http://www.jimmunol.org/content/172/9/5329.full.pdf

Latent Membrane Protein 2A, a Viral B Cell
Receptor Homologue, Induces CD5 + B-1 Cell
Development
This information is current as
of July 7, 2017.
Akiko Ikeda, Mark Merchant, Lori Lev, Richard Longnecker
and Masato Ikeda
J Immunol 2004; 172:5329-5337; ;
doi: 10.4049/jimmunol.172.9.5329
http://www.jimmunol.org/content/172/9/5329
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References
The Journal of Immunology
Latent Membrane Protein 2A, a Viral B Cell Receptor
Homologue, Induces CD5ⴙ B-1 Cell Development1
Akiko Ikeda, Mark Merchant,2 Lori Lev, Richard Longnecker,3 and Masato Ikeda
The latent membrane protein 2A (LMP2A) of EBV plays a key role in regulating viral latency and EBV pathogenesis by functionally mimicking a constitutively active B cell Ag receptor. When expressed as a B cell-specific transgene in mice, LMP2A drives
B cell development, resulting in the bypass of normal developmental checkpoints. In this study, we have demonstrated that
expression of LMP2A in transgenic mice results in B cell development that exclusively favors B-1 cells. This switch to B-1 cell
development occurs at the pre-B-cell stage of normal B cell development in the bone marrow, a B cell stage much earlier than
appreciated for B-1 commitment. This finding indicates that all pre-B cells have the capacity to assume a B-1 cell phenotype if they
encounter the appropriate signal during normal development. Furthermore, these studies offer insight into EBV latency and
pathogenesis in the human host. The Journal of Immunology, 2004, 172: 5329 –5337.
Department of Microbiology-Immunology, Northwestern University Feinberg School
of Medicine, Chicago, IL 60611
Received for publication September 25, 2003. Accepted for publication February
26, 2004.
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 U.S. Public Health Service Grants CA62234,
CA73507, and CA93444 from the National Cancer Institute (to R.L.) and by Grant
DE13127 from the National Institute of Dental and Craniofacial Research (to R.L.).
M.I. is a Special Fellow of the Leukemia and Lymphoma Society of America. R.L.
is a Stohlman Scholar of the Leukemia and Lymphoma Society of America.
2
Current address: Genentech, 1 DNA Way, MS No. 37, South San Francisco, CA
94080.
3
Address correspondence and reprint requests to Dr. Richard Longnecker, Department of Microbiology-Immunology, Northwestern University Feinberg School of
Medicine, Chicago, IL 60611. E-mail address: [email protected]
4
Abbreviations used in this paper: LMP2A, latent membrane protein 2A; BCR, B cell
Ag receptor; ITAM, immunoreceptor tyrosine activation motif; TgE, transgenic line
E; Tg6, transgenic line 6; Tg⌬ITAM, ITAM mutant transgenic mice; PtC, phosphatidyl choline; Btk, Bruton’s tyrosine kinase; BLNK, B cell linker protein; CLL, chronic
lymphocytic leukemia; NHL, non-Hodgkin’s lymphoma; H-RS, Hodgkin and ReedSternberg.
Copyright © 2004 by The American Association of Immunologists, Inc.
ability of LMP2A to interact with the protein tyrosine kinases Lyn
and Syk. In addition to mimicking a constitutive BCR signal,
LMP2A is also capable of blocking BCR-induced signaling by
recruiting Lyn and Syk (7, 8). The recruitment of Lyn and Syk by
LMP2A results in the constitutive phosphorylation of these kinases
independent of ligand binding. Further evidence that LMP2A is
co-opting the BCR signaling pathway was found when LMP2A
was shown to constitutively localize to lipid rafts. Therein,
LMP2A is thought to form self-aggregates that inhibit the translocation of the BCR into lipid rafts, thus blocking subsequent activation of the BCR (9). Together these data show that LMP2A
imparts multiple functions, which allow it to both mimic the BCR
signal required for B cell survival and also eliminate the ability of
the BCR to properly activate infected B cells.
Transgenic mice expressing LMP2A downstream of the ␮ Ig
heavy-chain enhancer and promoter (E␮) have highlighted the
ability of LMP2A to impart developmental and survival signals to
developing and mature B cells (5). In E␮LMP2A transgenic line E
(TgE), which expresses high levels of LMP2A, there is a developmental alteration characterized by a bypass of BCR expression
resulting in BCR-negative (BCR⫺) B cells in peripheral lymphoid
organs (10). The lack of BCR expression in TgE mice results from
the absence of Ig heavy chain rearrangement (10). Normally, B
cells lacking a cognate BCR rapidly undergo apoptosis; however,
in TgE mice, BCR⫺ cells persist. In E␮LMP2A transgenic line 6
(Tg6), which expresses lower levels of LMP2A, there are no dramatic differences in B cell development compared with wild-type
animals other than a slight reduction in the number of BCR⫹ B
cells in the spleen of LMP2A transgenic mice, indicating that there
is likely an expression threshold that must be overcome to observe
a developmental phenotype (10). Furthermore, LMP2A requires
the association and activation of Syk to alter B cell development
because LMP2A transgenic mice bearing mutations in the ITAM
of LMP2A show a B cell phenotype indistinguishable from that of
wild-type mice (11). These results indicate that LMP2A positively
mediates developmental signals in B cells and that Syk plays a key
role in mediating LMP2A signaling. However, the unusual BCR⫺
peripheral B cell subset in the E␮LMP2A⫹ mice has not been fully
characterized with respect to other B cell subsets.
Two distinct mature B cell populations, B-1 and B-2, are present
in the mouse and human periphery. B-1 cells are found mainly in
the peritoneal and pleuropericardial cavities, whereas B-2 cells are
0022-1767/04/$02.00
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T
he latent membrane protein 2A (LMP2A)4 is an EBVencoded protein that has been implicated in regulating
viral latency and pathogenesis within infected cells (1).
Upon primary infection, the virus enters a latent life cycle characterized by the expression of nine latency-associated viral proteins, including LMP2A (2, 3). LMP2A is one of the most consistently detected mRNAs found in humans infected with EBV,
suggesting that LMP2A plays an important role in regulating viral
persistence and in the development of EBV-associated diseases
(4). Our studies have demonstrated that LMP2A functionally mimics a constitutively active B cell Ag receptor (BCR) and modulates
BCR-mediated signal transduction during B cell development (5)
and activation (6).
Consistent with a role in functionally imitating BCR signaling,
LMP2A has been shown in EBV-immortalized human lymphoblastoid cell lines to interact with cellular proteins and localize to
subcellular compartments in a fashion similar to that of an activated BCR. The amino-terminal tail of LMP2A contains several
tyrosine residues important for these functions of LMP2A. Two of
these tyrosines encode a conserved immunoreceptor tyrosine activation motif (ITAM), which has been shown to be critical for the
5330
Materials and Methods
Mice
Wild-type C57BL/6 mice were obtained from The Jackson Laboratory (Bar
Harbor, ME). Construction of E␮LMP2A TgE, Tg6, and ITAM mutant
transgenic mice (Tg⌬ITAM) have been previously described (5, 11). For
analysis of flow cytometry and cell proliferation, 4- to 8-wk-old mice were
used. All animals were housed at the Northwestern University Center for
Experimental Animal Resources in accordance with university animal welfare guidelines.
Isolation of primary lymphoid cells and flow cytometry
Bone marrow cells were flushed from femur and tibia using IMDM (Life
Technologies, Rockville, MD). Spleens and lymph nodes were dissociated
between frosted slides in RPMI 1640 medium (BioWhittaker, Walkersville, MD) to prepare single cell suspensions. Peritoneal cells were obtained by peritoneal lavage with HBSS. Peripheral blood was drawn from
ophthalmic veins. RBCs were lysed in RBC lysis buffer (150 mM NH4Cl,
10 mM KHCO3, 0.1 mM Na2EDTA (pH 7.2)). Cells were suspended in
staining buffer (1⫻ PBS, 10 mM HEPES, 1% FBS, and 0.1% sodium
azide). For surface staining, 1 ⫻ 106 cells in 100 ␮l were incubated with
previously optimized concentrations of FITC-, PE-, PerCP-, APC-, and
biotin-labeled Abs at 4°C for 30 min. For a second step when required,
cells were resuspended in 100 ␮l of appropriate secondary reagent and
incubated at 4°C for 30 min. Cells were washed with 1⫻ PBS and analyzed
by using FACSCalibur (BD Biosciences, Mountain View, CA) and
CellQuest Pro analysis software (BD Biosciences). The Abs and secondary
reagents used were anti-CD5, anti-B220, anti-CD43, anti-CD23, anti-c-Kit,
anti-CD24, anti-BP-1 Ag, anti-IgM, and streptoavidin-PerCP (BD Biosciences). All data represent cells that fall within the lymphocyte gate
determined by forward and side scatter. A total of 5,000 –10,000 events in
the lymphocyte gate were analyzed.
Cell culture
A total of 1 ⫻ 106 bone marrow cells were cultured in 3 ml of IMDM, 30%
FBS, 100 ␮M 2-ME, 2 mM L-glutamine, and 10 ng/ml recombinant mouse
IL-7 (R&D Systems, Minneapolis, MN). After 4-day culture, medium was
changed to 3 ml of fresh medium supplemented with or without IL-7. Cells
were cultured for an additional 2 days and were used for flow cytometric
analysis.
Proliferation assay
Splenic and peritoneal B cells were purified by positive selection with
anti-CD19 (Miltenyi Biotec, Auburn, CA). Purified B cells were cultured
in RPMI 1640 medium supplemented with 10% FBS. Cells (0.5 ⫻ 105/
well) in triplicate were stimulated with various concentrations of LPS or
PMA in a 96-well tissue culture plate for 24 h. After stimulation, cells were
labeled with [3H]thymidine (1 ␮Ci/well) for 12 h. A microplate cell harvester (Filter Mate 196; Packard Instrument, Meriden, CT) was used to
collect cells, and a microplate scintillation counter (TopCount NXT; Packard Instrument) was used to assess proliferation.
Serum IgM levels
Total IgM levels in mouse serum were quantified by radial immunodiffusion at Prairie Diagnostic Services (Saskatoon, SK, Canada).
Results
LMP2A⫹ BCR⫺ B cells display the surface markers of CD5⫹
B-1 subset
Previous studies with LMP2A transgenic mice have shown that, in
addition to the loss of IgM expression, CD19 expression is high in
splenic B cells from TgE mice (16). High CD19 expression is also
found on B-1 cells within the peritoneal cavity (17). This commonality suggests that TgE B cells may share other features of B-1
lineage cells. To test this idea, TgE splenic B cells were analyzed
by flow cytometry for expression of the lymphocyte differentiation
markers CD5 and B220. TgE is a representative LMP2A transgenic line expressing high levels of LMP2A (10). In wild-type
mice, B-1 cells (CD5⫹ and B220⫹) are readily detected in the
peritoneal cavity and represent only a small proportion of splenic
B cells (Fig. 1A, WT). Approximately 10% of the peritoneal B
cells from wild-type mice are CD5⫹ (Fig. 1 and Table I). This is
somewhat lower than what is observed for other strains such as
BALB/C (data not shown), in which the number can be as high as
60% (18). In contrast, splenic and peritoneal B cells from TgE
mice are both predominantly CD5⫹ and B220⫹ (Fig. 1A, TgELMP2A), indicating that most B cells in TgE mice display a B-1a
phenotype. In addition, as would be expected from the previously
described TgE-LMP2A phenotype (5), the majority of these cells
are surface Ig negative (data not shown).
To further characterize the origin and trafficking of the splenic
CD5⫹ population cells in TgE mice, bone marrow B cells from
wild-type and TgE mice were examined (Fig. 1A, WT and TgELMP2A). Interestingly, bone marrow B cells from TgE mice also
exhibited a significant increase in CD5⫹ B cells (Fig. 1A). CD5⫹
and B220⫹ B cells were also the predominant population of B cells
in both the inguinal lymph node and in the peripheral blood in
TgE-LMP2A mice, whereas the majority of B cells in wild-type
mice were conventional B-2 cells (CD5⫺ and B220⫹; Fig. 1B, WT
and TgE-LMP2A). Increases in absolute numbers of CD5⫹ B cells
were also observed in each organ of TgE mice (Table I). These
results indicate that LMP2A signaling allows for the development
of a unique population of CD5⫹ B cells most closely related to the
B-1a lineage.
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mainly found in spleen, lymph nodes, and peripheral blood. B-1
cells are subdivided into CD5⫹ (B-1a) or CD5⫺ (B-1b) cells and
are distinguishable from B-2 cells by cell surface marker expression (IgMhigh, IgDlow, B220low, CD43⫹, and CD23⫺) (12, 13). B-1
cells are more likely to produce autoantibodies and may be involved in T cell-independent responses to common environmental
Ags. Another unique property of B-1 cells is the capacity for selfreplenishment. B-2 cells are responsible for T cell-dependent responses to exogenous Ags and for generating memory B cells.
Developmentally, immature B cells emigrate from the bone marrow to the splenic periarteriolar sheath, forming transitional type 1
B cells (14). Only a small percentage of type 1 cells develop into
transitional type 2 cells that reside in the spleen primary follicle.
Type 2 B cells then differentiate into follicular B-2 cells localized
in the primary lymphoid follicle or marginal zone B cells in the
perifollicular marginal zones (14). Despite the wealth of information in regard to the development of B-2 cells, the source of B-1
cells has been controversial. Initially, it was thought that B-1 cells
develop as a separate lineage that is distinct from conventional B-2
cells. Thus, B-1 cells develop in early life as a distinct population
(the lineage hypothesis) (15). However, recent studies suggest that
B-1 cells result from a pathway potentially available to all developing B cells. In this model, a different strength and context of
BCR signaling are required for the development of each B cell
subset (the differentiation hypothesis) (15).
In this study, the peripheral and bone marrow B cell subsets in
LMP2A transgenic mice were analyzed to clarify how LMP2A
alters B cell development. This analysis indicates that LMP2A
expression results in the exclusive generation of B cells that have
a phenotype indicative of the CD5⫹ B-1 subset or B-1a cell type.
The development of these cells is independent of a functional
BCR, indicating that LMP2A signals replace those normally derived from the BCR, resulting in a developmental switch to the B-1
cell lineage. These data support the differentiation model of CD5⫹
B-1 cell development because signals derived from LMP2A during
early B cell development are capable of switching B cells normally
committed to B-2 cell development to become B-1 lineage cells.
Furthermore, these data suggest that signals derived from LMP2A
during latent infection may help EBV establish a unique B cell
reservoir, a step that could be important during early pathogenesis
of the virus.
LMP2A INDUCES CD5⫹ B-1 CELL DEVELOPMENT
The Journal of Immunology
5331
High levels of LMP2A expression are required for
LMP2A-mediated differentiation of CD5⫹ B-1 cells
Previous studies have indicated that high levels of LMP2A expression are required for the generation and survival of BCR⫺ B
cells in the periphery of LMP2A transgenic lines, whereas lower
levels of LMP2A expression result in transgenic lines with no
dramatic developmental alteration, except for a reduction in the
normal number of BCR⫹ peripheral B cells (10). To determine
whether high levels of LMP2A expression are required for the
generation of cells bearing a B-1 lineage phenotype, B cells from
the spleen, peritoneal cavity, and bone marrow from the Tg6 line,
a representative low-expressing LMP2A transgenic line, were analyzed. B cells harvested from the spleen and bone marrow from
Tg6 transgenic mice do not show an increase in CD5⫹ cells when
compared with TgE or wild-type mice (Fig. 1A, WT, TgELMP2A, and Tg6-LMP2A). Absolute numbers of CD5⫹ B cells in
each organ of Tg6-LMP2A mice were not significantly different
when compared with wild type (data not shown). These data indicate that the high expression levels of LMP2A in TgE mice are
required for the alteration of B cell development resulting in the
generation of B-1a-like cells and that the lower levels of LMP2A
expression as observed in Tg6 mice are insufficient to drive B cells
toward the B-1a lineage.
Syk is required for LMP2A-mediated differentiation of CD5⫹
B-1 cells
We have shown that the Syk protein tyrosine kinase interaction
with LMP2A is required for the LMP2A-mediated bypass of B cell
developmental checkpoints (11). To investigate whether Syk is
also essential for LMP2A-mediated B-1a development, B cells
were analyzed for LMP2A Tg⌬ITAM. The LMP2A transgene in
these mice contains a mutation in the LMP2A ITAM that prevents
the association of LMP2A with Syk. This transgenic line, as well
as others, expresses levels of LMP2A comparable with those of the
TgE line (11). B cell development is normal in Tg⌬ITAM spleen
and bone marrow B cell populations. To determine whether the
interaction of Syk with LMP2A is important for the generation of
CD5⫹ B-1 lineage B cells in TgE mice, flow cytometry was performed on spleen, bone marrow, and peritoneal cavity cells collected from wild-type and Tg⌬ITAM mice. In Tg⌬ITAM mice,
there was a complete absence of the unique CD5⫹ and B220⫹ B
cell populations present in the spleen and bone marrow samples of
TgE mice (Fig. 1A, compare Tg⌬ITAM-LMP2A with TgELMP2A). Absolute numbers of total and CD5⫹ B cells in each
organ of Tg⌬ITAM mice did not show any difference when compared with wild type (data not shown). These results indicate that
the interaction of Syk with LMP2A is essential for LMP2A-mediated B-1a development.
TgE B cells are CD5⫹, CD43⫹, and CD23⫺, which is
characteristic of B-1a cells
To further verify the B-1a lineage phenotype of B cells from TgE
mice, B220⫹ gated B cells from the spleen, peritoneal cavity, and
Table I. Cell numbers in LMP2A transgenic micea
Bone marrow
Wild type (n ⫽ 7)
TgE-LMP2A (n ⫽ 6)
Spleen
Wild type (n ⫽ 13)
TgE-LMP2A (n ⫽ 12)
Peritoneal cavity
Wild type (n ⫽ 5)
TgE-LMP2A (n ⫽ 5)
Total Cells
B220⫹CD5⫹
B220⫹CD5⫺
64.6 ⫾ 13.3
53.3 ⫾ 9.9
5.5 ⫾ 0.2
16.3 ⫾ 0.7b
39.2 ⫾ 1.7
14.2 ⫾ 0.8b
83.9 ⫾ 1.85
60.5 ⫾ 16.0c
4.6 ⫾ 0.4
12.8 ⫾ 1.5b
38.9 ⫾ 2.8
3.9 ⫾ 0.5b
3.6 ⫾ 1.1
2.9 ⫾ 0.5d
0.37 ⫾ 0.02
0.81 ⫾ 0.04c
1.52 ⫾ 0.09
0.28 ⫾ 0.02b
a
The absolute number (⫻106) of each cell population was determined by flow
cytometry. Values are given as the mean ⫾ SD. Student’s t tests were calculated as
transgenic vs wild-type mice.
b
p ⬍ 0.001.
c
p ⬍ 0.005.
d
p ⬍ 0.05.
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FIGURE 1. CD5 expression on B cells isolated from TgE-LMP2A mice. A, Total cells isolated from spleen, peritoneal cavity, and bone marrow of wild
type (WT), high-LMP2A-expressing transgenic (TgE-LMP2A), low-LMP2A-expressing transgenic (Tg6-LMP2A), and ITAM-mutant-LMP2A transgenic
(⌬ITAM-LMP2A) mice were stained with CD5 and B220. Lymphocyte populations determined by forward and side scatter were plotted with CD5 and
B220. B, Lymph node and peripheral blood cells from WT and TgE-LMP2A mice were similarly stained with CD5 and B220. The percentages of CD5⫹
or CD5⫺ B cells to total lymphocytes are indicated. Representative experiments are shown from eight TgE-LMP2A, four Tg6-LMP2A, or three
⌬ITAM-LMP2A.
5332
LMP2A⫹ B-1a cells are committed in bone marrow at the large
pre-B stage
CD5 expression in TgE bone marrow B cells suggests that
LMP2A-mediated B-1 development starts within bone marrow. To
determine the exact stage at which the commitment to B-1 cells is
made, CD5 expression was examined in pro-, pre-, and immature
B cells from TgE bone marrow. Typically, CD43 and IgM are used
to developmentally stage bone marrow B cells, but because TgE
bone marrow B cells do not express Ig and B-1 cells are positive
for CD43, we instead chose to use c-Kit, CD24, and BP-1, which
distinguish different B cell developmental stages. As previously
described, different B cell developmental subsets can be distinguished by the following expression profile: early pro-B (c-Kit⫹,
CD24⫺), late pro-B (c-Kit⫹, CD24low), pre-B (c-Kit⫺, CD24high,
BP-1⫹), and immature B (c-Kit⫺, CD24high, BP-1⫺) (12). The
percentage of both early and late pro-B cells from B220⫹ gated
cells was the same in wild-type and TgE bone marrow, indicating
that TgE mice display normal pro-B cell development (Fig. 3A,
WT and TgE-LMP2A). As expected, CD5 expression in these
pro-B populations was negative in TgE and wild-type mice (Fig.
3A, WT and TgE-LMP2A). These data indicate that LMP2A-mediated B-1 lineage commitment does not occur at or before the
pro-B stage.
Next, pre-B and immature B cell stages were examined by staining TgE and wild-type bone marrow cells for CD24 and BP-1 (Fig.
3B). Expression of CD5 was observed in TgE pre-B cells
(CD24high and BP-1⫹), as well as in TgE immature B cells
(CD24high and BP-1⫺), indicating that CD5 expression begins during the pre-B cell stage. Interestingly, the BP-1⫹ population of
cells dramatically decreased in TgE mice. The nature of this dramatic decrease in pre-B cells is not known, but may be due to more
rapid transition through this B cell developmental stage. It is also
possible that LMP2A dramatically alters normal B cell developmental signals due to LMP2A expression and/or lack of a
cognate BCR.
To further examine LMP2A-mediated B-1 lineage development,
large pre-B cells were selectively cultured in medium containing
IL-7. IL-7 is a cytokine required for the survival of the early B cell
lineage, and culture conditions have been established and extensively used to selectively expand primary large pre-B cells (20,
21). Upon removal of IL-7 from culture, large pre-B cells can
progress into the small pre-B stage, with a small portion of these
FIGURE 2. B-1 immunophenotypes of TgE-LMP2A B cells. Cells isolated from spleen (A), peritoneal cavity (B), and bone marrow (C) from wild-type
(WT) and high-LMP2A-expressing transgenic (TgE-LMP2A) mice were stained simultaneously with B220, CD5, and either CD43 or CD23. B cells
(B220⫹) were gated from total lymphoid population and plotted with CD5 and CD43 (upper panels) or CD5 and CD23 (lower panels). The percentages
of B-1 cells (CD5⫹ CD43⫹ or CD5⫹ CD23⫺) and B-2 cells (CD5⫺ CD23⫹) to total B cells are indicated. One of four comparable experiments is shown.
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bone marrow were analyzed for CD43, CD23, and CD5 expression. The majority of splenic B cells from wild-type mice display
a B220⫹, CD5⫺, CD43⫺, and CD23⫹ surface marker expression
profile (Fig. 2A, WT). In contrast, TgE splenic B cells display a
surface marker expression profile characteristic of B-1a cells, with
the majority of the B cells staining B220⫹, CD5⫹, CD43⫹, and
CD23⫺ (Fig. 2A, TgE-LMP2A). This population was also negative
for Mac-1 (data not shown), confirming that the B cells from the
TgE mice had a splenic B-1 phenotype, because Mac-1 is negative
in the splenic B-1 cells but positive in the peritoneal cavity B-1
cells (19). In contrast with conventional splenic B-1 cells, which
are usually IgMhigh and IgDlow, TgE B cells lack expression of
IgM or IgD due to the previously characterized block in IgH rearrangement (5).
Because TgE splenic B cells appeared to resemble B-1-like lineage cells, we were interested in analyzing the cell surface marker
expression profile of peritoneal B cells. As expected, a large fraction of the B cells from the peritoneal cavity from wild-type mice
were B220⫹, CD5⫹, CD43⫹ and CD23⫺, confirming that these
cells display the expected B-1 cell surface profile (Fig. 2B, WT).
Likewise, B cells from the peritoneal cavity from TgE mice were
almost entirely B220⫹, CD5⫹, CD43⫹, and CD23⫺, confirming
their B-1 lineage phenotype (Fig. 2B, TgE-LMP2A). Similar to the
spleen, there was a dramatic increase in the number of these cells
when compared with wild-type mice (compare WT and TgELMP2A in Fig. 2, A and B). Interestingly, the peritoneal B cells
from TgE mice were positive for Mac-1, which is comparable with
B-1 cells found in the peritoneum in wild-type mice (data not
shown).
Because TgE B cells appear to display a B-1-like expression
profile in the spleen and the peritoneum, we were interested in
analyzing the bone marrow to determine whether the apparent
skewing toward the B-1 lineage occurs there, as suggested by previous data (Fig. 1). In the TgE mice, there was an appearance of B
cells in the bone marrow that had a B-1-like phenotype, as indicated by B220⫹, CD5⫹, CD43⫹, and CD23⫺ cell populations
(Fig. 2C, TgE-LMP2A). This population of cells was almost entirely absent in the bone marrow of wild-type mice when compared
with the TgE mice (Fig. 2C, WT and TgE-LMP2A). In summary,
the expression of lymphocyte differentiation markers indicates that
the majority of B cells from the spleen, peritoneal cavity, bone
marrow, inguinal lymph node, and peripheral blood from TgE
mice have a B-1a phenotype.
LMP2A INDUCES CD5⫹ B-1 CELL DEVELOPMENT
The Journal of Immunology
5333
LMP2A). Interestingly, TgE bone marrow cells developed normally through the pre-B stage, in that cultured cells from TgE bone
marrow maintained high BP-1 expression and showed a decrease
in cell size upon removal of IL-7 from the culture medium (Fig. 4,
TgE-LMP2A). Thus, in TgE bone marrow, pre-B development
was normal and both large and small pre-B cells were positive for
BP-1, which is similar to wild type. This clearly indicates that the
decrease in the BP-1⫹ population (Fig. 4B) is not due to LMP2A
down-regulating BP-1 expression, but rather is due to a reduction
in the pre-B population.
FIGURE 3. CD5 expression on pre-B and immature B cells but not on
pro-B cells. A, Bone marrow B cells (B220⫹) from wild-type (WT) and
high-LMP2A-expressing transgenic (TgE-LMP2A) mice were gated into
early pro-B cells (c-Kit⫹ CD24⫺) and late pro-B cells (c-Kit⫹ CD24low) in
the top panels. CD5 expression in these populations is indicated with histograms (middle and bottom panels). The numbers indicate the percentage
of pro-B and pre-B cells to total B cells (top panels) and CD5⫹ cells to
early or late pro-B cells (middle and bottom panels). B, Total bone marrow
lymphoid cells were gated with CD24high expression to select pre-B
(B220⫹ BP-1⫹), immature B (B220⫹ BP-1⫺), and mature B cells
(B220high) in the top panels. CD5 expression is similarly indicated with
histograms (middle and bottom panels). The numbers indicate the percentage of pre-B and immature B cells to total B cells (top panels) and CD5⫹
cells to these populations (middle and bottom panels). One of three comparable experiments is shown.
cells proceeding further into the immature B cell stage (22). This
developmental progression is characterized by a decrease in the
BP-1high expressing population with a subsequent decrease in cell
size (Fig. 4, WT). Bone marrow cells cultured from TgE mice
contained large pre-B cells that already expressed CD5 (Fig. 4,
compare 9% and 10% for WT with 43% and 34% for TgE-
LMP2A⫹ B-1 cells proliferate in response to LPS but not
to PMA
It is known that B-1 and B-2 cells respond differentially to mitogens (15), providing an important means to evaluate the B-1 phenotype of the B cells in the TgE mice. Peritoneal B-1 cells proliferate in response to LPS and to phorbol esters (PMA), whereas
B-2 cells proliferate only in response to LPS and not to PMA (23).
Recently, studies have revealed that the response of B-1 cells to
mitogens differs with respect to whether they were derived from
the spleen or the peritoneal cavity (24). Splenic B-1 cells appear to
function much like B-2 cells in that they respond to LPS but not to
PMA, unlike peritoneal B-1 cells. Therefore, to functionally characterize TgE B-1-like cells, thymidine incorporation assays were
performed on purified B cells from spleens isolated from TgE and
wild-type mice using LPS or PMA as mitogens. TgE splenic B-1like cells responded to LPS significantly more than did wild-type
B-2 cells (Fig. 5). In contrast, PMA caused proliferation of both
wild-type and TgE peritoneal B cells (Fig. 5). However, neither
wild-type splenic B cells nor TgE splenic B-1-like cells responded
to PMA (Fig. 5). This is similar to the increase in proliferation that
was observed in Ig-expressing B-1 cells (24). To our surprise, TgE
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FIGURE 4. CD5 expression on large and small pre-B stage B cells.
Bone marrow B cells from wild-type (WT) and high-LMP2A-expressing
transgenic (TgE-LMP2A) mice were cultured with medium supplemented
with recombinant murine IL-7. After 4-day culture, medium was changed
to fresh medium with IL-7 (filled histogram) or without IL-7 (open histogram). Cells were incubated for an additional 2 days and analyzed for CD5
expression (top panels), BP-1 expression (middle panels), and forward
scatter (bottom panels). The numbers indicate the percentage of CD5⫹
cells to total B220⫹ gated lymphoid cells. One of three comparable experiments is shown.
5334
LMP2A INDUCES CD5⫹ B-1 CELL DEVELOPMENT
peritoneal B-1 cells did not respond to LPS alone, which contrasts
with typical splenic B-1 cells. The mechanism of LMP2A-mediated B-1 development in the peritoneal cavity remains to be elucidated. In summary, the phenotype of TgE splenic B-1-like cells
is consistent with that of Ig-expressing splenic B-1 cells.
Serum IgM levels increase in LMP2A mice
Although the majority of splenic B cells in TgE mice are B220⫹
IgM⫺, we consistently observe a very small percentage of B220⫹
IgM⫹ B cells in TgE spleens. Typically, this number is between
10% and 30% of the B220⫹ IgM⫹ B cells (Fig. 6A, TgE-LMP2A;
compare 7% B220⫹ IgM⫹ with 23% B220⫹ IgM⫺). To further
characterize the nature of these cells, CD5 expression was analyzed on B220⫹ IgM⫺ and B220⫹ IgM⫹ B cells from the spleen.
Compatible with our earlier results, CD5 expression was greatly
increased in TgE B220⫹ IgM⫺ B cells (Fig. 6A, TgE-LMP2A). Interestingly, there was also enhanced CD5 expression in TgE B220⫹
IgM⫹ B cells, indicating that the IgM⫹ B cells in TgE spleens also
display a B-1a-like phenotype (Fig. 6A, TgE-LMP2A). Because TgE
animals were capable of making surface IgM, albeit at low levels, we
wanted to analyze serum IgM levels in TgE mice because B-1 cells
are known to be the main source for natural IgM production. Previous
studies have shown high levels of serum IgM in Src homology 2containing protein tyrosine phosphatase-1⫺/⫺ (25) and CD22⫺/⫺ (26,
27) mice, in which there is a considerable increase in the number of
B-1 cells in the periphery. Similar to these knockout mice, TgE mice
also have high levels of serum IgM (Fig. 6B), providing additional
functional data supporting the observation that TgE B cells display a
B-1a phenotype. It is interesting to note that the small number of B
cells that do manage to recombine their Ig genes and express IgM still
have a B-1 phenotype, indicating that the LMP2A signal is dominant
to the BCR signal.
Discussion
We have demonstrated that LMP2A TgE mice exhibit an unusual
distribution and development of B-1a cells. In wild-type adult
mice, B-1 cells are found in the peritoneal cavity, represent only a
small proportion of splenic B cells, and are absent from lymph
nodes and peripheral blood. In TgE mice, however, B-1-like cells
are the principal B cell population in bone marrow, spleen, and
periphery. Most interesting is the existence of a large B-1-like
subset in the TgE bone marrow, indicating that LMP2A redirects
FIGURE 6. Increase of serum IgM levels in high-LMP2A-expressing
transgenic (TgE-LMP2A) mice. A, Total splenic cells from wild-type (WT)
and TgE-LMP2A mice were stained with B220 and IgM. Gated lymphoid
populations are plotted with B220 and IgM, and percentages of B220⫹
IgM⫺ and B220⫹ IgM⫹ populations are indicated (top panels). CD5 expression in B220⫹ IgM⫺ (middle panels) and B220⫹ IgM⫹ populations
(bottom panels) are indicated with histograms. B, Total serum IgM levels
in WT (n ⫽ 8) and TgE-LMP2A (n ⫽ 7) mice were measured by radial
immunodiffusion. Mean values are indicated by bars.
B cell development to favor B-1a differentiation. This developmental alteration in TgE mice is dependent on the association of
LMP2A with Syk, indicating that normal BCR signaling pathways
initiated by Syk are used for this phenotype. In addition, this phenotype is only observed in transgenic mice with high expression
levels of LMP2A, indicating that strength of signal may be important for the LMP2A-mediated B-1a-like lineage commitment.
These studies clearly indicate that progenitor B cells in the bone
marrow are capable of differentiating into B-1 cells in the bone
marrow microenvironment if stimulated with the appropriate signals such as the signals derived from LMP2A.
B-1 cells in normal mice are frequently autoreactive and include
cells with specificities for IgG (rheumatoid factor), ssDNA, and
the common membrane phospholipid phosphatidyl choline (PtC),
specificities all of which are rare among B-2 cells (12, 13). The
B-1 subset also harbors cells specific for bacterial carbohydrate
Ags and phosphoryl choline. Two main hypotheses have been proposed to explain the generation of B-1 cells: the lineage model and
the differentiation model. The lineage model is based on cell transfer experiments of progenitor cells. Fetal liver can reconstitute
both the B-1 and B-2 compartments of irradiated mice, whereas
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FIGURE 5. Proliferation of LMP2A⫹ splenic B-1 cells in response to
LPS but not to PMA. Purified splenic and peritoneal B cells from wild-type
(WT) and high-LMP2A-expressing transgenic (TgE-LMP2A) mice were
cultured for 24 h with 25 ␮g/ml LPS or 100 nM PMA. Cultures were
labeled with 1 ␮Ci of [3H]thymidine for 12 h and incorporation of radioactivity was measured. Results represent the mean of triplicate cultures and
SDs are indicated by bars. One of three comparable experiments is shown.
The Journal of Immunology
mice display two distinct phenotypes. LMP2A transgenic mice
expressing higher levels of LMP2A in the bone marrow, such as
TgE, show a dramatic phenotype characterized by the development
of B cells that lack expression of IgM (10). LMP2A transgenic
mice expressing lower levels of LMP2A in the bone marrow, such
as Tg6, exhibit fairly normal B cell differentiation (10). In this
study, we have shown that high expression of LMP2A, as observed
in TgE B cells, correlates with the selective differentiation toward
a B-1-like lineage, whereas low levels of LMP2A expression, as
observed in Tg6 B cells, do not show an increase in skewing toward B-1 differentiation.
The LMP2A signal in many ways mimics a normal BCR signal.
Our previous studies have shown that LMP2A self-aggregates in
lipid rafts in the plasma membrane and constitutively associates
with Src family protein tyrosine kinases and the Syk tyrosine kinase, using specific phosphotyrosine motifs found in the LMP2A
amino terminus (7, 8). In addition, LMP2A, like the BCR, requires
Btk and BLNK, indicating similar cellular requirements for both
BCR and LMP2A signaling (16, 39). Indeed, the results obtained
in the ⌬ITAM-LMP2A mice reveal that Syk and its downstream
pathway are also essential for the LMP2A-mediated B-1 phenotype. However, unlike the signals thought to stem from a developing BCR, those stemming from LMP2A are thought to be constitutive, resulting in B cells that are chronically stimulated.
Commitment of cells into the B-1 lineage in the bone marrow has
been suggested from several lines of study, and our current study
provides further evidence that this can occur. In VH12 mice, bone
marrow, a large portion of VH12 Ig⫹ B cells, are CD5⫹ and defined as circulating mature cells (32). This CD5⫹ population may
contain some portion of pre-B or immature stage of B-1 cells. In
another example, VH11/Vk9 bone marrow reconstitutes splenic
B-1 population in irradiated mice (33). In addition, CD5⫹ cells
emerge from in vitro culture of VH11/Vk9 bone marrow cells in the
presence of IL-7 (33). These studies suggest that bone marrow
cells expressing B-1-specific Ig have a potential to progress into
the B-1 subset. Our data further support the finding that bone marrow B cells can express CD5 and commit into the B-1 lineage at
the pre-B stage as the result of LMP2A expression.
Commonly, it has been thought that B-1 lineage commitment
occurs at a transitional stage in the periphery (40, 41). Therefore,
the B-1 commitment at the pre-B stage shown in our study is
earlier than generally thought. The LMP2A transgene is cloned
downstream of E␮. In ␮-chain expression, ␮0 transcripts are detected at the early pro-B stage before productive rearrangement
(42). The rearranged ␮-chain is complexed with surrogate light
chain as the pre-BCR at the clonal proliferating pre-B stage. It is
likely that expression of LMP2A protein also starts at the early
pro-B stage, like other transgenes regulated by E␮. Therefore, it is
likely that LMP2A can substitute for the function of pre-BCR at
the pre-B stage.
Can the B-1 lineage commitment observed in TgE mice be generalized into B-1 differentiation mediated through normal developmental signals? During normal B cell development, it is thought
that the pre-BCR signals as soon as it is expressed on the cell
surface because pre-BCR-expressing cells are rarely isolated from
the bone marrow (43). Thus, it is commonly believed that an assembled functional pre-BCR is all that is required for pre-BCR
signaling and that specific Ags are not necessary. If the ␮-chain
fails to assemble with surrogate light chain, pro-B cells are not
positively selected to progress into pre-B cells (44). Despite this,
the majority of B cells in the bone marrow of TgE mice display a
B-1 phenotype, whereas most B-1-specific Ig transgenic mice
demonstrate no B-1 commitment within the bone marrow. This
difference can be explained in several ways. First, it is possible that
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adult bone marrow is generally limited to the generation of B-2
cells (28, 29). Progenitors for B-1 cells are selectively lost when
neonatal B cells are depleted by treatment with anti-IgM, whereas
B-2 recovery begins as soon as the treatment Ab disappears (30).
Thus, the lineage model suggests that B-1 cells arise from progenitor cells found in the fetal liver and are maintained long-term by
self-replenishment in the peritoneal cavity. The first indication that
B-1 cells might arise from differentiation of a common B cell progenitor cell arose from experiments in which continuous BCR
cross-linking in the absence of T cell help caused splenic B-2 cells
to develop into B-1-like CD5⫹ cells as a consequence of thymusindependent type 2 Ag-like activation (31). Recent Ig-transgenic
studies support such a differentiation model and have indicated the
importance of particular BCR specificity in generating B-1 or B-2
cells. In B-1-specific Ig transgenic mice (VH12 (anti-PtC), VH11
(anti-PtC), and VH3609 (anti-Thy-1)), transgene-expressing B
cells are predominantly the B-1 subset in spleen and peritoneal
cavity (32–34). In contrast, B cells are limited to the B-2 subset in
B-2-specific Ig transgenic mice (3-83 (anti-H-2k/H-2d) and HyHEL10 (anti-hen egg lysozyme)) (33, 35). Importantly, VH3609
mice reveal that the generation of B-1 cells is dependent upon Ag
association with the BCR. Introduction of the VH3609 transgene
results in an increase in CD5⫹ B cells reactive with T cell glycoprotein Thy-1 (34). However, when VH3609 mice are crossed onto
a Thy-1⫺/⫺ background, anti-Thy-1 Ab-producing B cells and
transgene-expressing B-1 cells are absent in the periphery (36).
Thus, engagement of the BCR by Ag, in this case Thy-1, is necessary for B-1 development.
Inducible disruption of the BCR complex in mature B cells results in the absence of all mature B cell subsets, revealing that
BCR signaling is required for both B-1 and B-2 B cell differentiation (37). In mice lacking key intracellular signaling intermediates (i.e., Bruton’s tyrosine kinase (Btk), B cell linker protein
(BLNK), Vav, phospholipase C ␥2, phosphatidylinositol 3-kinase
p85␣, protein kinase C ␤, and CD19), B-1 cells are dramatically
reduced in the peritoneal cavity (12, 13). Conversely, the loss of
inhibitory regulators, such as Src homology 2-containing protein
tyrosine phosphatase-1, CD22, and CD72, is associated with increased numbers of peritoneal B-1 cells (12, 13). Interestingly,
changes of B-1 cell levels are commonly correlated in splenic and
peritoneal B cells in these knockout/transgenic mice. These studies
lead to a model in which a stronger BCR signal is required for the
generation of peritoneal B-1 cells. Indeed, the observations that
mice bearing B-1 cells engineered to respond to specific Ags develop B-2 cells when there are defects in normal BCR-mediated
signal transduction or when there is a lack of the Ig-specific Ag
indicate that the quality of the signal through the BCR drastically
affects B cell development. In VH12/xid mice, deficiency of Btk
blocks the increase of B-1 cells that is observed in the wild-type
background VH12 mice (38). VH12/Vk4/xid mice develop B-2
cells despite the expression of B-1-specific VH12/Vk4 Ig (38). In
addition, selective B-2 B cell generation is found in VH3609/Thy1⫺/⫺ mice that lack expression of specific Thy-1 Ag for VH3609
Ig (36). These studies clearly indicate that signals with appropriate
strength and timing are required for B-1 lineage commitment.
Taken together, these data suggest that an intermediate BCR signal
favors B-2 differentiation, whereas a stronger signal favors B-1
cell differentiation.
Because there is a lack of animal models for EBV infection and
latency, transgenic models have been indispensable to clarify the
role of specific viral proteins in viral infection and subsequent
latency. To investigate the involvement of LMP2A in B-1 cell
development, three representative lines of LMP2A transgenic mice
were used. Previous studies have shown that LMP2A transgenic
5335
5336
with the generation of CLL-related malignancies via skewing toward the B-1 lineage during development.
In conclusion, our current studies using LMP2A transgenic mice
show that B-1 lineage commitment can occur in bone marrow B
cell progenitors. These findings imply that the quantity and quality
of BCR-like signals are what drive the B-1, B-2 lineage commitment decision. Furthermore, our results may shed light on a novel
association between EBV and some human cancers displaying a
B-1 lineage phenotype.
Acknowledgments
We thank the members of the Longnecker laboratory for providing advice
and help, Patrick Dennis for maintaining and providing Tg⌬ITAMLMP2A mice, and Michelle Swanson-Mungerson for providing invaluable
assistance for bone marrow cell culture and proliferation assay.
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LMP2A mediates a stronger signal than does the BCR. Second,
LMP2A competes with the BCR and blocks normal BCR signaling
(45). Our in vivo studies indicate that LMP2A provides a constitutive BCR-like signal (6, 46). Thus, in TgE mice, a strong
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similarity of cellular factors that are required downstream of
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(11), BLNK (39), and Btk (16). However, we cannot formally
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development in a manner that is independent of signals that mimic
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LMP2A to mediate a stronger signal for the B-1 development.
EBV is a ubiquitous human oncogenic herpesvirus associated
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known associations, there has been an interest in determining
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may be one such example. The surface expression of CLL cells is
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believed to be the transformed counterparts of normal B-1 cells.
Transformation into high-grade large cell non-Hodgkin’s lymphoma (NHL), designated Richter’s syndrome, occurs in ⬃3–10%
of CLL patients (49, 50). In most cases, the high-grade component
is considered to represent a blastic transformation of the CLL cells
(51). However, in rare cases, the high-grade component includes
characteristics of Hodgkin’s disease (52). Although EBV has not
been detected in CLL cells, 16% of NHL (53) and 92% of Hodgkin
and Reed-Sternberg (H-RS)-like cells (54) are positive for EBV,
suggesting that EBV may be involved in the high-grade transformation of CLL. However, it is not known whether the H-RS cells
arise from transformation of the underlying CLL cells or from a
different pathological process. Nevertheless, in a few of these
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data reveal that LMP2A of EBV can skew B cells toward the
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related diseases.
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populations in the tonsils where infectious virus is produced, EBV
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viral genome results in virus that can still infect human B cells
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