Notch2 Haploinsufficiency Results in
Diminished B1 B Cells and a Severe
Reduction in Marginal Zone B Cells
This information is current as
of June 17, 2017.
Colleen M. Witt, Woong-Jai Won, Vincent Hurez and
Christopher A. Klug
J Immunol 2003; 171:2783-2788; ;
doi: 10.4049/jimmunol.171.6.2783
http://www.jimmunol.org/content/171/6/2783
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References
The Journal of Immunology
Notch2 Haploinsufficiency Results in Diminished B1 B Cells
and a Severe Reduction in Marginal Zone B Cells1
Colleen M. Witt,* Woong-Jai Won,* Vincent Hurez,† and Christopher A. Klug2*
Recent studies have implicated a role for Notch in the generation of marginal zone (MZ) B cells. To further investigate the role
of Notch in the B cell lineage, we have analyzed the effects of reduced Notch2 signaling in mice expressing one functional allele
of Notch2 (Notch2ⴙ/ⴚ). Notch2ⴙ/ⴚ mice have reduced B1 B cells of the peritoneal cavity and show a severe reduction in MZ B
cells of the spleen. The reduction in MZ B cells was not due to the disruption of splenic architecture, disregulated terminal
differentiation, nor to increased apoptosis within the MZ B cell compartment. Rather, our data suggest that Notch2 haploinsufficiency leads to impaired development of MZ B cells, possibly by impacting the formation of immediate MZ B precursors. These
results provide evidence that Notch2 plays a determining role in the development and/or the maintenance of B1 B and MZ B
cells. The Journal of Immunology, 2003, 171: 2783–2788.
T
ies, taken with our findings that Notch2 protein is preferentially
expressed in B1 B and MZ B cells,4 implicate a role for Notch2 in
the generation of mature B cell subsets.
Previously, an inactivating mutation within the Notch2 locus
was generated by the targeted in-frame insertion of the Escherichia
coli lac-Z gene into the ankryin repeat region of Notch2 (10). This
mutation, which completely abolishes the signaling function of
Notch2, results in embryonic lethality at approximately embryonic
day 10.5. Mice that are heterozygous for this mutation
(Notch2⫹/⫺) are viable and provide a means to assess the effects of
reduced Notch2 signaling on the development of mature B cell
populations. To this end, Notch2⫹/⫺ and age- and sex-matched
wild-type littermates were analyzed using a combination of flow
cytometric and immunohistological approaches. Our results show
that haploinsufficiency of Notch2 leads to a reduction in both the
frequency and absolute number of B1 B cells in the peritoneal
cavity. In addition, Notch2⫹/⫺ mice have a severe reduction in the
frequency and absolute number of MZ B cells. The reduction in
MZ B cells is not due to disrupted splenic architecture, disregulated terminal differentiation, nor increased apoptosis within the
MZ B or transitional immature B cell populations, but rather due
to impaired development of MZ B cells. This study provides evidence that Notch2 plays a direct and determining role in the generation of both the MZ and B1 B cell subsets.
*Department of Microbiology, Division of Developmental and Clinical Immunology,
and †Department of Pathology, University of Alabama, Birmingham, AL 35294
Materials and Methods
Received for publication March 14, 2003. Accepted for publication July 3, 2003.
Notch2⫹/⫺ mice, previously described (10), were backcrossed for ⬎10
generations onto the C57BL-Ly-5.2 background. All mice used in these
experiments were either 4- or 12-wk-old females, as indicated. Control
mice used for analyses of Notch2⫹/⫺ mice were age-matched wild-type
female littermates.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by a Howard Hughes faculty development award to
C.A.K. (53000281) and a Basic Mechanisms in Lung Disease training grant to
C.M.W. (HL07553).
2
Address correspondence and reprint requests to Dr. Chris Klug, Department of
Microbiology, Division of Developmental and Clinical Immunology, University of
Alabama, WTI387, 1824 6th Avenue, Birmingham, AL 35294-3300. E-mail address:
[email protected]
Abbreviations used in this paper: MZ, marginal zone; ␤-gal, ␤-galactosidase; FDG,
fluorescein di-␤-D-galactopyranoside; FO, follicular; MZP, MZ precursor; TR1, transitional 1 stage; TR2, transitional 2 stage.
3
4
C. M. Witt, V. Hurez, C. S. Swindle, Y. Hamada, and C. A. Klug. Activated Notch
2 potentiates CD8 lineage maturation and promotes the selective development of B1
B cells. Submitted for publication.
Copyright © 2003 by The American Association of Immunologists, Inc.
Mice
Flow cytometry and Abs
For analyses of the B1 B cell population, peritoneal cavity cells were isolated by lavage, and single cell suspensions of harvested spleens were
prepared according to standard protocols. FACS analysis was done using
the following Abs: FITC-conjugated anti-B220 and PE-conjugated antiCD5 (BD PharMingen, San Diego, CA). Splenic populations were analyzed using FITC-conjugated CD23 and PE-conjugated CD21 (BD PharMingen) and anti-mouse IgM coupled to Cy5 (Jackson ImmunoResearch,
West Grove, PA). Cells were resuspended in propidium iodide, and viable
0022-1767/03/$02.00
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he generation of mature B cell subsets occurs through a
series of differentiative steps that includes the expression
of a functional surface Ig receptor and successful passage
through selective checkpoints (1, 2). Once mature, each subset
exhibits unique functional properties, which in their totality provide an optimal defense against a diverse array of foreign pathogens. Although much insight has been gained into the functional
properties of mature B cells, the mechanisms that drive the generation and selection of immature B cells into the various mature
B cell subsets remain unclear.
Mammalian Notch proteins (Notch1– 4) are highly conserved
type 1 transmembrane receptors that regulate cell fate decisions in
numerous developmental contexts (3, 4). Until recently, most of
what is known of Notch function in hemopoiesis has come from
studies of Notch1 that have demonstrated its critical role in T cell
development (5– 8). However, recent studies have shown that the
conditional inactivation of RBP-J␬, a primary mediator of Notch
signaling, leads to the loss of marginal zone (MZ)3 B cells (9),
suggesting a role for Notch signaling beyond the T cell lineage.
Also, our recent studies have shown that expression of an activated
form of Notch2 in hemopoietic progenitors blocks the development of conventional B2 cells and promotes the selective development of B1 B cells,4 a mature B cell subset that shares many
phenotypic and functional properties with MZ B cells. These stud-
2784
NOTCH2 IS NECESSARY FOR MZ B CELL DEVELOPMENT
cells were electronically gated and analyzed on FACSCalibur (BD Biosciences, San Jose, CA).
Notch2 protein expression
Notch2⫹/⫺ mice express an in-frame fusion of Notch2 and the E. coli
␤-galactosidase (␤-gal) protein driven by the endogenous Notch2 promoter, permitting the analysis of Notch2 protein expression by assaying
␤-gal activity within cells. Single cell suspensions from the spleens of
Notch2⫹/⫺ and wild-type littermates, serving as negative controls, were
stained with Abs (described below), washed, and loaded with fluorescein
di-␤-D-galactopyranoside (FDG) using FluoReporter lacZ Flow Cytometry
kit (Molecular Probes, Eugene, OR), as described in the manufacturer’s
protocol. Cells were resuspended in propidium iodide, and viable cells
were electronically gated and analyzed on MoFlo (Cytomation, Fort Collins, CO). Notch2 protein expression was determined by assessing ␤-gal
activity within specific subpopulations, indicated by the production of fluorescein upon reaction of FDG with E. coli ␤-gal. Abs included biotinylated anti-CD23 and PE-conjugated CD21 (BD PharMingen), streptavidinconjugated Texas Red (Southern Biotechnology Associates, Birmingham,
AL), and anti-mouse IgM coupled to Cy5 (Jackson ImmunoResearch).
Detection of apoptosis by TUNEL
Immunofluorescence analysis of splenic sections
Cryosections from Notch2⫹/⫺ and wild-type littermates were prepared at
4-␮m thickness, fixed in acetone, and blocked with 10% horse serum.
Sections were stained with MOMA-1 (rat IgG2a; a gift from G. Kraal,
Vrije Universiteit Medical Center, The Netherlands) and developed with
biotinylated goat anti-rat IgG, followed by incubation with strepavidin
7-amino-4-methylcoumarin-3-acetic acid (Vector Laboratories, Burlingame, CA). Sections were stained with ERTR-9 (also provided by G.
Kraal), washed, and incubated with a mixture of PE-conjugated anti-rat
IgM (BD PharMingen) for development of ERTR-9 and with Alexa 488conjugated anti-mouse IgM (MB86). Sections were washed and mounted
with Fluormount G (Southern Biotechnology Associates) and viewed with
a Leica/Leitz DMRB fluorescence microscope equipped with appropriate
filter cubes (Chromatechnology, Battleboro, VT). Images were acquired
with a C5810 series digital color camera (Hamamatsu Photonic System,
Bridgewater, NJ).
Results
Notch2⫹/⫺ mice have a reduction in peritoneal B1 B cells
To investigate the effects of Notch2 haploinsufficiency on B1 B
cell development, we performed flow cytometric analyses of this
population in Notch2⫹/⫺ mice, which had been backcrossed for
greater than 10 generations onto wild-type C57BL/6 mice. Age-
Notch2⫹/⫺ mice have a severe reduction in MZ B cells
Follicular (FO) B cell and MZ B cell subsets in the spleen reside
in distinct anatomical locations and display unique phenotypic and
functional properties (11). Flow cytometric analysis was used to
assess the effects of Notch2 haploinsufficiency on these cells from
Notch2⫹/⫺ and control littermates. Analyses were performed at 4
wk of age, early in the development of mature splenic B cell subsets, and at 12 wk of age when splenic subsets are well established.
Analyses at 12 wk of age showed that while the overall frequencies
of splenic B cells in Notch2⫹/⫺ mice were similar to their wildtype littermates (Fig. 2A), there was a dramatic reduction in the
frequencies of MZ B cells in Notch2⫹/⫺ mice, with an average
frequency of 1.7 ⫾ 0.7% in Notch2⫹/⫺ mice vs 9.2 ⫾ 1.2% in
wild-type littermates ( p ⬍ 0.001; n ⫽ 14 for each genotype; Fig.
2, A and B). Coincident with the severe reduction in this subset was
an increase in the relative frequencies of FO B cells, although this
increase did not reach statistical significance. Similar results were
obtained from analyses of younger mice, with the exception that
the disparity in frequencies of MZ B cells was less severe in mice
analyzed at 4 wk of age (5.6 ⫾ 1.1% in wild type vs 1.1 ⫾ 0.6%
in Notch2⫹/⫺; n ⫽ 14 for each genotype; Fig. 2B). The absolute
numbers of MZ B cells in young mice were 4-fold reduced as
Table I. Absolute numbers of mature B cell subsetsa
Peritoneal Cavity
⫹/⫹
⫹/⫺
⫹/⫹
⫹/⫺
4 wk
4 wk
12 wk
12 wk
Spleen
B1a (⫻105)
B1b (⫻105)
B2 (⫻105)
MZ (⫻105)
FO (⫻107)
9.6 ⫾ 1.7
8.7 ⫾ 2.0
13.3 ⫾ 2.0
7.1 ⫾ 1.4b
5.4 ⫾ 0.5
4.4 ⫾ 0.8
6.5 ⫾ 0.5
4.2 ⫾ 1.2b
2.6 ⫾ 1.6
3.0 ⫾ 0.7
5.0 ⫾ 0.9
5.6 ⫾ 1.5
22.4 ⫾ 7.4
5.6 ⫾ 1.2b
30.4 ⫾ 5.4
3.6 ⫾ 1.1b
2.2 ⫾ 0.4
3.0 ⫾ 0.5
2.5 ⫾ 0.6
2.4 ⫾ 0.7
a
Compiled data from two independent experiments of n ⫽ 14 for each genotype. Absolute numbers were calculated by multiplying the frequency of each B cell subset by
the total cell number. Means ⫾ SD are shown.
b
Values of p ⬍ 0.001 compared with controls.
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Splenocytes were stained with the above-described Abs and fixed in 2%
paraformaldehyde for 60 min at room temperature, washed, and resuspended in 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice.
Permeabilized cells were washed, resuspended in 50 ␮l of TUNEL reaction
mixture (In Situ Cell Death Detection Kit; Roche, Basel, Switzerland), and
incubated in the dark for 60 min at 37°C under humidified conditions. For
negative controls, cells were incubated in TUNEL reaction mixture without
addition of terminal transferase. For positive controls, fixed and permeabilized cells were treated with DNase I (3 U/ml in 50 mM Tris-HCL, pH
7.5, 1 mg/ml BSA) for 10 min at room temperature before incubation with
TUNEL reaction mixture. Subpopulations were analyzed by flow cytometry for the detection of FITC-labeled cells.
and sex-matched wild-type littermates served as controls. Although FACS analyses at 4 wk of age showed only a slight decrease in the relative frequencies and absolute numbers of
B220lowCD5intermediate cells (B1a) in the peritoneal cavity of
Notch2⫹/⫺ mice (data not shown and Table I), analyses at 12 wk
of age showed that the relative frequencies were ⬃50% of those of
control littermates (23.4 ⫾ 5.7% for Notch2⫹/⫺ vs 42.4 ⫾ 6.5%
for controls; p ⬍ 0.001; Fig. 1, A and B). Because the overall
cellularity of the peritoneal cavity was similar in Notch2⫹/⫺ mice,
these frequencies represented a ⬃2-fold reduction in the absolute
numbers of B1a B cells (Table I). Similarly, there was a reduction
in the frequencies of B220lowCD5low B1b B cells (12.4 ⫾ 4.3 for
Notch2⫹/⫺ vs 19.0 ⫾ 3.1% for controls; p ⬍ 0.001), which resulted in ⬃1.6-fold decrease in the absolute numbers of B1b B
cells (Table I). Similar results were obtained when peritoneal cavity cells were analyzed using anti-IgM and anti-Mac-1 costaining
(data not shown). Analyses of the B2 B cell population of the
peritoneal cavity showed increased frequencies in Notch2⫹/⫺ mice
as compared with controls, although values did not reach statistical
significance (Fig. 1B and Table I). The observation that Notch2
haploinsufficiency leads to a reduction in B1 B cells is consistent
with our recent studies,4 which showed that expression of activated
Notch2 in hemopoietic progenitors leads to the selective development of these cells. Collectively, these studies provide the first
evidence that the Notch2 family member plays a role in the development and/or the maintenance of the B1 B cell subset.
The Journal of Immunology
2785
compared with the more severe 8-fold reduction observed in mice
with fully developed MZ B cell compartments (Table I).
One explanation for the loss of MZ B cells in Notch2⫹/⫺ mice
is that Notch2 may be necessary for the maintenance of this B cell
subset. In this case, Notch2 haploinsufficiency could lead to their
disregulated terminal differentiation into plasma cells. However,
analyses of spleens (Fig. 2C) and bone marrow showed no significant increase in the frequencies or absolute numbers of IgM⫹Syn1high or IgA⫹Syn-1high natural plasma cells in unimmunized
Notch2⫹/⫺ mice as compared with their wild-type littermates (data
not shown). Taken with the observation that Ig serum levels were
not elevated in Notch2⫹/⫺ mice (data not shown), these results
suggest that the severe loss of MZ B cells in Notch2⫹/⫺ mice was
not due to disregulated terminal differentiation of MZ B cells.
Splenic architecture is grossly normal in Notch2⫹/⫺ mice
Another factor that could contribute to the reduction of MZ B cells
in Notch2⫹/⫺ mice might be the disruption of splenic architecture,
which may be necessary for MZ B cell recruitment and/or retention. To address this possibility, splenic cryosections from
Notch2⫹/⫺ and control littermates were stained with Abs against
markers that delineate the follicles and the MZ. Analysis by fluorescence microscopy revealed no gross abnormalities in
Notch2⫹/⫺ splenic architecture, as follicles appeared intact with
MZ macrophages (ERTR-9⫹ cells) present forming the MZ outer
boundary (Fig. 3A). MOMA-1⫹ metallophilic macrophages were
also present, forming the characteristic inner ring of the MZ that
surrounds FO B cells (IgM⫹ cells) (Fig. 3B). However, it should
be noted that while MOMA-1⫹ metallophilic macrophages were
present in the MZ of Notch2⫹/⫺ spleens, less intensely stained
MOMA-1⫹ cells were also observed dispersed throughout the red
pulp (Fig. 3C). Similar results were also observed in analyses of
splenic cryosections from RAG-1⫺/⫺ mice (J. Kearney and F.
FIGURE 2. Notch2⫹/⫺ mice have a severe reduction in MZ B cells. A,
Representative plot of splenic cells from Notch2⫹/⫺ and control littermates
analyzed at 12 wk of age stained with anti-IgM, anti-CD23, and anti-CD21.
The frequencies of MZ and FO B cells among IgM⫹ gated cells are shown.
B, Compiled frequency data for each of the splenic B cell subsets shown in
A from two independent experiments at each time point; n ⫽ 14 for each
genotype. C, Representative plot of IgM⫹Syn-1high plasma cells is shown.
Similar results were obtained with anti-IgA anti-Syn-1 costaining.
Martin, personal communication). Analysis at higher magnification of MOMA-1⫹ cells localized to the red pulp showed them to
be large and of myeloid-like morphology (Fig. 3D). Because the
MOMA-1-reactive Ag expressed on metallophilic macrophage is
unknown, it is not clear whether the distribution of MOMA-1⫹
cells represents the abnormal localization of metallophilic macrophages or the aberrant expression of the MOMA-1 Ag on resident
cells of the red pulp. Regardless, the MZ appeared to be overall
intact, making it unlikely that the loss of MZ B cells in Notch2⫹/⫺
mice was due to disrupted splenic architecture.
MZ B cells are developmentally impaired in Notch2⫹/⫺ mice
Upon emigration from the bone marrow, newly formed B cells
enter the spleen and progress through transitional stages (TR1 and
TR2), where it is believed the MZ vs FO B cell fates are determined (12). The frequencies of Notch2⫹/⫺ TR1 B cells, which
represent the earliest arrivals into the spleen, were similar to those
of wild-type littermates (Fig. 4A), indicating that there was no
gross impairment in the emigration of immature B cells into the
spleen. Importantly, while the overall frequencies of TR2 B cells
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FIGURE 1. Haploinsufficiency of Notch2 leads to a reduction in B1 B
cells. A, Representative plot of peritoneal cavity cells from Notch2⫹/⫺ and
control littermates analyzed at 12 wk of age stained with anti-B220 and
anti-CD5. Frequencies of B220lowCD5intermediate (B1a), B220lowCD5low
(B1b), and B220highCD5⫺ (B2 B) cells within the lymphoid compartment
of the peritoneal cavity are shown. Similar results were obtained with antiIgM anti-Mac-1 costaining. B, Compiled frequency data for each of the B
cell subpopulations shown in A from two independent experiments of n ⫽
14 for each genotype. ⴱ, Denotes values for p ⬍ 0.001.
2786
NOTCH2 IS NECESSARY FOR MZ B CELL DEVELOPMENT
were also similar in Notch2⫹/⫺ and wild-type littermates, a small
population of CD23⫹IgMhighCD21high cells that fall within the
TR2 population, which are believed to be the immediate precursors
of MZ B cells (MZP) (13–15), was markedly reduced (Fig. 4A).
Furthermore, flow cytometric analysis of Notch2 protein expression showed that while Notch2 protein levels were low in TR2
cells when gated as a whole population, Notch2 was highly expressed in the CD23⫹IgMhighCD21high subpopulation (MZP) (Fig.
4B). These results suggest that Notch2 may be functioning in developing MZP and that Notch2 haploinsufficiency leads to defects
in their formation and/or their survival. To address the latter possibility, splenic subpopulations were analyzed using a TUNEL assay to detect the presence of apoptotic cells. As shown in Fig. 4C,
we detected no increase in apoptosis in the CD23⫹IgMhigh
CD21high precursor population or in the TR2 population as a whole
(Fig. 4C). Nor was any increase in apoptosis detected in TR1 stage
B cells or in the MZ B cell compartment itself (Fig. 4C). These
results, taken with all of the above, suggest that Notch2 haploin-
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FIGURE 3. The splenic architecture in Notch2⫹/⫺
mice is grossly normal. A, Cryosections of 12-wk-old
Notch2⫹/⫺ and same-sex wild-type littermate spleens
stained with ERTR-9PE, showing the presence of MZ
macrophages as characteristic perifollicular ring. B,
Cryosections stained with ERTR-9 (red), the metallophilic macrophage-specific Ab MOMA-1 (blue), and
anti-IgM (green), showing intact FO and MZ. C,
MOMA-1 staining shows MOMA-1⫹ cells dispersed
throughout the red pulp of Notch2⫹/⫺ spleens. D,
MOMA-1 staining of red pulp at higher magnification.
MOMA-1⫹ cells appear to have myeloid-like morphology. A–D, Representative results from two independent experiments of n ⫽ 2 for each genotype.
sufficiency leads to impaired formation of MZ B cells, possibly by
impacting the generation of MZP during the transitional stages of
immature B cell development.
Discussion
In this study, we have shown that mice expressing only one functional allele of Notch2 have ⬃2-fold reduction in the frequency
and absolute number of B1 B cells. The observation that B1 B cell
numbers are similar to those of controls at early time points and
diminish with the age of the mice suggests that the maintenance
and/or self-renewing capacity of these cells are affected by haploinsufficiency of Notch2. This does not exclude the possibility that
the development of B1 B cells is also affected by reduction in
Notch2 dosage. Although the majority of B1 B cells in the adult
mouse are derived during fetal B lymphopoiesis, small numbers
continue to be generated from B cell progenitors in the bone marrow during adult life (16). Although it is not clear to what degree
The Journal of Immunology
adult-derived B1 B cells contribute to the overall B1 B cell population size, it is possible that Notch2 haploinsufficiency impairs
adult B1 B cell development, thereby contributing to the reduction
of this population in Notch2⫹/⫺ mice. This possibility is consistent
with our recent Notch2 gain-of-function studies, which showed
that Notch2 protein is highly expressed in B1 B cells and that
expression of an activated form of Notch2 in hemopoietic progenitors leads to the exclusive development of these cells.4 Collectively, these findings provide the first evidence of a link between
the Notch2 signaling pathway and the development and/or maintenance of the B1 B cell subset.
The reduction in B1 B cells in Notch2⫹/⫺ mice was also associated with a severe reduction in MZ B cells of the spleen, a mature
B cell subset that shares several phenotypic and functional properties with B1 B cells (11, 17). The observation that Notch2 haploinsufficiency affects MZ B cells is consistent with recent studies
that showed that the conditional inactivation of RBP-J␬, a primary
effector protein for the Notch signaling pathway, also resulted in
the loss of the MZ B cell compartment (9). The severe reduction
in MZ B cells in Notch2⫹/⫺ mice indicates that other Notch family
members do not effectively compensate for reduced Notch2 function in MZ B cell development. These results suggest that Notch2
signaling through RBP-J␬ is necessary for the formation of the MZ
B cell subset.
Of particular interest is the observation that the B1 B cell population was not affected by the conditional inactivation of RBP-J␬,
which is in contrast to the reduction in these cells observed in
Notch2⫹/⫺ mice. Several studies have documented alternative
Notch signaling pathways that do not depend on the actions of
RBP-J␬ (18 –22). Therefore, the differential phenotypes obtained
from the inactivation of RBP-J␬ and Notch2 haploinsufficiency
may reflect the use of an RBP-J␬-independent Notch2 signaling
pathway in the development and/or the maintenance of B1 B cells.
The fact that the reduction in B1 B cells is in association with
reduced MZ B cells suggests not only a role for Notch2 in the
development of these subsets, but also supports the notion that
these subsets share at least some common elements in their developmental pathways. Although the mechanisms that drive the selection of immature B cells into the various mature B cell subsets
are still being defined, there is growing evidence that MZ B and B1
B cells are positively selected into their respective compartments
(1, 23). Support for this model comes from recent studies that
showed that newly formed immature B cells, expressing a transgenic B cell receptor enriched in the MZ B cell compartment, were
positively selected into the MZ B cell pool, and that their enrichment was achieved by their preferential proliferation and survival
during selective checkpoints (24). According to this model, a potential role for Notch2 may be to facilitate positive selection
through enhanced proliferation and/or survival of immature B cells
with BCR specificities suited to either the MZ B or the B1 B cell
pools. Such a role is consistent with the known function of Notch
in cellular proliferation (25–27) and with numerous other studies
that have demonstrated Notch-mediated protection from apoptosis
(28 –31). In addition, NF-␬B activity is often involved in the promotion of cell survival (32–36), and the loss of certain NF-␬B
family members results in impaired MZ B cell development
(37– 41). Thus, recent evidence that the Notch signaling pathway targets NF-␬B activity (42– 44) may reflect an important
connection between Notch, NF-␬B activity, and MZ B cell development. Alternatively, it has been proposed that the strength
of signaling received through the BCR in immature B cells
determines their selection into either the FO B, MZ B, or B1 B
cell populations (15). This view raises the possibility that
Notch2 signaling modulates the strength of BCR signaling in
such a way as to drive the selection of immature B cells into
either the MZ or B1 B cell pools. Finally, cell fate determined
by strength of signal or by BCR specificity coupled to positive
selection are not mutually exclusive models, and evidence suggests that aspects of both models are likely to be used in the
generation of mature B cell subsets. Therefore, it remains possible that Notch2 functions at more than one level, by both
instructive and permissive means, to promote the development
of the MZ B and B1 B cell subsets.
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FIGURE 4. Notch2⫹ MZP are diminished in Notch2⫹/⫺ mice. Representative plot of splenic cells from Notch2⫹/⫺ and same-sex wild-type
littermates analyzed at 12 wk of age stained with anti-IgM, anti-CD23, and
anti-CD21. Top panel, Frequencies of IgMhighCD21⫹ TR2 B cells among
CD23⫹ gated cells are shown in rectangular gate. CD23⫹IgMhighCD21high
MZP are shown in circular gate. Bottom panel, Frequencies of
IgM⫹CD21low/⫺ TR1 B cells among CD23⫺ gated cells are shown. B,
Notch2 protein expression in CD23⫹IgMhighCD21⫹ TR2 B cells (top) and
in CD23⫹IgMhighCD21high MZP subpopulation (bottom). Notch2⫹/⫺ mice
express an in-frame fusion of Notch2 and E. coli ␤-gal from the endogenous Notch2 promoter. Splenocytes from Notch2⫹/⫺ and wild-type littermates, serving as negative controls, were stained as described in A and
loaded by hypotonic shock with FDG and analyzed by FACS. Notch2
protein-expressing cells are indicated by the production of FITC upon reaction of FDG with ␤-gal. Histogram overlays of negative control (filled)
and Notch2⫹/⫺ (open) cells gated on the TR2 population as a whole (top),
and of the MZP subpopulation within the TR2 population (bottom), are
shown. C, Representative results using TUNEL assay to identify frequencies of apoptotic cells by flow cytometry. Histograms are overlays of negative control (dashed), FITC⫹ cells (solid) that identify apoptotic cells
within each gated population. Stained cells treated with DNase I served as
positive controls (data not shown) and were ⬎95% FITC⫹. Results are
representative of three independent experiments of n ⫽ 2 for each
genotype.
2787
2788
Acknowledgments
We thank Lisa Jia for invaluable technical help. We are grateful to Dr.
Yoshio Hamada for his kind gift of Notch2⫹/⫺ mice, and to Dr. Pete Burrows for helpful comments and advice. We especially thank Dr. John Kearney for his technical expertise and many helpful discussions during the
preparation of this manuscript.
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NOTCH2 IS NECESSARY FOR MZ B CELL DEVELOPMENT