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Division of Biology and University of California, San Diego Cancer Center, University of California, San Diego, La Jolla, CA 92093
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the BCR is the focal receptor for B cell differentiation, it
also integrates signals provided by the coreceptor molecules (e.g.,
CD19, CD21, CD22, CD72, and Fc
RIIB). By modulating Ag recognition
and signal transduction through the BCR, the B cell coreceptors provide
information as to the nature of the Ag as well as the context in which
the Ag is encountered. Of particular note in this capacity is the B
cell-restricted cell surface protein CD19. CD19 is a 95-kDa
transmembrane protein bearing two extracellular Ig domains and an
extensive cytoplasmic tail (4, 5). Onset of CD19
expression occurs at the earliest stages of B cell development before
the expression of the pre-BCR. On mature B cells, CD19 is found in a
complex with the complement receptor (CD21), the tetraspan membrane
protein TAPA-1 CD81 (CD81), and Leu13 (6, 7).
Moreover, CD19 associates with members of the BCR complex and is
rapidly phosphorylated following BCR cross-linking
(8, 9, 10). CD19 coligation with surface Ig (sIg) greatly
augments B cell activation (11), presumably through the
enhanced recruitment or activation of associated kinases and adaptor
molecules. Thus, CD19 behaves as an essential downstream element of BCR
signaling and is thought to functionally link CD21 with the BCR
following corecognition of C3d-bearing Ags (12).
The importance of CD19 in the development and function of B cells was demonstrated through the targeted deletion of the CD19 gene (13, 14). Mice rendered CD19 deficient present multiple B cell defects, including decreased B cell numbers in the periphery as well as a severe reduction in B-1 cells. CD19-/- mice are also functionally impaired in responding to T cell-dependent Ags as evidenced by a sharp reduction in primary and secondary Ab responses and a lack of germinal center (GC) formation. Recently, it has been shown that CD19 is essential for the generation of marginal zone (MZ) B cells (15, 16). In this study, we provide strong evidence that modulation of cell survival is a key property of CD19 function in vivo and accounts for some of the phenotypes observed in CD19-/- mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD19-/- mice on BALB/c (IgHb) (The Jackson Laboratory, Bar Harbor, ME) background (10-generation backcross) were maintained under pathogen-free conditions and handled in accordance with the guidelines set forth by the Animal Subjects Program at the University of California, San Diego. BALB/c (IgHb)-congenic mice were used as wild-type controls and BALB/c (IgHa) wild-type mice were used as recipients for adoptive transfer experiments. Bcl-2 mice (Eµ-bcl-2-22) were purchased from The Jackson Laboratory.
Cell culture
Splenic B cells were purified by depletion of adherent cells bound to plastic and lysis of T cells using anti-CD4-, anti-CD8-, and anti-Thy1.2-specific Abs and rabbit complement (Cedarlane Laboratories, Westbury, NY). Cells were cultured at 1 x 106 cells/ml in RPMI 1640 with 10% FCS, penicillin/streptomycin, L-glutamine, nonessential amino acids, sodium pyruvate (Cellgro; Mediatech, Hernden, VA) and 2-ME (Life Technologies, Rockville, MD). Cells were stimulated with anti-IgM F(ab')2 or anti-IgG F(ab')2 (Jackson ImmunoResearch, West Grove, PA), and apoptosis was measured by flow cytometry using propidium iodide (PI). Apoptotic cells were identified by a sub-G0-G1 peak after PI staining in 1 mM Tris, 0.1% Triton X-100, 0.1% sodium citrate, 0.1 mM EDTA, and 50 µg/ml PI (pH 8).
Flow cytometry
After harvesting organs, single-cell suspensions were prepared and RBCs were lysed with ACK buffer (0.15 M NH4Cl, 1 mM KMCO2, 0.1 mM Ng2 EDTA, pH 7.4). Cells (1 x 106) were stained for 15 min on ice with diluted Ab, washed with PBS containing 1% FCS, and incubated with streptavidin-conjugated fluorochromes when necessary. Abs against the following surface markers were obtained from BD PharMingen (San Diego, CA): CD24 (heat stable Ag, HSA)-FITC, CD24(HSA)-biotin (bio), IgD-FITC, IgM-PE, IgM-bio, CD19-PE, B220-allophycocyanin, B220-PE, CD5-bio, GL7-FITC, bromodeoxyuridine (BrdU)-FITC, and streptavidin-allophycocyanin. Anti-IgD bio was purchased from Southern Biotechnology Associates (Birmingham, AL) and anti-B220 TriC was obtained from Caltag Laboratories (Burlingame, CA).
Adoptive transfer
Recipient mice (BALB/c/IgHa) were lethally irradiated (1000 rad) and administered antibiotics (neomycin, 1 mg/ml, and polymixin B, 0.1 mg/ml; Life Technologies) in the drinking water postreconstitution. Bone marrow cells from wild-type and CD19-/- mice were depleted of B cells using Mini-MACS columns (Miltenyi Biotec, Auburn, CA) and anti-B220-conjugated magnetic beads. B cell-depleted bone marrow cells (1 x 107) were injected into the lateral tail vein of irradiated recipients. To generate bone marrow chimeras, B cell-depleted bone marrow from age-matched wild-type and CD19-/- mice were mixed before injection. Chimeric mice were analyzed 8 wk postirradiation.
Splenic/lymph node B cell transfer
Wild-type and CD19-/- B cells from age-matched mice were purified, admixed at a 1:1 ratio, labeled with 5 µM CFSE (Molecular Probes, Eugene, OR) for 10 min, washed, and injected into the tail vein of wild-type BALB/c mice. Organs were harvested at 5, 18, or 35 days posttransfer and analyzed by flow cytometry.
Autoreconstitution
Wild-type BALB/c, CD19-/- mice, Bcl-2-transgenic, and CD19-/-/Bcl-2-transgenic mice were sublethally (500 rad) irradiated and allowed to reconstitute for 12–14 days before flow cytometric analysis of bone marrow and spleen cell populations.
BrdU treatment
Age-matched wild-type BALB/c and CD19-/- mice were administered BrdU in their drinking water (1 mg/ml, changed twice weekly) for up to 12 wk. Mice were sacrificed following 6, 9, or 12 wk of labeling and analyzed by flow cytometry for the presence of BrdU-positive B cells. Student’s t test was used to determine the level of significance for differences between sample means of wild-type and CD19-/- mice at each time point.
Immunohistology
Spleens from CD19+/-, Bcl-2, CD19-/-, and CD19-/-/Bcl-2 mice were frozen in OCT compound above liquid nitrogen and stored at -90°C. Ten-micrometer spleen sections were fixed in acetone and allowed to dry. Sections were blocked with 5% FBS in PBS and incubated with the primary Ab for 1 h or overnight. Secondary reagents included anti-rat Ig FITC or streptavidin-Cy3 (Zymed Laboratories, San Francisco, CA). The MOMA-1 Ab was purchased from Bachem (King of Prussia, PA) and peanut agglutinin (PNA)-FITC from Vector Laboratories (Burlingame, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Early in life the composition of the B cell compartment is
dynamic, whereas the majority of recirculating B cells in adult animals
are relatively long-lived and not rapidly replenished by newly formed
cells from the bone marrow (17). We now show that CD19
contributes to the maintenance of the peripheral B cell pool. This
notion was initially suggested by the finding that B cell numbers were
reduced in adult CD19-/- mice
(13). This deficiency is more striking early in ontogeny
where we observe a 3- to 4-fold reduction in B cells from neonatal
spleens (Fig. 1
a). At this
stage of ontogeny, B cells bearing the B-1 cell phenotype
(IgMhigh, CD5+(B1a
subtype), CD23-) are well represented in
wild-type mice, but reduced in CD19-/- mice.
Interestingly, however, the reduction in B cell numbers we observed in
neonatal CD19-/- mice is not restricted to B
cells bearing the B-1 cell phenotype, but also applies to conventional
(B-2, CD23+) cells and thus indicates a general
impairment in B cell lymphopoiesis.
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b), suggesting that there is an increase
in the relative number of
IgMhighIgDhigh
(transitional-2) B cells in CD19-/- mice. To
determine whether CD19-/- mice exhibit a
developmental block in peripheral B cell maturation, expression of
additional mature B cell surface markers was determined (Fig. 1b). A similar number of CD19-/-
splenic B cells expressed the surface marker 493, which has been
associated with immature and transitional B cells (20, 21). In addition, CD19-/- and wild-type
B cells expressed similar levels of L-selectin, MHCII, and HSA. These
data suggest that B cells from CD19-/- mice
undergo normal maturation, but may express higher levels of sIgM as an
outcome of selection. Because compensatory effects may obscure the
severity of the CD19 defect, we chose to directly evaluate B cell
formation in chimeric mice bearing mixed populations of
CD45R/B220-depleted bone marrow cells from adult
CD19-/- and wild-type mice. Animals were
lethally irradiated and reconstituted with bone marrow from BALB/c or
BALB/c-congenic CD19-/- mice or combinations
thereof. The B cell compartment of recipient mice was analyzed 8 wk
postreconstitution to guarantee recovery of the peripheral immune
system. Fig. 1c shows representative flow cytometric data of
cells from the bone marrow, spleen, lymph node, and peritoneal cavity
of mice reconstituted with 50:50, 80:20, or 20:80 ratios of
CD19-/- and wild-type bone marrow cells. In all
organs, we observe a greater presence of CD19-positive B cells in
excess of the 50:50 ratio at which they were injected. This
trend is first noted at the immature sIgM-positive stage in the
bone marrow and continues in the periphery where the majority of
IgD-positive mature B cells are also CD19 positive. Thus, these data
illustrate a clear advantage for CD19-expressing B cells in the
formation and maintenance of peripheral B cell subsets.
In contrast to the transfer of fetal liver cells, transfer of bone
marrow cells does not lead to efficient reconstitution of B-1 cells
(22). In our bone marrow reconstitution experiments,
CD19-positive
IgMhighIgDhigh and
IgDlowIgMhigh cells are
present in the peritoneal cavity (Fig. 1b and data not
shown). This latter population bears the phenotype of immature or
transitional B cells (IgMhigh,
CD23neg, IgDlow) that is
also shared by B-1b cells; however, it is unlikely to represent B-1
cells as it is uniformly CD5 negative (data not shown).
CD19-/- IgDneg B cells
were greatly underrepresented in the peritoneal cavity of all recipient
mice (Fig. 1c). The severity of this defect may indicate an
inability of newly generated CD19-/- B cells to
enter or survive in the peritoneal cavity.
CD19-/- B cells exhibit increased turnover and reduced survival
The inability of CD19-/- B cells to
effectively compete with wild-type B cells suggests a role for CD19 in
promoting cell survival. To address this issue, we used long-term
BrdU-labeling procedures and flow cytometric analysis. Mice were
administered BrdU continuously in their drinking water for 6, 9, or 12
wk. Thus, all unlabeled peripheral B cells were generated before
administering BrdU. A representative labeling profile is shown in Fig. 2a where, following 9 wk of
labeling, all developing B cells in the bone marrow and transitional B
cells in the spleen are labeled, but a fraction of recirculating and
follicular B cells remain unlabeled. Comparing wild-type and
CD19-/- mice after 6 or 9 wk of labeling, we
detected a significantly higher percentage of labeled mature B cells
(B220high, HSAlow) in the
bone marrow, spleen, and lymph nodes of CD19-/-
mice (Fig. 2b). At 12 wk of BrdU labeling, the percentage of
labeled cells in CD19-/- and wild-type mice
appeared to normalize; which may reflect a countereffect of reduced B
cell generation in CD19-/- mice. Overall, the
differential labeling kinetics indicated that there was a relatively
smaller fraction of CD19-/- B cells whose
survival spanned the labeling period. Using 50% labeling as a relative
index of B cell longevity, it was evident that
CD19-/- B cells turn over more rapidly than
their wild-type counterparts (5.5 vs 8 wk).
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c). In this study, we observe a dramatic
loss of CD19-/- B cells in the spleen and blood
by day 18 posttransfer and very few CD19-/- B
cells in the peritoneal cavity. This effect is due to reduced survival
rather than reduced proliferation of CD19-/- B
cells, since the vast majority of transferred cells retain uniform
high-intensity CFSE labeling regardless of CD19 expression (Fig. 2d). The lymph node and bone marrow showed less of a
disparity in recovery of donor B cells at the selected time points.
This distinction could reflect the heterogenous nature of the
transferred splenic B cells from wild-type animals, which is also
distinct from CD19-/- animals insofar as they
lack MZ B cells. Since the lymph node contains a more homogeneous
population of mature B cells, we repeated the cotransfer experiment
using lymph node B cells and extended the analysis to 35 days (Fig. 2c). Here, we observed a dramatic reduction of
CD19-/- B cells in the spleen, bone marrow, and
lymph nodes of recipient mice. Thus, these results are consistent with
the BrdU-labeling experiments and together suggest that CD19-dependent
survival is an intrinsic property of mature recirculating B cells. BCR-induced cell survival is CD19 dependent
To understand the basis of CD19-mediated B cell survival, we
assessed the in vitro growth properties of purified splenic B cells
from CD19-/- and wild-type mice. Unlike the in
vivo situation, where recirculating B cells are exposed to a milieu of
cognate and soluble ligands that act in part to promote longevity, B
cells are short-lived in culture unless supplied with growth factors or
mitogens. CD19-/- and wild-type B cells display
similar survival properties in the absence of exogenous stimuli that
protect cells from apoptosis (Fig. 3a). Because B cell survival
in vivo is dependent upon continued expression of the BCR
(3), we investigated whether such a signal was in turn
dependent upon downstream signaling by CD19. Fig. 3b shows a
dose-response curve using agonistic anti-IgM
F(ab')2 with CD19-deficient and wild-type splenic
B cells and measuring cell cycle progression and survival 36 h
after initiation of culture. CD19-/- B cells
show reduced proliferation in response to anti-IgM stimulation
(Fig. 3b), as reported (13). However, BCR
stimulation promotes survival of splenic B cells, even at levels
suboptimal for mitogenic stimulation (Fig. 3b). Strikingly,
although CD19-/- and wild-type B cells show
similar rates of spontaneous apoptosis in the absence of sIg
cross-linking or the presence of an irrelevant Ab, BCR-mediated rescue
from apoptosis is at least 10-fold more effective in the presence of
CD19. Thus, cell cycle analysis (Fig. 3b) and thymidine
incorporation studies (data not shown) support the hypothesis that the
relative reduction in viable CD19-/- cells at
low levels of BCR stimulation is mainly attributed to increased cell
death and not reduced proliferation. The BCR-mediated rescue we
observed is likely indicative of the low-level tonic signaling through
the BCR that is required for the maintenance of the mature B cell pool
and suggests that in the absence of CD19 higher levels of BCR
cross-linking are necessary to promote cell survival.
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Surface Ig engagement on mature B cells up-regulates expression of
the Bcl-2 homologues Bcl-XL and A1 (23, 24),
whereas CD19 engagement has been shown to selectively up-regulate Bcl-2
expression (25). In vitro, constitutive expression of
Bcl-2 is sufficient to prevent spontaneous apoptosis of
CD19-/- and wild-type B cells (Fig. 3a). Therefore, we sought to determine whether promotion of
B cell survival by ectopic overexpression of Bcl-2 was sufficient to
rescue the developmental and functional defects associated with CD19
deficiency, or whether these defects were intrinsic to the lack of
CD19-induced signaling events distinct from survival. We show that B
cell-specific expression of a Bcl-2 transgene leads to
increased formation of sIgMhigh cells in the bone
marrow of both wild-type and CD19-/- animals
(Fig. 4a). In wild-type
animals, these immature/transitional B cells are particularly sensitive
to negative selection and express low levels of Bcl-2 and
Bcl-xL and A1 (26, 27). Hence,
egress from the marrow to the periphery is normally accompanied by a
dramatic loss in B cell numbers (28), which can be
attenuated through increased expression of Bcl-2. Curiously, a
significant proportion of the sIgMhigh cells in
the bone marrow of Bcl-2-transgenic mice coexpress high levels of sIgD,
a population that is thought to be restricted to the spleen
(29). To determine whether these cells are newly generated
or recirculating B cells, we performed a kinetic analysis of B cell
differentiation following sublethal irradiation (Fig. 4b).
After 14 days of autoreconstitution, the majority of splenic B cells in
CD19+/- and CD19-/- mice
bear the phenotype of transitional B cells
(IgMhigh, IgDlow,
HSAhigh; Figs. 4b and
5 and data not shown). However, in the
presence of forced Bcl-2 expression, a large population of mature B
cells (IgMpos, HSAlow) is
present in CD19+/- mice and, to a much lesser
extent, CD19-/- mice (Figs. 4b and
5). This population is also present 2 days postirradiation, suggesting
that they represent radioresistant cells present before treatment
(Figs. 4b and 5). Interestingly, radioresistance appears to
require both elevated levels of Bcl-2 and progression to the mature B
cell stage, since immature/transitional B cells overexpressing Bcl-2
remain sensitive to this level of irradiation. Altogether, these
findings indicate that the reduced numbers of peripheral B cells in
CD19-/- mice cannot be wholly explained by a
failure to up-regulate Bcl-2 at the immature-mature B cell
transition.
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a, summarized in Fig. 5), such that B cell numbers in
the spleen of adult CD19-/- animals approximate
that of heterozygous counterparts. However, cell numbers did not reach
the levels seen in Bcl-2-transgenic mice that retain CD19 expression.
This partial rescue is also selective for B-2 cells since B-1 cells are
not restored in the peritoneal cavity of Bcl-2-transgenic mice on the
CD19-/- background (Fig. 4a). To
determine whether the Bcl-2-mediated increase in B-2 cells is
accompanied by a recovery of B cell function in
CD19-/- mice, immunizations were performed with
SRBCs (Fig. 6) and the T
cell-dependent Ag, DNP-keyhole limpet hemocyanin (data not
shown). If CD19-/- B cells are sufficiently
activated but do not survive within the GC environment, then we may
expect to see a rescue of GC formation on the Bcl-2-transgenic
background. Examination of frozen spleen sections 7 days
postimmunization revealed a high incidence of well-formed GC in
wild-type and Bcl-2-transgenic mice (Fig. 6); however, provision of
Bcl-2 was not sufficient to rescue GC B cell differentiation in the
absence of CD19. This finding suggests that CD19 is necessary early in
the recognition of and/or response to protein Ags leading to the
generation of GC B cells. Overall, these findings suggest that ectopic
expression of Bcl-2 can compensate for CD19-associated defects in the
maintenance of recirculating follicular B cells, but cannot substitute
for Ag-induced differentiation signals.
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MZ B cells are key participants in the capture of and response to
blood-borne Ags in the spleen (30). This B cell subset
appears late in ontogeny and may represent an Ag-selected population
primed to respond to further antigenic challenge (15, 31).
The size of the MZ B cell population shows some strain variation;
however, CD19-/- mice on the BALB/c, 129, or
mixed C57BL/6/129 background all present a deficiency in MZ B cells
(Refs. 15, 16 and data not shown). To determine whether
this deficiency results from impaired survival of cells destined for
the MZ B cell compartment, we performed immunohistochemical staining
for IgM and IgD in spleens of wild-type, Bcl-2, and
Bcl-2/CD19-/- mice. Remarkably, overexpression
of Bcl-2 on the CD19-/- background led to a
recovery of the MZ B cell population as evidenced by the formation of
an outer ring of
IgMposIgDneg cells adjacent
to the marginal sinus (Fig. 7a). These findings were
corroborated by immunohistochemical staining for metallophilic
macrophages and B cells using Abs against MOMA-1 and CD45R/B220,
respectively (data not shown), and resolution of the MZ B cell
population (CD21high,
CD1dhigh) by flow cytometry (Fig. 7b).
Electronic gating on the
CD21highCD1dh population
shows that the vast majority of the cells are also CD23 negative, which
is consistent with their designation as MZ B cells and distinct from
CD23-positive follicular B cells (Fig. 7b). These data
suggest that either selection into or maintenance in the MZ B cell
compartment is dependent upon a CD19-mediated survival signal. This
trait is in common with follicular B cell homeostasis, but distinct
from CD19-dependent generation of B-1 and GC B cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD19 acts as a downstream effector for sIg and CD21 (CR2) (33). Thus, B cell recognition of internal and foreign Ags alike will result in the recruitment and phosphorylation of CD19, resulting in augmented B cell activation. This augmentation is apparently necessary for B cells to respond to paucivalent protein Ags, but is dispensable for responding to most, but not all, T cell-independent Ags (13, 14, 34, 35, 36). To date, the role of CD19 in B cell generation and maintenance has not received significant attention, despite the observation that CD19-/- mice show a reduction in peripheral B cell numbers (13). Therefore, to assess the contribution of CD19 to the generation and maintenance of mature B cells, we directly compared the ability of CD19-/- and wild-type B lineage cells to differentiate and survive in an irradiated syngeneic host. In this competitive situation, CD19-/- B cells exhibited a clear disadvantage in contributing to the peripheral B cell pool. The inability of CD19-/- B cells to compete effectively with wild-type B cells could be explained in part by a shorter intrinsic life span. This hypothesis is supported by long-term BrdU-labeling studies where we demonstrated a much higher rate of turnover for mature (B220high, HSAlow) CD19-/- B cells than their wild-type counterparts. However, after 12 wk of BrdU administration, the percentage of labeled CD19-/- B cells was similar to that of wild type. This finding suggests that another parameter, such as reduced generation of CD19-/- B cells, may contribute significantly as the labeling period persists and gradually leads to a reduced number of B cells entering the periphery. Therefore, we corroborated these studies with direct labeling and cotransfer of wild-type and CD19-/- B cells. Here, we observe a selective and rapid loss of CD19-/- B cells from all lymphoid tissues. Thus, we document a novel role for CD19 in promoting B cell survival in vivo. Such a role may underlie the therapeutic basis of targeted B cell ablation studies using anti-CD19/genistein conjugate Abs which mediate specific inhibition of CD19-associated tyrosine kinase activity and result in the directed apoptosis of human B cell leukemias and lymphomas (37).
Because some level of BCR-mediated signaling is required for the continued survival of naive and memory B cells in the absence of overt Ag (3, 38), we reasoned that CD19 may be necessary to propagate this survival signal. Support for this idea was provided by the demonstration that CD19-/- B cells are indeed impaired in BCR-mediated rescue from spontaneous apoptosis in vitro. This effect occurs at levels of anti-IgM stimulation that does not induce significant cell cycle progression, and thus should also be considered in the context of reported defects in proliferation (13) that are ameliorated by costimulation with survival factors such as IL-4 (14). In vivo, it would be expected that CD19-/- B cells are hyporesponsive to tonic signaling through the BCR, thus precipitating their rapid loss in the absence of compensatory up-regulation of sIg levels, as observed (18, 19), or perhaps increased receptor affinity for some internal ligands, as implicated by the recent work of Shih et al. (36).
Protection from apoptosis is often achieved by the specific induction of pro-survival factors of the Bcl-2 family. Indeed, Roberts and Snow (25) have shown that signaling through CD19 may selectively augment the accumulation of Bcl-2 in resting B cells. Therefore, we determined whether forced expression of Bcl-2 could lead to a rescue of survival-related deficiencies in CD19-/- mice. Normally, Bcl-2 expression is tightly regulated during B cell development with high levels found in pro-B cells and mature B cells, but low levels in pre-B cells and immature B cells (27, 39). This expression pattern is distinct from Bcl-xL and A1, which are up-regulated upon (pre-) BCR engagement or induced during the immature/mature B cell transition, respectively (23, 24). Apart from expression profiles, it is unclear whether these Bcl-2 homologues possess properties distinct from Bcl-2. Mice that overexpress Bcl-2 in the B lineage have an overabundance of B cells and serum Ab, develop autoimmune disease, and have increased GC formation and memory B cell generation (40, 41). We find that provision of Bcl-2 leads to a partial rescue in the number of follicular B cells in CD19-/- mice, suggesting compensation for a survival factor(s) normally induced in the presence of CD19. These findings are consistent with the results of Lam et al. (3), who showed that BCR-dependent survival of mature recirculating B cells can be partially compensated by overexpression of Bcl-2 (3) and, along with our in vitro studies, suggests that B cell persistence in vivo requires CD19 to efficiently propagate tonic survival signals induced by the BCR.
In addition to basal signaling through the BCR complex, successful
passage through a given checkpoint in B cell differentiation requires
signals through the (pre-) BCR and coreceptor molecules that are of
sufficient strength and quality to induce cell cycle progression and
differentiation. Recent evidence from gene-targeted and Ig-transgenic
mice suggests that MZ B cells also represent an Ag-selected population
(42). Selection into the MZ B cell compartment is strictly
dependent on Pyk2 and CD19 function (15, 16, 43), whereas
Btk appears to play an ancillary role that may be modulated by CD19
signaling (15, 44, 45, 46). Although responses of MZ B cells
to T cell-independent Ags is facilitated by complement activation and
opsonization (43, 47), MZ B cell formation is not impaired
in the absence of B cell complement receptors (CD21/CD35) or C3
(43, 45). Thus, the requirement for CD19 in MZ B cell
generation is by association with sIg or an unknown ligand.
Overexpression of Bcl-2 on the CD19-/-
background results in a selective recovery of the MZ B cell subset,
suggesting that the precursors to MZ B cells may be present in
CD19-/- mice but are not activated sufficiently
to up-regulate survival factors such as Bcl-2 and thus fail to further
differentiate and colonize the MZ. Alternatively, it has been noted
recently that, similar to B-1 cells, MZ B cells are particularly
long-lived and less reliant upon the bone marrow for replenishment in
the adult animal (48, 49). Thus, this nonrecirculating
population may not be sustained in the absence of CD19. Downstream
effectors of CD19 signaling that may account for this function include
members of the NF-
B family that are known to be required for MZ B
cell formation (50, 51, 52, 53), perhaps through the activation of
target genes such as Bcl-xL and A1, in addition
to Bcl-2. Alternatively, we have shown that CD19 promotes activation of
the pro-survival kinase Akt (54), suggesting the
involvement of a phosphatidylinositol 3-kinase-dependent
survival pathway. Characterization of such regulators of MZ B cell
survival awaits further investigation.
The data presented here underscore CD19 as an important regulator of cell fate decisions by virtue of promoting cell survival. Nonetheless, it would be inaccurate to classify CD19 simply as a pro-survival molecule. In point of fact, we show that while overexpression of Bcl-2 selectively rescues MZ B cell formation and promotes B cell homeostasis, it does not rescue GC formation or the B-1 B cell population. In the case of B-1 cells, subthreshold signaling through the BCR in the absence of CD19 may preclude commitment to the B-1 cell pathway. This differentiative signal cannot be circumvented simply by increasing cell survival. The CD19-CD21 coreceptor complex also regulates commitment to the GC B cell pathway (33). GC formation is a complement-dependent process requiring cytokines and cognate interactions with CD4 T cells (55). CD19 likely participates in this process by augmenting signals induced by corecognition of Ag by sIg and CD21. In this context, recent work from Pierce and colleagues (56, 57) suggests that CD19/CD21 may be required both for sustained signaling by the BCR and for efficient directed processing of Ags for presentation to T cells. CD19 has also been functionally associated with CD40 (13, 58), which may help explain the absence of GCs in CD19-/- mice. Thus, the role of CD19 in B cell biology is complex and multifaceted, likely depending upon the stage of B cell differentiation and the nature of the Ag encountered, much like the BCR itself.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert C. Rickert, Division of Biology and University of California, San Diego Cancer Center, University of California, San Diego, 9500 Gilman Drive, MC 0322. La Jolla, CA 92093-0322. E-mail address: rrickert{at}ucsd.edu
3 Abbreviations used in this paper: BCR, B cell receptor; MZ, marginal zone; BrdU, bromodeoxyuridine; sIg, surface Ig; GC, germinal center; PNA, peanut agglutinin; PI, propidium iodide; HSA, heat-stable Ag; bio, biotin.
Received for publication June 6, 2002. Accepted for publication October 23, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B is required for the development of marginal zone B lymphocytes. J. Exp. Med. 192:1175.B-inducing kinase. J. Immunol. 165:804.B/p52 present with defects in humoral responses, germinal center reactions, and splenic microarchitecture. J. Exp. Med. 187:147.This article has been cited by other articles:
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  C. D. Milne, S. A. Corfe, and C. J. Paige Heparan Sulfate and Heparin Enhance ERK Phosphorylation and Mediate preBCR-Dependent Events during B Lymphopoiesis J. Immunol., March 1, 2008; 180(5): 2839 - 2847. [Abstract] [Full Text] [PDF]
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M. L. Janas, D. Hodson, Z. Stamataki, S. Hill, K. Welch, L. Gambardella, L. C. Trotman, P. P. Pandolfi, E. Vigorito, and M. Turner The Effect of Deleting p110{delta} on the Phenotype and Function of PTEN-Deficient B Cells J. Immunol., January 15, 2008; 180(2): 739 - 746. [Abstract] [Full Text] [PDF]
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 M. Thomas, M. Calamito, B. Srivastava, I. Maillard, W. S. Pear, and D. Allman Notch activity synergizes with B-cell-receptor and CD40 signaling to enhance B-cell activation Blood, April 15, 2007; 109(8): 3342 - 3350. [Abstract] [Full Text] [PDF]
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R. C. Lindsley, M. Thomas, B. Srivastava, and D. Allman Generation of peripheral B cells occurs via two spatially and temporally distinct pathways Blood, March 15, 2007; 109(6): 2521 - 2528. [Abstract] [Full Text] [PDF]
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E. M. Fuentes-Panana, G. Bannish, F. G. Karnell, J. F. Treml, and J. G. Monroe Analysis of the Individual Contributions of Ig{alpha} (CD79a)- and Igbeta (CD79b)-Mediated Tonic Signaling for Bone Marrow B Cell Development and Peripheral B Cell Maturation J. Immunol., December 1, 2006; 177(11): 7913 - 7922. [Abstract] [Full Text] [PDF]
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 Y. Ma and A. C. Ross The anti-tetanus immune response of neonatal mice is augmented by retinoic acid combined with polyriboinosinic:polyribocytidylic acid PNAS, September 20, 2005; 102(38): 13556 - 13561. [Abstract] [Full Text] [PDF]
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E. Diamant, Z. Keren, and D. Melamed CD19 regulates positive selection and maturation in B lymphopoiesis: lack of CD19 imposes developmental arrest of immature B cells and consequential stimulation of receptor editing Blood, April 15, 2005; 105(8): 3247 - 3254. [Abstract] [Full Text] [PDF]
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 A. R. Ferguson, M. E. Youd, and R. B. Corley Marginal zone B cells transport and deposit IgM-containing immune complexes onto follicular dendritic cells Int. Immunol., October 1, 2004; 16(10): 1411 - 1422. [Abstract] [Full Text] [PDF]
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 Z. Keren, E. Diamant, O. Ostrovsky, E. Bengal, and D. Melamed Modification of Ligand-independent B Cell Receptor Tonic Signals Activates Receptor Editing in Immature B Lymphocytes J. Biol. Chem., April 2, 2004; 279(14): 13418 - 13424. [Abstract] [Full Text] [PDF]
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) and CD19-/- (•) mice were fed BrdU continuously
for 6, 9, or 12 wk (three mice per time point). Statistical
significance is indicated by displayed p values.
c, A 50:50 mixture of wild-type and
CD19-/- splenic or lymph node B cells was labeled with
CFSE and injected i.v. Bar graph (three mice per group) shows
percentage of CD19-positive CFSE-labeled cells recovered from various
organs 5 or 18 days after injection. BM, bone marrow; Spl, spleen; LN,
lymph node; PEC, peritoneal cavity; PBL, peripheral blood leukocytes.
d, Flow cytometric analysis of cells harvested from
spleen 18 days postinjection and showing largely uniform staining for
CFSE.
