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Ontario Cancer Institute and Department of Immunology, University of Toronto, Toronto, Ontario, Canada
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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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Pro-B cells generating a productive HC gene rearrangement can express
an early form of the Ig receptor, termed the pre-B cell receptor
(pre-BCR), which contains the µ HC, the surrogate light chain
proteins 
5 and VpreB, the signaling components Ig-
and Ig-ß,
and several unidentified glycoproteins (7, 8, 9). Several lines of
evidence have indicated that pro-B cells that make a functional HC gene
rearrangement and express pre-BCR are triggered to enter a stage of
rapid cell division before differentiating into quiescent small pre-B
cells (1, 3, 10, 11). The importance of pre-BCR in regulating the pro-B
to pre-B cell transition is demonstrated by studies of mice deficient
in expression of pre-BCR components. Mice deficient in expression of HC
as well as mice deficient in expression of 5 show arrest of B cell
development at the pro-B cell stage (3, 12, 13). Mice able to produce
cytoplasmic but not membrane-bound HCs (14) show a similar arrest in B
cell development (3). Expression of Ig HC transgenes in
recombinase-activating gene (RAG)-deficient mice restores
differentiation to the pre-B cell stage (5, 15), and this restoration
requires the presence of 5 (16). Studies using mutant HC transgenes
demonstrate that association of the HC with Ig- and Ig-ß is
necessary for promoting the pro-B to pre-B cell transition in
RAG-deficient mice (17, 18). Thus, while the critical importance of
pre-BCR signals is well established, the mechanisms through which these
signals promote B cell differentiation are not understood.
Cytokine receptors are also involved in regulating the B cell developmental cascade. B cell progenitors in the bone marrow are in intimate contact with the processes of reticular stromal cells (1), and such stromal cells have been shown to promote survival and differentiation of B cell progenitors in vitro (19, 20, 21, 22, 23). Several cytokines produced by stromal cells have been implicated in the positive or negative regulation of early B cell development. Of these, IL-7 (24) is unique in its ability to induce clonal proliferation of primary B cell progenitors in vitro (22, 25, 26). The importance of IL-7 in B cell development in vivo was underscored by studies examining gene-targeted mice deficient in IL-7 or IL-7R (27, 28). In these studies, loss of the IL-7-IL-7R interaction led to a severe impairment of development beyond the pro-B cell stage. IL-7R is expressed on both pro- and pre-B cells, but not on sIgM+ B cells (29, 30, 31).
In this study we examine the interplay between pre-BCR and IL-7R signals at the level of biologic responses of primary pro-B cells. Our results provide clear evidence that responses through the IL-7R are modulated during pro-B cell differentiation in a pre-BCR-dependent fashion. Our results suggest that fine tuning of IL-7 responses may provide a mechanism for positive selection of pre-BCR+ pro-B cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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µmT mice (32) were generated in the laboratory of Dr. K. Rajewsky (Institute for Genetics, Cologne, Germany) and were obtained through Dr. L. Shultz (The Jackson Laboratory, Bar Harbor, ME). RAG-2-/- (13) and RAG-2-/- HC186 transgenic (tg) (5) mice were provided by Dr. F. Alt (The Children’s Hospital, Boston, MA). RAG-2+/- mice were generated by crossing RAG-2-/- mice with C57BL/6 mice. C57BL/6 mice were purchased from The Jackson Laboratory. All mice were bred and maintained at the Wellesley Hospital Research Institute animal facility (Toronto, Canada) and were used between 6 and 10 wk of age.
Cell purification and culture
Bone marrow cell suspensions were prepared and resuspended at
2 x 107 cells/ml in PBS and 5% FCS. Fc receptors
were blocked by incubation with the FcBLOCK Ab (PharMingen, San Diego,
CA). Cells were then stained with the following labeled Abs (all from
PharMingen): FITC-labeled anti-
light chain (mAb R8-140),
phycoerythrin (PE)-labeled anti-CD43 (mAb S7), and biotin-labeled
anti-CD19 (mAb 1D3). Staining with the biotin-labeled Ab was
revealed by second-step staining with quantum red-streptavidin (Sigma,
St. Louis, MO). Cell sorting was performed using a
FACStarPlus (Becton Dickinson, Mountain View, CA)
using gates similar to those indicated in Fig. 1
. Sorted populations were routinely
>95% pure.
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Abs and cell staining procedures
For determination of cell surface and cytoplasmic expression of
µ HC, cells cultured under the indicated conditions were surface
stained with biotin-labeled anti-IgM (mAb 33-60), followed by
quantum red-streptavidin (Sigma). Cells were then fixed in 1%
paraformaldehyde for 20 min at room temperature. After washing, cells
were permeablized with 0.2% Tween-20 in PBS for 15 min at room
temperature. FITC-labeled goat anti-mouse µ-chain Abs (Jackson
ImmunoResearch Laboratories, West Grove, PA) were then added, and the
cells were incubated on ice for 20 min. Cells were washed twice with
PBS containing 2.5% FCS and 0.2% Tween-20 and then analyzed on a
FACScan flow cytometer (Becton Dickinson). For the experiment shown in
Fig. 5B, cells were doubly stained with PE-labeled
anti-CD2 (mAb RM2-5; PharMingen) and FITC-labeled anti-IgM (mAb
33-60), and the CD2+IgM- and
CD2-IgM- cells were sorted on the
FACStarPlus (Becton Dickinson).
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-chain expression, bone marrow cells were
stained for four-color FACS analysis using allophycocyanin-labeled
anti-B220 (mAb RA3-6B2; PharMingen), PE-labeled anti-CD43,
biotin-labeled anti-IL-7R -chain (mAb A7R34 (29); gift from Dr.
S. Nishikawa), and either FITC-labeled anti-CD19 (mAb 1D3;
PharMingen) or FITC-labeled anti- light chain. Staining with the
biotin-labeled Ab was revealed by second-step staining with quantum
red-streptavidin. Four-color-stained cells were analyzed on a FACS
Calibur flow cytometer equipped with CellQuest software (Becton
Dickinson). |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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light
chain-CD43+ pro-B cells from normal or
HC-deficient bone marrow (Fig. 1
) and assessed the ability of these
cells to proliferate and differentiate in response to IL-7. HC-deficient pro-B cells have a specific defect in low threshold IL-7 responses
Since the mechanism leading to selective expansion of
pre-BCR+ pro-B cells is not known, we hypothesized that
pre-BCR-dependent signals might be required for pro-B cells to undergo
proliferative responses in the presence of physiologic levels of IL-7.
We thus compared the ability of pro-B cells from normal and
HC-deficient mice to proliferate in the presence of a wide range of
IL-7 concentrations (Fig. 2). Strikingly,
we found that HC-deficient pro-B cells exhibited a specific deficiency
in the ability to respond to low levels of IL-7. This effect was most
pronounced in the picogram per milliliter range of IL-7 concentrations,
where the HC-deficient cells respond with up to 10-fold lower levels of
proliferation than normal pro-B cells. In contrast, HC-deficient pro-B
cells proliferate to an equal or greater extent than normal pro-B cells
in the nanogram per milliliter range of IL-7 concentrations (Fig. 2).
The IL-7-responding cells in these HC-deficient cultures expressed the
B-lineage-specific markers CD19, B220 (>95% positive), and BP1
(>80% positive; data not shown), confirming their B-lineage identity.
Thus, the deficiency in IL-7 responsiveness exhibited by HC-deficient
pro-B cells is not a complete loss of IL-7 responsiveness, but, rather,
represents a shift in the IL-7 dose-response threshold.
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). These data suggest that both normal
and HC-deficient pro-B cell populations contain cells with a high IL-7
dose-response threshold, but only the normal population contains cells
with a low IL-7 dose-response threshold. In contrast to the
CD43+ pro-B cell population, the CD43- pre-B
cell population from control mice showed no significant proliferation
over background at any IL-7 concentration (Fig. 2B), in
accord with previous findings (2, 30). Together these results suggest
that pre-BCR-expressing pro-B cells undergo a transient modulation of
the IL-7 dose-response threshold, followed by a complete loss of IL-7
responsiveness during differentiation to the pre-B cell stage. Culture of pro-B cells under low IL-7 conditions leads to selective outgrowth of cytoplasmic and surface µ+ cells
The differential ability of normal and HC-deficient pro-B cells to respond to picogram per milliliter IL-7 concentrations suggests that IL-7, present at such limiting levels, may represent a critical signal for selective expansion of pro-B cells that successfully rearrange and express one of their HC loci. We thus compared cytoplasmic and surface expression of µ HC in the cell populations responding to nanogram per milliliter and picogram per milliliter IL-7 concentrations.
Pro-B cells isolated from normal or HC-deficient bone marrow were
cultured in bulk in the presence of 24 ng/ml or 24 pg/ml IL-7. After 4
days of culture, cell recoveries for control and HC-deficient pro-B
cell cultures were similar at 24 ng/ml IL-7, while at 24 pg/ml
approximately 10-fold fewer cells were recovered from HC-deficient
cultures than from control cultures (Fig. 3A). Recovered cells were
stained with Abs against surface IgM and then fixed, permeabilized, and
stained for cytoplasmic µ (cµ; Fig. 3B). Normal pro-B
cells cultured for 4 days with 24 ng/ml IL-7 have evident
cµ+ and cµ- populations. As expected, the
cµ- population lacks expression of surface IgM (Fig. 3B), but continues to express CD19 (data not shown). A small
fraction of the cµ+ cells expresses IgM on the cell
surface (Fig. 3B). After a further 3 days of culture with 24
ng/ml IL-7, a greater proportion of the cells are cµ-,
and a greater proportion of the cµ+ cells now express IgM
on the cell surface. In contrast, normal pro-B cells cultured with 24
pg/ml IL-7 lack the distinct cµ- population present in
the 24 ng/ml IL-7 cultures. These cultures also have a higher frequency
of surface IgM+ cells, which is especially apparent on day
7 of culture. These results indicate that culture of normal pro-B cells
in the presence of picogram per milliliter IL-7 concentrations
selectively expands µ+ cells, while culture with nanogram
per milliliter IL-7 concentrations maintains both µ+ and
µ- B cell precursors. These data are consistent with the
hypothesis that, under limiting conditions, the IL-7 signal alone can
support selective expansion of functionally rearranged B cell
precursors.
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To examine the effect of enforced pre-BCR expression on pro-B cell
IL-7 responses we examined RAG-2-/- pro-B cells
carrying a rearranged µ transgene (RAG-2-/-
HCtg) (5). On day 4 of culture, the RAG-2-/-
HCtg cultures showed an approximately twofold lower level of
proliferation than RAG-2-/- cultures in the
presence of nanogram per milliliter levels of IL-7 (Fig. 4A). In contrast, at picogram
per milliliter IL-7 concentrations, the
RAG-2-/- HCtg pro-B cells show a three- to
sixfold higher response than RAG-2-/- pro-B
cells. The CD43- pre-B cell population generated in the
HCtg bone marrow was also examined and was unresponsive to IL-7 at any
of the concentrations tested (Fig. 4A). These data
demonstrate that expression of Ig HC in the absence of conventional Ig
LCs is sufficient to modulate the IL-7 dose-response threshold at the
pro-B cell stage and shut down IL-7 responsiveness at the pre-B cell
stage.
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B). Interestingly, the
response of the RAG-2-/- HCtg pro-B cells
peaked earlier than that of the RAG-2-/- pro-B
cells, with the highest response on day 3 and a marked decline by day
5. In contrast, proliferation of RAG-2-/- pro-B
cells did not reach maximal levels until day 4 of culture, and no
significant decrease occurred on days 5 and 6 (Fig. 4B). Pre-BCR expression limits the duration of IL-7-induced clonal expansion
One hypothesis to explain the effect of enforced pre-BCR
expression on the kinetics of the IL-7 response is that expression of
pre-BCR leads to differentiation to an IL-7-unresponsive stage during
the culture period. This hypothesis would predict that during culture
with IL-7, pre-BCR-expressing cells will gradually lose the ability to
clonally expand in response to IL-7, while pre-BCR-deficient cells will
retain this ability. To test this prediction, we cultured
RAG-2+/-, RAG-2-/-, or
RAG-2-/- HCtg pro-B cells for 4 days in the
presence of high levels of IL-7 and then determined the frequency of
cells retaining the ability to clonally expand in IL-7. We found that
RAG-2-/- cultures retain a substantially
greater frequency of cells clonable in IL-7 than RAG-2+/-
or RAG-2-/- HCtg cultures (1/6 to 1/9 vs 1/27
to 1/40 or 1/48 to 1/90, respectively; Fig. 5A).
Since IL-7 responsiveness is clearly lost during the pro-B to pre-B
cell transition in vivo (Figs. 2 and 4), we hypothesized that the
pre-BCR-dependent loss of IL-7 responsiveness during in vitro culture
could represent an analogous differentiation event. We thus sought to
determine whether the IL-7-unresponsive cells arising in normal pro-B
cell cultures could be separated from the cells retaining IL-7
responsiveness by virtue of a pre-B vs pro-B cell phenotype. We used
CD2 as a marker for pre-B cells (5), since the starting pro-B cell
populations are CD2-, and CD2 expression clearly
discriminates a pre-B cell population arising in IL-7 cultures of
normal, but not pre-BCR-deficient, cells (data not shown). Day 4
cultures were sorted into CD2-/IgM- or
CD2+/IgM- fractions, and it was found that the
CD2+ population has completely lost the ability to form
clones in the presence of IL-7, while the CD2- population
contains a high frequency of cells clonable in IL-7 (Fig. 5B). These results indicate that culture of normal pro-B
cells with IL-7 leads to development of a pre-B cell population that
becomes IL-7 unresponsive.
What is the fate of pre-BCR-expressing pro-B cells that differentiate
into IL-7-unresponsive pre-B cells during culture with IL-7? To address
this issue we conducted experiments designed to follow the fate of
individual pro-B cell clones isolated from RAG-2+/-,
RAG-2-/-, or RAG-2-/-
HCtg bone marrow. Pro-B cells were plated at limiting dilution in the
presence of nanogram per milliliter concentrations of IL-7 on the day
of purification from bone marrow, and individual clones were identified
on day 4 and reassessed on days 7 and 11 of culture. This analysis
revealed striking differences between the different genotypes.
RAG-2-/- pro-B cell clones largely survived and
continued to increase in size throughout the 11-day culture period
(Fig. 5C and data not shown). In contrast, >90% of the
RAG-2-/- HCtg clones identified on day 4 ceased
proliferating and died by day 11 (Fig. 5C). This result is
in accord with data indicating an inability to established long term
proliferating pre-B cell cultures from RAG-2-/-
HCtg bone marrow (5). RAG-2+/- clones showed an
intermediate pattern, with approximately 70% of the clones dying by
day 11.
Together these results support the hypothesis that expression of pre-BCR on pro-B cells ultimately leads to differentiation to a pre-B cell stage, where proliferation in response to IL-7 is arrested. In the absence of further survival and/or differentiation signals, this growth arrest appears to end in cell death.
Loss of IL-7 responsiveness at the pre-B cell stage, but not
pre-BCR-dependent modulation of IL-7 responsiveness at the pro-B cell
stage, is associated with altered expression of IL-7R -chain
To determine whether the observed pre-BCR-dependent alterations in
IL-7 responsiveness could be accounted for by regulation of IL-7R
expression, we examined surface expression of the IL-7R -chain in
RAG-2+/-, RAG-2-/-, and
RAG-2-/- HCtg bone marrow by multicolor flow
cytometry (Fig. 6). Examination of the
entire CD19+ B-lineage compartment indicates that
IL-7R+ cells express similar levels of IL-7R on their
surface in the three mouse genotypes. However, the pre-BCR-deficient
RAG-2-/- mice show a significantly higher
frequency of IL-7R+ cells, presumably due to the absence of
later developmental stages that are IL-7R- (29, 30). When
the B220+CD19+CD43+ pro-B
populations are compared, the frequencies of IL-7R+ cells
observed in the three genotypes are more similar, with
RAG-2+/- pro-B cells having 44.3–55.3%
IL-7R+ cells, RAG-2-/- pro-B cells
showing 62.9–65.8%, and RAG-2-/- HCtg cells
showing 32.4–48.6% IL-7R+ cells (Fig. 6A). The
levels of IL-7R expressed on the surface of IL-7R+ pro-B
cells is undistinguishable among the three genotypes. These results
indicate that the differential ability of pre-BCR-expressing and
pre-BCR-deficient pro-B cells to respond to picogram per milliliter
IL-7 concentrations is unlikely to be due to differential expression of
IL-7R -chain.
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-chain expression in B-lineage subpopulations in
the control RAG-2+/- bone marrow indicates an interesting
pattern of expression during B cell differentiation (Fig. 6B). IL-7R is expressed at the highest levels on
CD43+ pro-B cells, with CD43- pre-B cells
showing substantially decreased levels of expression. Consistent with
this result, expression of the HC transgene in
RAG-2-/- mice, which restores development of
pre-B cells (5), leads to down-regulation of IL-7R expression in the
B-lineage compartment (Fig. 6A). These findings indicate
that the loss of IL-7 responsiveness at the CD43- pre-B
cell stage (Figs. 2B and 4A) correlates with
reduced expression of IL-7R -chain. light chain+ B
cells show no detectable expression of IL-7R (similar to control
staining; data not shown), as has been demonstrated previously
(29, 30, 31). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The data presented here clearly show that the ability of pro-B
cells to respond in the presence of nanogram per milliliter levels of
IL-7 is not dependent on pre-BCR expression. This conclusion is in
apparent conflict with earlier work examining B cell progenitors from
scid mice (33, 34), which concluded that expression of the
HC is required to generate IL-7-responsive cells. Given the results of
our study and others (5) demonstrating IL-7 responses in RAG-deficient
B cell progenitors, it appears that the scid mutation may
interfere with the development of IL-7-responsive pro-B cells for
reasons other than their lack of pre-BCR expression. Indeed, the
present results show that pro-B cells from HC-deficient mice often have
greater levels of proliferation than normal pro-B cells in the
presence of high levels of IL-7. This increase is probably
related to the failure of HC-deficient pro-B cells to differentiate to
an IL-7-unresponsive stage (Fig. 5), which leads to a more sustained
response.
Pre-BCR-dependent modulation of IL-7 responsiveness during B-lineage differentiation
In contrast to the results at saturating IL-7 concentrations, we
find that expression of pre-BCR is required to generate pro-B cells
capable of responding to picogram per milliliter levels of IL-7. This
result defines a novel and potentially critical regulatory event in
early B cell differentiation that is dependent on pre-BCR expression.
The ability of pre-BCR to alter the threshold of ligand needed for
triggering through IL-7R could bear some analogy to other multireceptor
systems in lymphocytes such as Ag receptor-costimulatory molecule
systems. For example, the concentration of ligand required for
triggering through the BCR can be altered by up to 100-fold when
coreceptors such as CD19, CD22, or Fc
RIIb1 are coligated
(35). In these cases, the modulation of the triggering threshold is
thought to occur at least in part through direct alteration of
receptor-proximal signaling events by recruitment of regulatory protein
tyrosine phosphatases (35, 36). However, the present data do not
distinguish whether pre-BCR signals alter IL-7R signals directly
through a relatively simple biochemical cascade (as in the above
examples) or indirectly through a more complex cascade of
differentiative events that alter the signaling milieu within the cell.
It is possible that the pathway from pre-BCR expression to altered
responses through IL-7R involves several intermediate biochemical or
transcriptional events or even a discrete differentiation step. In any
case, it will be important to identify the regulatory target molecules
involved in modulating responses through IL-7R and determine whether
responses through other cytokine receptors, particularly the receptors
for IL-2, IL-4, IL-9, and IL-15, which share the common subunit
c with IL-7R (37), are modulated in a dose-dependent
manner during lymphocyte development.
As IL-7R is thought to mediate both cell survival and proliferation, it
will be interesting to determine whether the pre-BCR-dependent
modulation of IL-7 responsiveness is due to altered cell survival
signals, cell proliferation signals, or both. The present data
examining cell recoveries by trypan blue staining (Fig. 3A)
show an approximately 50% decrease in viable cell numbers for
pre-BCR-deficient pro-B cells after 4 days of culture in picogram per
milliliter IL-7 concentrations vs a fourfold increase in viable cells
in controls. However, this does not represent compelling evidence for
cell death over failure to proliferate, given that these are primary
cells that have been run through a FACS sorter before culture. Further
studies will be required to address this issue.
We also provide evidence that expression of pre-BCR leads to shortened
duration of IL-7-induced clonal expansion by causing differentiation to
an IL-7-unresponsive pre-B cell stage. Thus, expression of pre-BCR
appears to lead to two sequential alterations of IL-7 responsiveness:
1) the transient modulation of the IL-7 dose response at the pro-B cell
stage, followed by 2) the shutdown of IL-7 responsiveness at the pre-B
cell stage. These two regulatory events probably represent independent
downstream consequences of pre-BCR-dependent signals. Modulation of the
dose-response threshold at the pro-B cell stage does not appear to be
associated with changes in IL-7R expression and so is likely to be due
to altered signaling pathways. In contrast, the shutdown of IL-7
responsiveness at the pre-B cell stage correlates with decreased
expression of IL-7R, providing a potential mechanism for this
alteration in responsiveness. In this context, it is interesting that
our data indicate a possible trend showing a lower frequency of
IL-7R+ cells in RAG-2-/- HCtg pro-B
cells than in RAG-2+/- or RAG-2-/-
pro-B cells (Fig. 6). This apparent trend may be related to the
down-regulation of IL-7R expression during the pro-B to pre-B cell
transition, in that the early and high level expression of pre-BCR in
the heavy chain tg pro-B cells may lead to rapid down-regulation of
IL-7R concomitant with CD43 down-regulation, resulting in more frequent
inclusion of IL-7R-negative late pro-B cells in the CD43+
pro-B cell gate. Further studies will be required to clearly address
this issue.
Previous experiments assessing the effect of a µ transgene on growth factor responses of HC-deficient pro-B cells have given rise to diverging interpretations (5, 15). One study found that expression of a µ transgene greatly decreases proliferative responses when whole bone marrow cells are cultured with IL-7 for 5 days (5). Since the frequency of pro-B cells is reduced in HCtg bone marrow (Refs. 5 and 15 and data not shown), and our data suggest that the IL-7-induced proliferative response peaks earlier (day 3) with HCtg pro-B cells, this study may have underestimated the capacity of these cells to proliferate in response to IL-7. However, the conclusion that pre-BCR expression leads to eventual shutdown of IL-7 responsiveness is in complete accord with our results. Another study concluded that a µ transgene promotes the ability of HC-deficient pro-B cells to undergo contact-independent proliferation in the presence of a stromal cell line in the absence of exogenously added IL-7 (15). Interestingly, it has been shown that in some stromal cell lines, expression of IL-7 mRNA can be dramatically up-regulated by contact with pre-B cells (38). Thus, given the present results, one interpretation of the contact dependence experiment is that the requirement of HC-deficient cells for stromal cell contact simply reflects a requirement for higher levels of IL-7.
Role of IL-7 and pre-BCR in regulating the pro-B to pre-B cell transition
Since expression of pre-BCR appears to regulate IL-7
responsiveness, it could be possible that the role of HC in promoting
the pro-B to pre-B transition in vivo is simply to lower the threshold
of IL-7 responsiveness in pro-B cells sufficiently to allow survival
and continued differentiation in the presence of the limiting amounts
of IL-7 present in vivo. However, culture of HC-deficient pro-B cells
in the presence of saturating IL-7 levels does not rescue
differentiation to the pre-B cell stage, as assessed by surface marker
expression and the level of Ig light chain gene rearrangement (39),
suggesting that the extended survival and proliferation afforded by
superphysiologic IL-7 concentrations are insufficient to overcome the
requirement for pre-BCR. In contrast, normal pro-B cells clearly
undergo differentiation to the pre-B cell and B cell stages during
short term culture with IL-7 (Fig. 3B) (39).
Rolink and colleagues found that long term B cell precursor lines
undergo differentiation events when IL-7 is withdrawn from the
cultures, suggesting that culture in saturating IL-7 conditions may
inhibit differentiation of pro-B cells to pre-B and B cell stages (23).
Our present results using short term cultures indicate that this
putative inhibitory effect is at best partial, since normal pro-B cells
do differentiate into pre-B cells and IgM+ B cells in the
presence of ng/ml IL-7 levels, albeit at a lower frequency than
observed in the picogram per milliliter cultures (Fig. 3B
and data not shown). In light of the data indicating that
pre-BCR-deficient pro-B cells fail to proliferate in response to
picogram per milliliter concentrations of IL-7 (Figs. 2, 3A,
and 4), we consider it most likely that the striking difference in
frequency of µ- B cell precursors found in nanogram per
milliliter vs picogram per milliliter IL-7 cultures (Fig. 3B) is due to differential proliferation of the
µ- cells; however, we cannot rule out the possibility
that nanogram per milliliter IL-7 concentrations may also partially
inhibit the differentiation of these µ- cells.
Implications for B cell differentiation in the bone marrow
Several lines of evidence indicate that IL-7 may be present in limiting quantities at the sites of B lymphopoiesis in the bone marrow. Many stromal cell lines that support B lymphopoiesis in vitro produce very low levels of IL-7 mRNA and free soluble IL-7 (20, 21). In addition, IL-7 is bound by extracellular matrix components such as fibronectin and heparin sulfate (40, 41), which are produced by stromal cells. IL-7 bound to heparin sulfate was shown to be functionally inactivated (40), suggesting that IL-7 activity may normally be attenuated by binding to the extracellular matrix. There have also been suggestions that the IL-7 made by stromal cells is only released in small quantities and in a specifically regulated manner (42). Lastly, IL-7 tg mice (43, 44) and mice injected with rIL-7 (45) show a large increase in the number of pre-B cells in the bone marrow, suggesting that the amount of pro-B cell proliferation is normally limited by the low availability of IL-7.
If IL-7 is indeed present at limiting levels, cells able to respond to low IL-7 concentrations could have a large proliferative advantage in vivo. Thus, the ability of pre-BCR to modulate the IL-7 dose-response threshold could be critical in providing pre-BCR+ pro-B cells with a proliferative advantage over pre-BCR- pro-B cells, leading to the preferential accumulation of functionally rearranged HC alleles before light chain rearrangement (10, 31, 46, 47).
Together with data from the literature, the results presented
here suggest the following model for the role of pre-BCR in regulating
IL-7 responsiveness and differentiation (Fig. 7). IL-7R is expressed soon after
commitment to the B lineage; however, the earliest B-lineage-committed
progenitors do not respond to even high levels of IL-7 (48). In
contrast, heat-stable Ag+ early pro-B cells undergoing
DJH rearrangement develop the ability to proliferate in
response to superphysiologic levels of IL-7. However, our data suggest
that before pre-BCR expression, the array of signals transmitted
through the IL-7R is insufficient to trigger proliferation at the
presumably low concentrations of IL-7 present in the developmental
microenvironment. While these cells may not proliferate in response to
IL-7 in vivo, it is plausible that IL-7R-mediated signals could
play some role in mediating the survival and differentiation of early
pro-B cells, as has been previously suggested (28, 49).
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and Ig-ß (17, 50, 51).
We postulate that this signal directly or indirectly alters the milieu
of signaling mediators such that the threshold for IL-7 responsiveness
is lowered, allowing the cells to proliferate in response to the low
levels of IL-7 present in the microenvironment. After several rounds of
division, this proliferative response terminates in differentiation to
the pre-B stage and loss of the ability to proliferate in response to
IL-7 (2, 25, 52) (Fig. 5). Our data (Fig. 6) as well as those from
other studies (53) indicate that this loss of IL-7 responsiveness at
the pre-B cell stage correlates with reduced expression of IL-7R. Thus, through the collaborative action of pre-BCR and IL-7R signals, a pre-B cell pool enriched for cells expressing functional HC proteins would be generated before activation of LC rearrangement.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology, University of Washington, Seattle, WA 98195.
3 Address correspondence and reprint requests to Dr. Christopher J. Paige, Ontario Cancer Institute, 610 University Ave., Toronto, Ontario, Canada M5G 2M9.
4 Abbreviations used in this paper: HC, immunoglobulin heavy chain; LC, immunoglobulin light chain; BCR, B cell receptor; RAG, recombinase-activating gene; tg, transgenic; PE, phycoerythrin; cµ, cytoplasmic µ.
Received for publication March 30, 1998. Accepted for publication August 7, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5 and Vpre-B) and the immunoglobulin µ chain form a complex that is transported to the cells surface. J. Exp. Med. 172:973.5 surrogate light chain on the surface of µ heavy chain-negative early precursor B cell lines. J. Exp. Med. 178:469.5 protein in B cell development. Cell 69:823.[Medline]
and Igß can independently induce the precursor B cell transition and allelic exclusion. J. Exp. Med. 182:1389.. Blood 74:1936.RIIb1. Curr. Opin. Immunol. 8:378.[Medline]
chain: a functional component of the interleukin-7 receptor. Science 262:1877. chain transmits distinct signals for proliferation and differentiation during B lymphopoiesis. EMBO J. 15:1924.[Medline]
5 is expressed on the surface of mouse pre-B cell lines and can function as a signal transduction molecule. Int. Immunol. 3:1129.L chain gene rearrangements and c-kit and IL-7 receptor expression in stromal cell-dependent pre-B cells. J. Immunol. 149:1973.[Abstract]
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