By
From the * National Institute for Medical Research, Mill Hill, London, NW7 1AA, United
Kingdom; and the Department of Immunology, University of Birmingham Medical School,
Birmingham, B15 2TT, United Kingdom
The tyrosine kinase Syk has been implicated as a key signal transducer from the B cell antigen receptor (BCR). We show here that mutation of the Syk gene completely blocks the maturation of immature B cells into recirculating cells and stops their entry into B cell follicles. Furthermore, using radiation chimeras we demonstrate that this developmental block is due to the absence of Syk in the B cells themselves. Syk-deficient B cells are shown to have the life span of normal immature B cells. If this is extended by over-expression of Bcl-2, they accumulate in the T zone and red pulp of the spleen in increased numbers, but still fail to mature to become recirculating follicular B cells. Despite this defect in maturation, Syk-deficient B cells were seen to give rise to switched as well as nonswitched splenic plasma cells. Normally only a proportion of immature B cells is recruited into the recirculating pool. Our results suggest that Syk transduces a BCR signal that is absolutely required for the positive selection of immature B cells into the recirculating B cell pool.
The initial development of mammalian B cells in the
bone marrow is characterized by the sequential rearrangement of heavy and light chain immunoglobulin genes
resulting in the production of large numbers of B cells
throughout life (1). These newly produced (immature) B cells
express antigen receptors (BCRs)1 in the form of membrane-bound IgM; they leave the marrow for the spleen and other
secondary lymphoid tissues, where most of them normally
die within a week (2). A small minority of immature B
cells are induced to mature into long-lived recirculating B cells (3). These express both IgM and IgD and migrate
between the follicles of lymphoid tissues for several weeks
(5). Comparison of VH gene usage between immature
and recirculating B cells shows that recirculating cells use
only a subset of the VH genes expressed in immature cells,
suggesting that there is selectivity in the recruitment of immature B cells (9). It has been proposed that these results may reflect positive selection of immature B cells into
the recirculating pool on the basis of binding of the BCR to endogenous selection molecules (14). A prediction
from this hypothesis is that signaling through the BCR is
essential for the selection of immature B cells into the recirculating pool.
Signal transduction through the BCR is dependent on the
associated nonpolymorphic receptor subunits CD79 We and others have previously reported the generation
of mice with a targeted disruption of the Syk gene (Syktm1Tyb),
which is generally a lethal defect when homozygous (Syk Generation of Fetal Liver Chimeras.
Fetal liver cells were harvested from embryos at day 16.5 of gestation generated by intercrossing mice that were Syk+/ Immunohistochemistry.
Cryostat sections (5 µm) of frozen splenic
tissue were prepared and stained as previously described (25). In the
live adult Syk Staining of Cells with Fluorescent Antibodies.
Bone marrow and
spleen cells were stained with monoclonal antibodies as previously described (23). Fluorescence was analyzed on a FACStar®
or FACSVantage® flow cytometer with Cellquest software (Becton Dickinson, Mountain View, CA). Dead cells were excluded on
the basis of low forward light scatter and only live cells falling
within the lymphocyte scatter gate are shown. Anti-B220-allophycocyanin, anti-IgMa-PE, anti-IgDa-FITC, and anti-CD43-biotin
were purchased from PharMingen, anti-3-83 idiotype (54.1; gift of
D. Nemazee) and anti-IgD (1.19) were purified from tissue culture
supernatants and conjugated to FITC or biotin by standard methods. Biotinylated antibodies were revealed with streptavidin-PE
(Biogenesis, Poole, UK) or streptavidin-RED613 (GIBCO-BRL,
Gaithersburg, MD).
We had
earlier shown, using irradiated mice reconstituted with
Syk
It was unclear from our preliminary study if
this developmental block is due to a malfunction within B
cells, or some other cell type required for the formation of
follicles, or both. To address this issue, chimeras were constructed by transferring Syk
In wild-type rodents, when the recirculating pool of B
cells is selectively depleted so that B lymphopoiesis continues unimpaired, the proportion of immature B cells recruited to become recirculating cells increases dramatically
until the pool is filled. This replenishment takes 4-5 d. This
study shows that most immature B cells have the potential
to become recirculating cells and are not deleted in some
central selection process within the marrow. This indicates that under normal circumstances there is competition between immature B cells for limited numbers of slots in the
recirculating pool (2). Furthermore, the slots in the recirculating pool become available because of competition between recirculating and immature B cells. This is shown in
experiments in which the recirculating pool of SCID mice
is partially reconstituted with recirculating cells (26). In
these chimeras, which produce no immature B cells, the
number of recirculating cells remains constant; their life-span is indefinite, rather than the few weeks for which they
persist in normal mice (5, 8), and they are not able to replicate to fully fill the partially reconstituted recirculating pool
(26). These data indicate that it is the presence of immature
cells that results in the physiological loss of recirculating cells. It is notable that Syk Although Syk It is possible that the block in maturation of Syk
The transition of immature to recirculating B cells is accompanied by
an increase in expression of the anti-apoptotic gene Bcl-2
(31, 32). Thus it was possible that Syk-deficient immature B cells failed to differentiate into recirculating B cells because, unlike control cells, they had not expressed Bcl-2 and
hence died prematurely. To address this, we generated radiation chimeras using Syk-deficient, 3-83-expressing fetal
livers containing the Eµ-Bcl-2-36 (Bcl-2Tg) transgene,
which constitutively expresses Bcl-2 in the B lineage (33).
Expression of Bcl-2 resulted in a greater accumulation of
immature Syk-deficient B cells both in the marrow and
spleen, and in the generation of some IgM+ IgDlow cells
which probably correspond to transitional B cells as described by Carsetti et al. (34; Fig. 3, a, c, and d). However,
we were unable to detect any Syk To confirm that the Bcl-2Tg had indeed increased the
life-span of Syk-deficient immature B cells, Syk
The finding that Syk deficiency causes a total block in
recruitment of immature B cells into the recirculating B
cell pool provides insight into the signals involved in this
maturation step. A similar, although incomplete, block in
maturation was described in mice carrying a CD79 What is the purpose of such a signal? The restricted VH
usage of recirculating B cells in contrast to immature B cells
has been used to suggest that a subset of immature cells is
positively selected into the recirculating pool on the basis
of BCR binding to endogenous selection molecules, although it cannot be excluded that this effect is due to selective deletion (9). In any case, since cells of the recirculating B cell pool typically express surface immunoglobulin,
it may be that selection ensures that only cells with a functional BCR are recruited into the recirculating B cell compartment; such a process would necessarily require a BCR
signal.
Mutations in a number of other signaling molecules have
been reported to affect the transition of immature B cells
into recirculating cells. Mice with a mutation in Btk, a tyrosine kinase involved in BCR signaling, contain some recirculating B cells, but have an accumulation of transitional
cells (38, 39). Furthermore, in the presence of normal competitor B cells, Btk-mutated B cells are completely blocked
in their development at the immature B cell stage (40).
Studies of mice deficient in the tyrosine phosphatase CD45
demonstrated that this too is involved in BCR signaling
(41). These mice have plenty of recirculating B cells,
but these cells are short-lived and have abnormally high
IgM and IgD expression; engagement of surface immunoglobulin in these mice partially rescues this phenotype (44).
These results are consistent with a requirement for BCR-mediated signals in the development of immature B cells into long-lived recirculating B cells.
The failure of Syk In conclusion, we have shown that the tyrosine kinase
Syk is absolutely required for the maturation of immature
B cells into the recirculating follicular B cell pool. Furthermore, although Syk-deficient immature B cells reach the
red pulp and outer T zone of the spleen, they fail to enter
follicles. The T zone or red pulp may be the site where immature B cells normally receive a BCR signal that drives
their positive selection into the recirculating pool.
and
CD79
(Ig
and Ig
; reference 17). In particular, CD79
/
contain peptide motifs termed ITAMs (immune receptor
tyrosine-based activation motifs) on their intracellular domains that are crucial for signaling. Cross-linking of the
BCR results in the phosphorylation of the tyrosine residues
in the ITAMs of CD79
/
and their association with, among
others, the protein tyrosine kinase p72Syk (Syk; references
18). This association is mediated by an interaction of
the SH2 domains of Syk with the ITAM phosphotyrosines
and leads to the activation of the Syk kinase, suggesting that
Syk may be an important signal transducer of the BCR.
This view was strengthened by disruption of the Syk gene
in a chicken B cell lymphoma, which led to a severe deficiency in BCR signaling (22).
/
;
references 23, 24). By reconstituting irradiated mice with Syk
/
fetal liver cells we observed reduced numbers of
cells making the transition from pro-B to pre-B cell, suggestive of a role for Syk in pre-BCR signaling (23). In addition, despite the presence of a small number of immature
Syk
/
B cells in the bone marrow, there were no detectable B cells in the spleen and lymph nodes (23). In this report we extend this analysis and demonstrate that although
marrow-derived immature Syk
/
B cells migrate to the
red pulp and outer T zone of the spleen, they fail to enter
B cell follicles. Furthermore, we show that they are completely blocked in their ability to mature into recirculating follicular B cells and that this defect is most likely due to a
role for Syk within the B cells themselves. These results
strongly suggest that a Syk-mediated BCR signal is required for the transition of immature B cells into the recirculating B cell pool and this may reflect a step at which
positive selection of B cells occurs.
/3-83µ
/H-2d/Ly9.2/IgHb and
Syk+/
/Eµ-bcl-2-36/H-2d/Ly9.2/IgHb. Genotypes of parents
and fetuses were determined by Southern blotting for Syk, 3-83, Eµ-bcl-2, H-2, and IgM and by flow cytometry for Ly9. Radiation chimeras were generated by injecting 1.5 × 106 fetal liver
cells intravenously into female BALB/c (H-2d/Ly9.1/IgHa) mice
that had received two doses of 500 rad from a 60Co source 3 h
apart. The recipient mice were given neomycin sulphate (0.16%)
in their drinking water for 4 wk after irradiation and were analyzed 2-4 mo after reconstitution.
/
mice, B and T cells were identified using either
rat anti-B220 (PharMingen, San Diego, CA) or rat anti-CD3 (Serotec Ltd., Kidlington, Oxford, UK), followed by biotinylated rabbit
anti-rat Ig (Dako Ltd., High Wycombe, UK). Donor B cells were
identified in tissue sections from radiation chimeras using biotinylated mouse anti-IgMb (PharMingen) or biotinylated anti-3-83
(54.1; gift from D. Nemazee, National Jewish Center for Immunology, Denver, CO). In both cases biotinylated antibodies were
revealed using streptavidin-ABComplex-alkaline phosphatase conjugate (Dako Ltd.) and napthol-ASMX-phosphate and Fast Blue BB
(Sigma Chemical Co., Poole, Dorset, UK) to give a blue precipitate
at the site of antibody binding. Sheep anti-mouse IgD or anti-mouse
IgM (The Binding Site, Birmingham, UK) were detected with peroxidase-labeled donkey anti-sheep Ig (The Binding Site) using hydrogen peroxide and diaminobenzidene to give a gold-colored precipitate at the site of antibody binding. Both secondary antibodies were absorbed with 10% normal mouse serum before use to
remove cross-reactivity. The thymidine analogue 5
-bromo-2-deoxyuridine (BrdU; Sigma Chemical Co.) in saline was administered as 2 mg intraperitoneally 2 h before death, to label cells in the S phase of the cell cycle. The incorporated BrdU was identified by treating stained slides for 20 min with 1 M HCl at 60°C to expose the
incorporated BrdU and destroy the activity of previously bound
immunohistological reagents. Anti-BrdU (Dako Ltd.) was then
added to the slides after they had been washed. This was detected
with biotinylated goat anti-mouse Ig (Dako Ltd.) followed by
Streptavidin-ABComplex-alkaline phosphatase. Enzyme activity
was demonstrated using naphtol-ASMX-phosphate-free acid (Sigma
Chemical Co.) with Fast Red TR salt (Sigma Chemical Co.).
Syk/
Immature B Cells Accumulate in the Outer T Zone
and Do Not Mature into Recirculating Follicular B Cells.
/
fetal liver, that there was a partial block at the pro-B
to pre-B cell transition, suggestive of a role for Syk in pre-BCR signaling (23). Furthermore, we noted that despite
the presence of a small number of immature Syk
/
B cells
in the marrow, no detectable recirculating B cells accumulated in the peripheral lymphoid organs, at least as judged
by flow cytometric analysis (23). To investigate further the
fate of Syk
/
immature B cells, we turned to the more
sensitive technique of immunohistology to look for the
presence of Syk
/
B lineage cells in the spleen, since this is
at least one of the sites to which immature B cells migrate
upon leaving the marrow (3, 4). Initially, analysis was performed on spleens of two Syk
/
mice that, exceptionally,
had survived to adulthood. Strikingly, this showed small
numbers of immature (IgM+IgD
) B cells lined up along
the edge of the T zone, but a total absence of both follicles
and recirculating (IgM+IgD+) B cells (Fig. 1). Thus Syk
/
immature B cells migrate from the marrow to the spleen
but do not mature into recirculating follicular B cells.
Fig. 1.
Immunohistochemistry of splenic B cells in an adult Syk/
mouse. Sections were stained with antibodies to IgD or IgM; these were revealed with peroxidase (brown) or with antibodies to CD3 or B220 revealed with alkaline phosphatase (blue) as indicated. Sections labeled "none" were
stained only with the peroxidase-coupled secondary antibody to reveal endogenous peroxidase activity. (a-c) Serial sections from the spleen of an exceptional Syk
/
mouse that survived to adulthood; small numbers of B220+ cells are found in the outer T zone (T) and a proportion of these are IgM+ (arrows in b), but none are IgD+ nor have they entered the follicles. The B220+IgM
cells in this spleen were shown to be CD3
(data not shown). Note in
c the brown staining of eosinophils which contain endogenous peroxidase activity; the same eosinophils are seen staining brown in a and b. (d-f ) Serial sections from a heterozygous littermate showing the development of normal follicles (F), which contain IgM+IgD+ recirculating B cells; IgM+IgD
plasma
cells are present in extra-follicular foci (E) in this spleen. Scale: black bar represents 100 µm. Analysis of a second Syk
/
mouse gave similar results.
[View Larger Version of this Image (78K GIF file)]
/
B Cells Is a Cell-autonomous Defect.
/
fetal liver into irradiated
wild-type mice which differed in their immunoglobulin
heavy chain allotype; this allotype difference allowed B
cells of host and donor origin to be distinguished. Flow cytometric analysis of these mice confirmed that they contained Syk
/
immature B cells in the marrow, but none
were detectable in peripheral lymphoid tissue (data not
shown). The spleens from 20 of these chimeras were examined by immunohistology, and all were found to have small
numbers of donor Syk
/
immature B cells (IgMb+IgD
)
in the T zones and red pulp, but no Syk-deficient B cells in the follicles (Fig. 2 a); the B cells in these follicles were exclusively host (IgMa+) cells (Fig. 2 a and data not shown).
Furthermore, analysis of lymph nodes from the same chimeras failed to find any donor Syk-deficient B cells, although again host IgMa+IgD+ cells were found in the follicles (Fig. 2 b). In contrast, chimeras made with Syk+/+ or
Syk+/
fetal liver had normal follicles filled with donor
IgMb+IgD+ B cells (data not shown). Effective reconstitution of the irradiated mice with Syk
/
donor hematopoietic cells could be clearly documented in the T lineage (data
not shown); however, there was always a small, although variable, level of residual host lymphopoiesis. The presence of
significant numbers of host (Syk+/+) follicular IgMa+IgD+
B cells, particularly in lymph nodes, demonstrates that wild-type B cells can mature in these Syk
/
chimeras; consequently the maturational arrest of the Syk
/
immature B
cells appears to be due to the absence of Syk within the B
cells themselves.
Fig. 2.
Immature Syk/
B cells migrate to the spleen but do not mature into follicular B cells. Immunohistochemistry of peripheral B cells in radiation chimeras whose nature is shown to the left above each micrograph; i.e., whether the reconstituting livers were Syk+/
or Syk
/
and if they contained the 3-83 or Bcl-2 transgenes (29, 33). All fetal liver donors were of the IgHb immunoglobulin allotype; all hosts were IgHa. 20 Syk
/
nontransgenic
chimeras and at least three chimeras from each group of transgenic donors were analyzed. The specificity of the antibodies used for staining the sections is
shown to the right above each micrograph; the molecule stained gold by immunoperoxidase is shown first, the one stained blue by immunoalkaline phosphatase is shown second and, where used, BrdU staining is in red. Cells double positive for gold and blue appear dark brown. Scale: black bar represents
100 µm. (a) Donor IgMb+ Syk
/
immature B cells (small arrows) are present in the T zone (T) and red pulp (R) of the spleen, but not in the partially filled follicles (F), which contain mature IgD+ (gold) cells of host origin. Donor IgMb+Syk
/
plasma cells in the red pulp are indicated by large arrows. (b) Section through a lymph node from a similar chimera to that in a. Well-developed primary follicles are present but no donor IgMb+Syk
/
B cells were detected in this or in any lymph node from similar chimeras. (c) Spleen section from a Syk+/
/3-83 chimera that, like the ones shown in d and e, had been
given BrdU for 12 h, 8 d before the spleen was taken. The follicular mantles are filled with mature 3-83+IgD+ (dark brown) donor B cells; occasional cells
(arrowed) have a red-stained nucleus, indicating that they had taken up BrdU during the 12-h pulse 8 d previously. Some 3-83+IgD
(blue) donor B cells can be
seen in the outer T zone and red pulp; some of these may be immature B cells, but none are BrdU+. The germinal center (G) is filled with donor-derived
3-83
IgHb+ B cells (data not shown) that arise from occasional rearrangement of endogenous Ig genes in the 3-83 transgenics. (d) Spleen section from a
Syk
/
/3-83 chimera. Small numbers of 3-83+IgD
donor B cells (blue and arrowed) can be seen in the outer T zone and red pulp; none of these are
BrdU+. As in a the follicles exclusively contain host IgD+ cells (gold) but no donor B cells. (e) Spleen section from a Syk
/
/3-83/Bcl-2Tg chimera. More
donor B cells are present in the T zone and red pulp than in the chimera in d not carrying Bcl-2Tg. Nevertheless, there were no donor B cells in follicles.
Some of the donor 3-83+ B cells had BrdU in their nuclei (arrowed and red). These must have been in cell cycle as pre-B cells 8 d previously, indicating an extended life span for these immature B cells.
[View Larger Version of this Image (149K GIF file)]
/
immature B cells fail to mature in
chimeras where host B cell production was insufficient to
fill the follicles completely. In these mice the mutant cells are
not being competitively excluded from follicles; rather they
have an intrinsic inability to mature.
/
B cells do not enter the recirculating
B cell pool, plasma cells of donor origin were present in the
red pulp of many chimeras (Fig. 2 a); some of these had
switched to IgG production (data not shown). This indicates
that Syk
/
immature B cells can be induced to become
plasma cells, apparently without first becoming recirculating cells, and suggests that some signals can be delivered
through the BCR in the absence of Syk. Isolated cells with
an immature B cell phenotype have been induced to become plasma cells in free cultures or splenic fragment cultures, but the possibility that the responding cells matured to recirculating cells in culture was not excluded (27, 28).
/
B Cells, but Does Not Overcome
the Maturational Block.
/
immature B cells was not complete, but
simply inefficient, and the failure to observe any Syk
/
recirculating cells was due to the reduced number of immature B cells produced in the marrow of the Syk
/
chimeras (23). To attempt to overcome this, we made further chimeras with Syk
/
and control fetal livers containing
the 3-83µ
(3-83) BCR transgene; this encodes IgM and
IgD specific for H-2Kk and H-2Kb, but not H-2Kd; the
background of both the donor and host in all these chimeras was H-2d (29). Although 3-83-expressing B cells were
abundant in the bone marrow of the Syk-deficient chimeras, all these cells expressed IgM with little or no IgD and
thus do not represent recirculating cells, which are typically
IgM+IgDhi (Fig. 3, a and d). In Syk
/
chimeras made without a BCR transgene, most B lineage cells in the marrow
are pro-B cells, and thus it was possible that the 3-83-expressing cells in the Syk
/
/3-83 chimeras would still be phenotypically pro-B cells, despite the expression of a rearranged
BCR (23). However, flow cytometric analysis demonstrated that most of the 3-83-expressing cells in these chimeras do not express CD43, a marker of pro-B cells that is
turned off as the cells mature into pre-B/immature B cells (Fig. 3 b; reference 30). Thus, the Syk
/
/3-83 chimeras
contained large numbers of Syk
/
cells with an immature
B cell phenotype. The mechanism whereby the expression
of the 3-83 BCR partially rescues the pro-B
pre-B cell developmental block is unclear. Despite the abundance of these immature cells in the bone marrow, very few Syk-deficient cells could be detected in the spleen and lymph
nodes of the chimeras and again these were immature in
phenotype (IgM+IgD
/low; Fig. 3, c and d, and data not
shown). Furthermore, immunohistology demonstrated that
in contrast to wild-type 3-83+ B cells, the few Syk-deficient cells were again confined to the outer T zone and red
pulp of the spleen (Fig. 2, c and d).
Fig. 3.
Positive selection of immature B cells. Flow cytometric analysis of cells
from radiation chimeras reconstituted with liver cells from Syk+//3-83 (Syk+/
),
Syk
/
/3-83 (Syk
/
), and Syk
/
/3-83/Bcl-2Tg (Syk
/
+ Bcl-2) fetuses or from
an adult Syk+/
/3-83/Bcl-2Tg (Syk+/
+ Bcl-2) mouse. In separate experiments chimeras made with Syk+/
/3-83/Bcl-2Tg fetal liver gave essentially the same results as presented in this figure (data not shown). Chimeras made with Syk+/+/3-83 fetal
liver gave results indistinguishable from Syk+/
/3-83 chimeras (data not shown). (a)
The first column from the left shows the expression of B220 and the transgenic BCR
(3-83) on bone marrow cells; B220+3-83+ cells are boxed and their fraction as a percentage of all lymphoid cells is shown. The second column shows the expression of
IgMa and IgDa by B220+3-83+ cells with gating to show immature (IgM+IgD
),
transitional (IgM+IgDlow), and mature (IgM+IgDhigh) B cells; the percentage for each
gated population is expressed as a fraction of all lymphocytes. (b) The first column
shows the gating on immature (B220low3-83low) cells that was used to generate the
histograms of CD43 expression on B220+3-83+ bone marrow cells shown in the second column; percentages represent the fraction of gated immature B220low3-83low
cells that are CD43
. (c) Expression of B220 and 3-83 in the spleen; B220+3-83+
cells are boxed and their fraction as a percentage of all lymphoid cells is shown. (d) Mean numbers ± SEM of B220+3-83+ cells in the bone marrow (per
femur) and spleen are shown. In the marrow, these have been subdivided into immature (IgM+IgD
), transitional (IgM+IgDlow) and mature
(IgM+IgDhigh) B cells using the gating shown in a. Data in (a-c) are representative of at least six mice of each genotype. Absolute cell numbers in the marrow were determined using eight Syk+/
/3-83 mice, six Syk
/
/3-83 mice, three Syk+/
/3-83/Bcl-2Tg mice, and four Syk
/
/3-83/Bcl-2Tg mice;
data for the spleen cell numbers was determined using four Syk+/
/3-83 mice, four Syk
/
/3-83 mice, three Syk+/
/3-83/Bcl-2Tg mice, and two
Syk
/
/3-83/Bcl-2Tg mice.
[View Larger Versions of these Images (55 + 23 + 27K GIF file)]
/
Immature B Cells.
/
recirculating
IgM+IgDhigh B cells (Fig. 3, a and d). Immunohistology confirmed that the Bcl-2 transgene had caused the accumulation of many more Syk-deficient B cells in the spleens of
Syk
/
/3-83/Bcl-2Tg chimeras, and once again they were
exclusively located in the outer T zone and red pulp, but
not in follicles (Fig. 2 e). Thus, the expression of Bcl-2 allows Syk-deficient immature B cells to proceed a little further in their differentiation into transitional cells and to
accumulate in larger numbers in the spleen, but is not sufficient to rescue the developmental block in their maturation
into recirculating follicular B cells.
/
/3-83 chimeras with and without Bcl-2Tg were given a 12-h pulse with the thymidine analogue BrdU to identify cells proliferating during the pulse that had subsequently gone out of
cell cycle. Pre-B cells exit the cell cycle ~24 h before first
expressing surface immunoglobulin (35) and immature and
recirculating B cells do not subsequently proliferate unless
they are recruited into an antibody response (4). In wild-type rodents, pulse-labeled immature B cells enter the spleen
on the second and third day after a 12-h BrdU pulse and
are lost within 7 d (4, 36). In this experiment, spleens of
the chimeric mice were examined 4 and 8 d after the BrdU
pulse. At 4 d, BrdU-labeled 3-83+ B cells were present in
roughly equal proportions in mice with and without Bcl-2Tg (Fig. 4). By 8 d, as expected, BrdU-labeled B cells
were largely absent from the chimeras without the Bcl-2Tg (Fig. 2 d) but were present in an undiminished proportion
of the B cells carrying the Bcl-2Tg (Fig. 2 e). Thus, in the
absence of Bcl-2Tg, Syk
/
immature cells turn over with
normal kinetics. However, in chimeras with Bcl-2Tg, the
life span of Syk-deficient immature B cells was significantly
extended.
Fig. 4.
Life span of Syk/
immature B cells is increased by expression of Bcl-2. Chimeric mice reconstituted with Syk
/
/3-83 fetal livers
with and without Bcl-2Tg were given a 12-h pulse of BrdU. Graph shows
the percentage of splenic 3-83+ B cells that were also labeled with BrdU
either 4 or 8 d after the BrdU pulse. At each time point, spleens from
three or four mice were analyzed by quantitative immunohistology as
previously described (4). The lines are drawn between median values.
The number of cells enumerated was 168-263 cells/spleen in the Syk
/
/
3-83 chimeras and 673-994 cells/spleen in the Syk
/
/3-83/Bcl-2Tg chimeras.
[View Larger Version of this Image (18K GIF file)]
(Ig
)
gene with a mutated intracellular signalling domain (37). Since
CD79
is a signaling component of the BCR that may act
by directly binding and activating Syk (18), we propose
that the maturation of immature B cells into recirculating
follicular B cells requires a BCR signal, and that Syk is an
essential transducer of at least part of this signal.
/
B cells to be recruited into the recirculating B cell pool is superficially reminiscent of the fate
of autoreactive B cells that develop in the presence of soluble autoantigen (45). These B cells accumulate in the outer
T zone of the spleen and do not compete with nonautoreactive immature B cells for signals to enter the recirculating
B cell pool. They differ from the Syk
/
B cells described
here, and indeed from normal immature B cells (3, 4), in
that they do not migrate to the red pulp as well as the T
zone. It seems that these antigen-engaged B cells in the
outer T zone are programmed to interact with cells that
can deliver accessory signals that induce B cells to migrate
to and proliferate in follicles or extrafollicular foci (46, 47). Such cells concomitantly become inefficient in competing
for signals that induce maturation into recirculating B cells
(45). In contrast, the Syk
/
immature cells described here
are not antigen-engaged, migrate to both the red pulp and
T zones of the spleen, and fail to enter follicles even in the
absence of significant competition. It is more likely that the
follicular exclusion reflects the lack of a Syk-mediated signal required for maturation into a recirculating B cell. In
this regard, it is interesting to note that B cells lacking the
putative chemokine receptor Burkitt's lymphoma receptor 1 (BLR1) are also unable to enter splenic follicles, but remain trapped in the outer T zone (48). Since the expression of
BLR1 is increased upon maturation from immature to recirculating B cells, it is possible that Syk
/
B cells fail to
enter splenic follicles because they have not induced the
expression of BLR1. However this cannot explain the failure of Syk-deficient B cells to enter lymph node follicles, since apparently this occurs normally in the BLR1 mutants.
Address correspondence to V.L.J. Tybulewicz, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK. Phone: 44-181-913-8699; FAX: 44-181-906-4477; E-mail: v-tybule @nimr.mrc.ac.uk
Received for publication 26 September 1997 and in revised form 24 October 1997.
The work in London was supported by the Medical Research Council and the work in Birmingham was supported by a Medical Research Council program grant.We thank C. Atkins for expert assistance in flow cytometric analysis; D. Nemazee for 3-83µ transgenic
mice and the hybridoma 54.1; S. Cory and M. Merkenschlager for permission to use and provision of the
Eµ-bcl-2-36 transgenic mice, respectively. We thank E. Schweighoffer for critical reading of the manuscript.
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