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* Department of Immunology, Faculty of Medicine, Technion, and
Endocrine Research Unit, Carmel Medical Center, Haifa, Israel
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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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The CD19 molecule is a positive regulator of the BCR, which lowers the
signaling threshold required for B cell activation upon BCR
ligation (23, 24). Through highly conserved nine tyrosine
residues in its cytoplasmic domain, CD19 is thought to act as an
adapter protein that recruits Src homology-2 domain-containing
signaling molecules such as Lyn, Vav, phosphoinositol 3-kinase, and
phospholipase C
2 (reviewed in Ref. 25). Expression of
CD19 is also required for maximal BCR-mediated phosphotidylinositol
3-kinase activation and Ca2+ mobilization
(26), implicating that deficiency of CD19 results in
incompetent BCR (27). CD19 is expressed from the earliest
stages of B cell development and regulates pre-BCR signaling
(28) and recombinase gene expression in pro-B cells
(29). CD19-deficient mice have relatively normal B cell
development in the bone marrow (BM) (30, 31). However,
these mice have 50–60% reduction in peripheral B cells, which are
hyporesponsive to most transmembrane signals (30, 31).
Interestingly, CD19-deficient mice have a severe loss of B-1 B cells,
whereas CD19-overexpressing mice have increased B-1 cell compartment
(30, 31, 32). These data imply a critical role for CD19 in
balancing the BCR signaling threshold required for mature B cell
activation, while its function in promoting B cell development is still
not clear.
Signaling competence of the BCR is important to promote B cell
maturation, as revealed by developmental stage-dependent differences in
BCR signaling (33, 34), and in mice deficient of Btk,
SH2-domain-containing leukocyte protein (SLP)-65, and Lyn
(14, 16, 35). Impaired B maturation (36) and
failure to populate the marginal zone compartment in spleen
(37) are described in different Ig transgenic (Tg) mouse
models deficient of CD19. In a recent study Reichlin et al.
(13) used mice lacking a functional Ig
cytoplasmic
domain to show that immature B cells expressing signaling-incompetent
BCR are developmentally arrested. We have previously shown that
developmental arrest is also imposed in immature B cells encountering
self-Ag in the BM (38, 39). These cells however, elevate
V(D)J recombination machinery and undergo receptor editing to alter the
BCR specificity (39, 40). Therefore, it is possible that
developmentally arrested immature B cells expressing
signaling-incompetent receptors undergo secondary V(D)J recombination
to express new receptors. This hypothesis is tested in the present
study using the 3-83Tg mice (that manifest excellent allelic exclusion
in vivo and in vitro (41, 42)) deficient in CD19
(31). Our results propose that immature 3-83Tg B cells
expressing signaling-incompetent receptors, deficient in CD19, fail
positive selection and are developmentally arrested. These B cells
undergo intensive secondary L chain V(D)J recombination to edit their
BCR. Therefore, we suggest that CD19 regulates the BCR signaling
threshold required for the establishment of L chain allelic exclusion,
positive selection, and maturation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice used were 3-83Tg B10.D2nSn/J, encoding a BCR reactive to
the mouse class I MHC Ags Kk and
Kb (a gift from Dr. D. Nemazee, Scripps
Institute, La Jolla, CA (43)) or non-Tg littermates.
CD19-deficient mice were a gift from Dr. R. Rickert, University of
California (San Diego, CA) (31). The 3-83Tg
CD19-/- mice were generated and backcrossed
>10 generations on an H-2d-selected B10.D2 background to ensure the
absence of the 3-83 cognate Ag as revealed by both tissue staining and
analysis of B cell development. Mice were housed and bred at the animal
facility of Technion, Faculty of Medicine (Haifa, Israel), and used at
4–10 wk of age. For some experiments, homozygous 3-83Tg
CD19-/- (3-83TgHz
CD19-/-) mice were generated and typed by
genomic Southern blot (39). To do so, genomic DNA was
digested with HindIII, electrophoresed on agarose gel,
blotted to a 
probe membrane (Bio-Rad, Richmond, CA), hybridized
with a [32P]J
1–5 DNA probe, and subjected
to phosphor imager analysis (FUJIFILM FLA 2000; Fuji, Stamford, CT) for
quantitation. The ratio of transgenic fragment (at 3.2 kb) and the
germline fragment (at 2.8 kb) was calculated and used to distinguish
3-83 homozygous from heterozygous mice. A restriction map of the
relevant DNA fragment was presented previously (39).
Cell culture
BM cultures for B cell precursors were prepared as we have previously described (39, 42). Briefly, BM cells, RBC depleted, were cultured for 5 days in the presence of 50–100 U/ml rIL-7. Cells grown in these primary cultures (>95% B220+) (42) were used directly for cellular and molecular analysis or for adoptive transfer experiments. In some experiments B cells were stained using Fab goat anti-mouse IgM FITC (Jackson ImmunoResearch Laboratories, West Grove, PA) and anti-mouse IgD JA12.5-biotin (42) and fractioned to IgM+/IgD+ and IgM+/IgD- using anti FITC and/or streptavidin MACS microbeads (Miltenyi Biotec, Auburn, CA). For some experiments intracellular levels of cAMP were determined by RIA as described previously (44).
RNA and DNA analyses
RNA and DNA from a single-cell suspension of BM, spleen, or
lymph nodes (LN) or from BM cultures were prepared as we have
previously described (39, 42). Quantitation of endogenous
V-J1 DNA rearrangements was determined by PCR assay using genomic
DNA template and normalized to 
-actin. PCR conditions and primer
sequences were described in detail previously (42). Levels
of recombinase-activating gene-2 (RAG-2) and B220 (CD45R) control gene
mRNA were determined by RT-PCR assay. PCR conditions and primer
sequences for RAG-2 were previously described (42). PCR
conditions for B220 are 30 s at 94°C, 30 s at 64°C, and
1.5 min at 72°C for 22 cycles. Primer sequences for B220 are: sense,
5'-ctgagcacaacagagaatgcccttc-3'; and antisense,
5'-ctgaagagatatctgtggtgctgac-3'. PCR products were run on gel,
transferred to membrane, and probed using
32P-labeled specific probes for RAG-2 and B220 as
previously described (42). Blots were exposed to x-ray
film and scanned with a phosphorimager. To obtain a semiquantitative
estimate of gene expression, the signal intensity of RAG-2 was
normalized to the B220 signal as previously described
(42).
Flow cytometry
Single-cell suspensions from BM, spleen, LN, or BM cultures were
stained for surface marker expression using FITC-, PE-, and
biotin-conjugated mAbs, visualized with streptavidin TriColor (Caltag
Laboratories, San Francisco, CA). Abs used for cell staining were goat
anti-mouse IgM (Caltag Laboratories); goat anti-mouse 
(Southern Biotechnology Associates, Birmingham, AL); mAbs to B220 and
RA3-6B2 (Caltag Laboratories); IgDa, AMS 9.1,
heat-stable Ag (HSA), and 30-F1 (BD PharMingen, San Diego, CA);
and IgMa, RS3, and anti-3-83 idiotype 54.1
(42). Data for three-color analysis were collected on a
FACSCalibur and analyzed using CellQuest software (BD PharMingen).
Ca2+ flux
Kinetic variations of intracellular Ca2+ were determined using the Ca2+ indicator Fluo-3. This fluorescein derivative undergoes a dramatic increase in fluorescence intensity upon Ca2+ binding (45) and has emission/excitation spectra similar to those of fluorescein (46). The use of Fluo-3 to analyze Ca2+ in hemopoietic cells upon stimulation has been described (46). To study Ca2+ influx, 2 x 106 B cell precursors were washed in MGB buffer (5 mM KCl, 147 mM NaCl, 0.22 mM KH2PO4, 1.1 mM Na2HPO4, 5.5 mM glucose, 0.3 mM MgSO4, 1 mM MgCl2, 10 mM HEPES (pH 7.4), and 0.1% BSA) and incubated in MGB buffer and 0.5 mg/ml Fluo-3/AM for 30 min. After washing, B cells were resuspended in MGB buffer and 1 mM CaCl2 and warmed to 37°C. Cells were stimulated with monoclonal anti-IgM Ab b.7.6 (10–20 µg/ml) (40), and calcium flux was measured by the fluorescence intensity of Fluo-3/AM using the green channel (525 nm) on a FACSCalibur. Data were acquired for 60 s before BCR cross-linking.
Serum Ab detection
Detection of allotype-specific IgMa Abs in
serum was performed by standard ELISA as previously described
(47). Plates were coated with RS3 anti
IgMa mAbs, and bound Abs were revealed by
anti-3-83 idiotype (54.1) or with polyclonal goat anti-mouse
IgM or goat anti-mouse (Southern Biotechnology Associates).
Amounts of Abs in serum were calculated using a reference standard
curve of purified IgMa Ab.
BM chimera and adoptive transfer
BM chimera were prepared as previously described
(48). Donor BM cells, T and B cells depleted (confirmed by
FACS), were i.v. injected into lethally irradiated (800 rad) recipients
of the indicated genetic background (5 x
106 cells/mouse). Four weeks after reconstitution
spleen and LN were inspected by flow cytometry for the presence of
donor-derived,
idiotype+/+ B cells.
For adoptive transfer experiments B cell precursors were grown in BM cultures, washed, and i.v. injected into sublethally irradiated (400 rad) B10.D2 recipients (5 x 106 cells/mouse). Five days after cell transfer spleens of recipient mice were inspected by flow cytometry for the presence of transferred cells using anti-idiotypic staining.
Statistical analysis
The statistical significance of differences between experimental groups was determined using unpaired two-tailed Student’s t test or one-way multiple range ANOVA, with differences considered significant at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In 3-83Tg mice B cell development and maturation occur rapidly
(42, 49). Nonetheless, 3-83Tg mice deficient in CD19
manifest a severe reduction (60–80%) in mature
IgM+ B cells in spleen (36) (Fig. 1
, A and B,
respectively) and accumulation of immature cells in the BM
(36) (data not shown), implying that immature 3-83Tg
CD19-/- B cells are developmentally arrested.
To determine whether developmentally arrested 3-83Tg
CD19-/- B cells undergo receptor editing, we
studied the induction of V(D)J recombination and the establishment of
allelic exclusion in 3-83Tg CD19-/- mice (Fig. 1). Although all splenic IgM+ B cells in 3-83Tg
CD19-/- mice express the Tg receptor, a large
population of cells coexpresses an endogenous L chain (1.6%; Fig. 1A). This population reflects >5% of the total
IgM+ B cells in the 3-83Tg
CD19-/- spleen (Fig. 1B), a value
that is not different from that in a non-Tg mouse (5–8%; Fig. 1B). We also found many endogenous V-J1 DNA
rearrangements in purified IgM+ 3-83Tg
CD19-/- splenic B cells, but not in 3-83Tg
splenic B cells (not shown). H chain allotypic staining revealed that
in both mice all splenic B cells express the transgenic encoded
IgMa receptor, and no
IgMb-positive B cells were found (not shown).
Similar results were found in LN (not shown). Furthermore, serum Ab
analysis revealed that 3-83Tg CD19-/- mice
produce large amounts of IgMa Abs, composed of
the transgenic H chain (µHa) and the endogenous
L chain (; Fig. 1C, lower panel). This is
accompanied by a marked decrease in the total amount of
idiotype+/IgMa Abs
(middle panel). In contrast, essentially no
IgMa/ were found in serum of 3-83Tg
mice.
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, D and E). In contrast, no
RAG-2 expression was found in spleen or LN, suggesting that lack of
allelic exclusion and induction of V(D)J recombination in 3-83Tg
CD19-/- mice are restricted to developing B
cells in the BM. We conclude that immature 3-83Tg B cells
expressing signaling-incompetent BCR, deficient in CD19, are
developmentally arrested and undergo V(D)J recombination to edit their
receptors. 3-83Tg CD19-/- B cell precursors do not maintain allelic exclusion in vitro
To further assess the development of immature 3-83Tg
CD19-/- B cells we used our in vitro BM culture
system (42). After 5 days of culture 3-83Tg and 3-83Tg
CD19-/- grow equally well, with no differences
in cell recovery (not shown), purity (>95%
B220+; not shown), or phenotype (IgM and
IgDa expression; Fig. 2A, left). However,
immature 3-83Tg CD19-/- B cells did not
maintain allelic exclusion in culture relative to the control 3-83Tg
cells, as reflected by a significant population of B cells coexpressing
endogenous L chain and the transgenic 3-83 receptor (4%; Fig. 2A, right). This is supported by the finding of a
significant 10-fold elevation in RAG-2 gene expression (Fig. 2B, upper panel) and a significant 10- to 15-fold
increase in endogenous V-J1 DNA rearrangements (Fig. 2B, lower panel) in 3-83Tg
CD19-/- cultures. Furthermore, RAG expression
was detected in both early
(IgM+/IgD-) and late
(IgM+/IgD+, transitional)
immature 3-83Tg CD19-/- B cells grown in
culture (Fig. 2C), perhaps suggesting that acquisition of
sIgD is not sufficient to turn off DNA recombination in these cells.
Thus, lack of allelic exclusion in 3-83Tg
CD19-/- B cells was shown both in vivo and in
vitro at the levels of DNA, RNA, and protein. Moreover, we found a 2-
to 3-fold increase in intracellular cAMP levels in 3-83Tg
CD19-/- B cell precursors relative to 3-83Tg
precursors (Fig. 2D). These observations are in agreement
with earlier studies showing that V(D)J recombination and RAG gene
expression are increased upon induction of intracellular second
messengers such as protein kinase A and cAMP (50, 51).
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Earlier studies showed that the expression level of BCR is
important to promote B cell development and to establish allelic
exclusion (52, 53, 54, 55). Fig. 3
shows that splenic 3-83Tg CD19-/- B cells
express significant 1.5- to 2-fold higher levels of sIgM receptors
relative to their 3-83Tg counterparts (left). In
contrast, 3-83Tg and 3-83Tg CD19-/- developing
B cells in BM in vivo (Fig. 3, middle) and in cultures (Fig. 3, right) express indistinguishable levels of sIgM. We
conclude that the difference in the BCR level of expression in mature B
cells may reflect a selection process in vivo rather then a
differential level of BCR synthesis between the two mice. Thus,
immature 3-83Tg CD19-/- B cells that fail
selection because of incompetent BCR are developmentally arrested and
undergo intensive V(D)J recombination to edit their receptors.
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To test whether the induction of V(D)J recombination in 3-83Tg
CD19-/- B cells reflects an intrinsic B cell
defect, we generated short term BM chimera. BM cells from 3-83Tg or
3-83Tg CD19-/- donor mice were used to
reconstitute lethally irradiated B10.D2 or non-Tg
CD19-/- (CD19-/-) mice.
Spleen cells of reconstituted mice were stained after 4 wk to detect
idiotype+/+
double-expressing cells as an indication of the lack of allelic
exclusion (Fig. 4). The results show that
in contrast to 3-83Tg BM chimera, mice reconstituted with 3-83Tg
CD19-/- BM produce significant
idiotype+/+
double-expressing cells regardless of the genotype of recipient mice
(Fig. 4A). We conclude that lack of allelic exclusion in
3-83Tg CD19-/- B cells is an intrinsic B cell
defect and does not reflect environmental changes resulting from CD19
deficiency. Importantly, because 3-83Tg cells develop equally well in
B10.D2 and CD19-/- chimera (Fig. 4), it is
unlikely that V(D)J recombination in 3-83Tg
CD19-/- mice is driven by self-Ag. Additional
phenotypic analyses revealed that mature 3-83Tg
CD19-/- B cells, developing in either B10.D2 or
CD19-/- chimera, had significantly 1.5- to
2-fold higher levels of BCR expression (Fig. 4B), but a
60–70% reduction in frequencies (Fig. 4). This supports our
hypothesis that the BCR level of expression is important in promoting
developmental progression of 3-83Tg B cells expressing
signaling-incompetent BCR, deficient in CD19.
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To test the hypothesis that high expression levels of BCR can
compensate for compromised signaling in promoting allelic exclusion,
selection, and maturation, we generated homozygous 3-83Tg
CD19-/- mice (3-83TgHz
CD19-/-). In these experiments, we adopted a
previously shown experimental maneuver, in which homozygosity of the
transgene loci enhances the level of transgene expression compared with
heterozygosity (52, 53). Bone marrow analysis of early
immature B220low/HSAlow B
cells showed that transgene homozygosity resulted in 10–15% elevated
levels of BCR expression (Fig. 5A; IgM mean fluorescence
intensity (MFI) in 3-83TgHZ CD19-/- is 69
relative to 58–60 in the 3-83Tg and 3-83Tg
CD19-/-). In later developmental stages in the
BM (B220low/HSAhigh; Fig. 5A) no significant difference in BCR expression levels was
found, probably reflecting the fact the BCR expression levels in the
3-83Tg mouse model are developmentally regulated (40). The
results in Fig. 5B (table) support our hypothesis by showing
that allelic exclusion was completely restored in the 3-83TgHz
CD19-/-. In a striking contrast to 3-83Tg
CD19-/- mice, RAG-2 gene expression in BM of
3-83TgHz CD19-/- mice was undetectable, and
essentially no
idiotype+/+
double-expressing B cells were found in their spleens (Fig. 5B). Similar results were found in BM cultures in vitro
(Fig. 5B). As expected, we found that 3-83TgHz
CD19-/- B cells expressed high levels of BCR
relative to 3-83Tg B cells, but not significantly higher then
heterozygous 3-83Tg CD19-/- B cells (Fig. 5C, left), and had a reduced immature B cell
compartment in the BM relative to 3-83Tg
CD19-/- (not shown). Also, analysis of HSA
expression revealed that splenic B cells from 3-83TgHz
CD19-/- and control 3-83Tg mice acquired the
mature HSAlow phenotype (MFI, 390 and 362,
respectively), whereas more splenic 3-83Tg
CD19-/- B cells expressed the immature
HSAhigh phenotype (MFI, 550; Fig. 5C,
right). Hence, our results suggest that high levels of BCR
expression can compensate for incompetent BCR, deficient in CD19, in
signaling for establishment of allelic exclusion maturation and
selection. In contrast, the failure to fulfil appropriate BCR signaling
capabilities results in developmental arrest and receptor editing. In
this experimental system high levels of BCR expression by splenic B
cells can be obtained through a selection process (in 3-83Tg
CD19-/-) or more efficiently upon transgene
homozygosity (in 3-83TgHz CD19-/-).
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To study the signaling capacity of immature 3-83Tg
CD19-/- B cells, we monitored
Ca2+ mobilization patterns upon BCR stimulation.
To do so immature 3-83Tg, 3-83Tg CD19-/-, and
3-83TgHz CD19-/- B cells grown in culture were
stimulated with anti-BCR Abs. The kinetics of
Ca2+ mobilization clearly show that immature
3-83Tg CD19-/- B cells poorly mobilize
Ca2+ in response to BCR cross-linking (Fig. 6). This is not dose-dependent, as
titration of the stimulating Ab, up to 20 µg/ml, had no effect (not
shown). Because immature 3-83Tg and 3-83Tg
CD19-/- B cells grown in culture were
indistinguishable in sIgM expression (Figs. 2A and 3), we
conclude that the poor Ca2+ mobilization is due
to the expression of signaling-incompetent BCR, deficient in CD19. This
is supported by the finding that immature 3-83TgHz
CD19-/- B cells completely restored their
Ca2+ mobilization response upon anti-IgM
stimulation (Fig. 6). Thus, a high BCR level of expression effectively
compensates for a compromised receptor in Ca2+
mobilization upon BCR stimulation.
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To test the possibility that immature 3-83Tg
CD19-/- B cells are developmentally arrested
and undergo receptor editing because they fail positive selection, we
used an adoptive cell transfer approach in which cell survival is
recapitulated as positive selection (56, 57, 58). In these
experiments, immature 3-83Tg, 3-83Tg CD19-/-,
and 3-83TgHz CD19-/- B cells grown in vitro
were adoptively transferred into non-Tg B10.D2 recipients. Five days
after cell transfer the spleen and LN of reconstituted mice were
analyzed for the presence of Tg B cells. The results presented in Fig. 7 clearly show that the selection and
survival of immature 3-83Tg CD19-/- B cells in
vivo were substantially reduced. The survival of immature 3-83Tg
CD19-/- B cells in the reconstituted mice was
2- to 3-fold lower than that in control immature 3-83Tg B cells in both
percentages (Fig. 7A) and absolute numbers (Fig. 7B). Careful analysis and comparison of the MFI of sIgM
revealed that selected 3-83Tg CD19-/- B cells
had significantly elevated (30–40%) BCR expression levels compared
with the selected 3-83Tg B cells (Fig. 7B). Similar results
were found in the LN (not shown). In contrast, immature 3-83TgHz
CD19-/- B cells had survival and selection
rates similar to those of control immature 3-83Tg B cells (Fig. 7), and
both had also acquired a high level of IgD (not shown). We conclude
that high levels of BCR expression can compensate for a
signaling-incompetent receptor, deficient in CD19, in signaling not
only for allelic exclusion, but also to promote maturation and positive
selection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because of signaling incompetence, B cell maturation is impaired, and B
cells are developmentally arrested at the immature stage (13, 14, 16, 35). In immature 3-83Tg CD19-/- B
cells this developmental arrest induces intensive V(D)J recombination
attempts to edit the BCR, thereby abrogating the strict allelic
exclusion imposed by the 3-83Tg receptor (Figs. 1 and 2). Similarly,
developmental arrest is imposed in immature autoreactive B cells
encountering self-Ag, and in proB cells expressing incompetent preBCR
(18, 20, 21, 22, 39, 40, 60). Both have been shown to undergo
secondary V(D)J recombination. Thus, receptor editing is a salvage
mechanism for self-reactive immature B cells and for developing B cells
expressing incompetent receptors. The possibility that V(D)J
recombination in 3-83Tg CD19-/- B cells is
induced by self-Ag is unlikely (Fig. 3). Other explanations for
secondary V(D)J recombination, which may account for normal-developing
B cells such as poor H and L pairing (61), out-of-frame
DNA rearrangements (4), and preferences/favoring of
specific V segments (19), are irrelevant for the 3-83Tg
model (42, 43). We therefore suggest that in 3-83Tg
CD19-/- mice receptor competence plays a
critical role in limiting B cell maturation and imposing developmental
arrest at the immature stage. Interestingly, when bred onto the
negatively selecting MHC class I H-2b background, the lack of CD19 did
not affect the ability of developing 3-83Tg B cell to undergo clonal
deletion and receptor editing (36). Similar results were
obtained in mice deficient in Btk (62), a BCR signaling
molecule acting downstream of CD19 in the BCR signaling cascade
(25). Thus, despite CD19 or Btk signaling deficiencies,
central tolerance in the 3-83Tg system is maintained, suggesting that
self-Ag signaling can be compensated by other effector molecules.
The fact that 3-83Tg CD19-/- B cells fail to
impose allelic exclusion argues that feedback inhibition by Ig proteins
alone is not sufficient to establish L chain allelic exclusion
(6, 63). This is because immature 3-83Tg and 3-83Tg
CD19-/- B cells are indistinguishable in BCR
expression levels in the BM in vivo (Fig. 4) (36) and in
vitro (Fig. 4). Therefore, it is possible that competent BCR signaling
in immature B cells is also required for suppression of V(D)J
recombination and to establish L chain allelic exclusion, as proposed
for the H chain loci (1, 18, 20, 21, 22). Many studies in both
normal and Ig Tg models suggest that BCR expression and signaling are
required to promote positive selection and survival of mature B cells
in the periphery (reviewed in Refs. 2 , 59 ,
and 64). In the 3-83Tg CD19-/-
mouse model positive selection and maturation of B cells were
restricted to B cells that were able to compensate for the compromised
BCR by coexpression of endogenous L chain or by expression of high
levels of BCR. Such a compensation mechanism has been described
previously (27). That lack of CD19 inhibits B cell
selection into the marginal zone has also been described in a different
Ig-Tg mouse model deficient in CD19 (37). This suggests
that the BCR signaling threshold regulates positive selection and
allelic exclusion. The ability of 3-83Hz
CD19-/- mice to restore L chain allelic
exclusion and selection strongly supports this hypothesis (Figs. 5 and 7). Similarly, recent studies using 3-83 H+L knockin mice or
anti-RBC H+L Tg mice clearly showed that underexpressed BCR failed
to promote positive selection or impose L chain allelic exclusion. Both
were restored in mice homozygous for the transgene, in which BCR
expression levels were elevated (52, 53). A model
proposing a role for BCR signaling in establishing L chain allelic
exclusion has been recently described, although no direct evidence has
been presented (65). The data described in this study
strongly support the proposed model. Thus, functional, non-self-BCR can
signal in immature B cells for positive selection and developmental
progression, concomitantly with establishment of L chain allelic
exclusion. The data we show in this study suggest that the CD19
molecule is an important regulator in determining the BCR signaling
threshold required for these processes. It also proposes that immature
B cells that fail positive selection because of receptor signaling
incompetence can activate the receptor-editing mechanism to alter
the BCR.
Interestingly, only L chain allelic exclusion was impaired in 3-83Tg CD19-/- mice, whereas endogenous H chain gene recombination was efficiently suppressed. This finding supports the hypothesis that allelic exclusion establishment at the H and L chain loci are independent (1, 3, 18, 19). It was previously shown that pre-BCR signaling and positive selection of pro-B cells are followed by changes in locus-specific V(D)J recombinase activity from the H chain locus to the L chain locus, indicating that this activity is developmentally regulated (3, 19). Thus, pro-B cells that are developmentally arrested continue to rearrange H chain genes (20, 21, 22), whereas developmentally arrested immature B cells continue to rearrange L chain genes (39, 40). Because 3-83Tg CD19-/- B cells are arrested at the immature stage, V(D)J recombination is directed to the L chain locus rather than to the H chain locus. This situation resembles tolerance process in B cell development, where self-reactive immature B cells are developmentally arrested upon self-Ag binding, and secondary DNA rearrangements are directed to the L chain loci rather then to the H chain locus (42, 64).
The functional competence of the BCR complex determines not only
developmental progression, but also survival and activation of B cells
(1, 59). Earlier studies showed that mature 3-83Tg
CD19-/- B cells had a diminished
Ca2+ response when stimulated with Ag, and many
of the cells did not respond at all (36). In contrast, the
signaling capacity (Ca2+ influx and
phosphotidylinositol 3-kinase activation) of mature non-Tg
CD19-/- B cells is controversial, and it
appears that different BCR stimuli account for the different results
(26, 36, 66). We show in this study that immature B cells
expressing signaling-incompetent 3-83Tg receptor, deficient in CD19,
have poor Ca2+ response upon BCR ligation (Fig. 6). However, Ca2+ mobilization is completely
restored in 3-83TgHz CD19-/-, suggesting that a
high level of BCR expression can effectively circumvent and compensate
for CD19 deficiency. It is now important to extend these studies and to
exclude the possibility that Fc receptor signaling is altered in the
absence of CD19 and contributes to the obtained results. In such
experiments anti-IgM F(ab')2
(36) or a 3-83-specific mimetic peptide (33)
should be used to stimulate the cells. Interestingly, a high BCR
expression level directs B cell development to the B1 compartment
(53, 54, 55), which is completely absent in
CD19-/- mice and in mice deficient in Btk or
SLP-65 (14, 16, 35). This proposes that the BCR signaling
threshold, which is regulated by CD19, Btk, and SLP-65 molecules, not
only determines developmental progression, but is also involved in
directing the B cell lineage.
The conclusions of this study were reached by taking advantage of a well-described Ig Tg mouse model and an IL-7 BM culture system. Because both experimental maneuvers perturb B cell development in several ways, as we have described (40, 42), it is important to confirm these results in a more physiological system. This system, however, should confound the difficulties of distinguishing V(D)J recombination normally expressed in developing B cells and recombination processes associated with the lack of positive selection. It will also be important to address whether the positive selection process we found in the adoptive transfer experiments also apply to normal immature BM B cells expressing nontransgenic BCR.
Finally, if BCR signaling is required to establish L chain allelic
exclusion and to promote maturation, it probably involves intracellular
second messengers, such as cAMP. Those aim to transduce signals to the
nucleus and to activate transcription factors regulating V(D)J
recombination (67, 68). Several studies have shown that
agents activating protein kinase A or increasing cAMP also elevate
V(D)J recombination in B cells (50, 51). How BCR or
pre-BCR signal for allelic exclusion is not known, nor have the
ligands, if any, that transduce these signals been identified. In
developing T cells, a high TCR level and suppression of RAG gene
expression and of TCR gene rearrangements are correlated with a
positive selection step, which is dependent on TCR-MHC interaction
(69). Several studies showed Ag-mediated rescue of B cells
in signaling-deficient or receptor-insufficient cells (70, 71). However, it is more likely that basal or tonic signaling of
an unligated BCR may itself provide sufficient signal to promote B cell
development, allelic exclusion, selection, and survival (1, 64). In contrast to T cells, B cells express coreceptors, such
as CD19, that act to balance such signaling and therefore may be
involved in regulating developmental progression, as we show in this
study. Further understanding of the signaling cascade that links BCR
signaling (or the basal level of signaling) with V(D)J recombination is
required.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Doron Melamed, Department of Immunology, Faculty of Medicine, Technion, Haifa 31096, Israel. E-mail address: melamedd{at}techunix.technion.ac.il
3 Abbreviations used in this paper: BCR, B cell receptor for Ag; BM, bone marrow; HSA, heat-stable Ag; LN, lymph node; MFI, mean fluorescence intensity; RAG, recombinase-activating gene; Tg, transgenic; SLP, SH2-domain-containing leukocyte protein.
Received for publication November 26, 2001. Accepted for publication April 3, 2002.
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