerberg, Carin Dahlberg, Marisa Baptista, Christopher J. Moran, Cynthia Detre, Marton
Keszei, Michelle A. Eston, Frederick W. Alt, Cox Terhorst, Luigi D. Notarangelo and Scott B. Snapper

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