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,
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Molecular Biology Institute, University of California, Los Angeles, CA 90095; and Departments of
Pediatrics and
Immunology, University of Washington, Seattle, WA 98195
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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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2 x
107 IgM+ BM immature B
cells are produced daily (3). Only 10–20% of these cells
survive to exit the BM and enter the spleen (4, 5). B
lymphocytes in the spleen were initially categorized into mature (M)
and immature, or transitional, cells. In contrast to immature B cells,
mature cells are long-lived (15–20 wk vs 3–4 days for immature cells)
and recirculate beyond the spleen to secondary lymphoid organs
(6). Specific patterns of surface marker expression
have been used to distinguish immature(heat-stable Ag
(HSA)highIgMhighIgDlowB220low)
from mature
(HSAlowIgMlowIgDhighB220high)
splenic B cells (4, 7).
Signaling through the B cell receptor (BCR) is required for the
development and maintenance of mature splenic B lymphocytes. Deletion
of the Ig cytoplasmic tail leads to a severe block in the generation of
peripheral B cells (8). Conditional ablation of the IgH
chain in the periphery leads to rapid death in all splenic B cell
populations (9), suggesting that an intact BCR is crucial
for the survival of splenic B cells. Additional experiments,
demonstrating a restricted BCR repertoire in mature splenic cells,
suggest that signals from the Ag receptor drive B cell selection and
differentiation into the pool of long-lived mature cells
(10, 11, 12, 13, 14, 15). Furthermore, mice defective in Bruton’s
tyrosine kinase (Btk) (16) and several other BCR signaling
molecules (including Syk, Lyn, phosphatidylinositol 3-kinase,
BLNK, phospholipase C
2, and VAV) (17, 18, 19, 20) each
exhibit reduced numbers of mature splenic B cells. Together, these
mouse models suggest two alternative, but not necessarily mutually
exclusive, possibilities: 1) BCR signaling in immature B cells is
required for their maturation into long-lived mature cells, and/or 2)
BCR signaling in mature B cells is required for their ongoing
survival.
Previous studies suggest that, in response to BCR cross-linking, HSAlow mature B cells proliferate, whereas HSAhigh immature splenic B cells are relatively nonresponsive and die (7, 21). An implication of these results is that the splenic immature B cell stage acts as a target for negative selection in the periphery (21). However, recent reports have subdivided the immature (HSAhigh) splenic B cell population into two distinct subsets: transitional 1 (T1) and transitional 2 (T2) B cells (22). In vivo experiments indicate that T1 cells give rise to T2 cells, whereas T2 cells can further differentiate into HSAlow mature follicular (M) B cells. Confocal microscopy reveals that T2 cells are situated within primary follicles adjacent to M cells, whereas T1 cells are located at the outer periarteriolar lymphoid sheath (PALS), outside of the follicle (22). Furthermore, a large fraction of T2 cells are in G2/M phase of the cell cycle, suggesting they are in a more activated state than the T1 immature subset. A recent report also indicates that T2 cells, but not T1 cells, actively proliferate in response to the novel B cell growth factor BAFF (BLyS, TALL-1, THANK, zTNF4) (23). These data suggest that immature splenic B cells are heterogeneous and that the T1 and T2 subsets may respond differentially to key developmental signals.
In the current report, we directly evaluated the BCR-dependent signaling function of highly purified T1 vs T2 splenic immature B cells. Our data demonstrate important differences in BCR responsiveness between T1 and T2 cells. Most notably, the T2 subset of HSAhigh immature B cells generates proliferative, antiapoptotic, and differentiation signals in response to BCR engagement. In contrast, the T1 subset is relatively unresponsive to BCR stimulation. These observations argue against a requirement for BCR signaling in T1 cell development but indicate that BCR signaling likely plays a critical role in T2 B cell survival and maturation. Taken together with previously published data, the distinct BCR-dependent responses of these two immature subsets are consistent with T1 cells being a target for B lineage negative selection and T2 cells playing a unique role in Ag-driven positive selection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c and BALB/xid mice were bred and maintained in the animal facilities of the MacDonald Research Laboratories and handled according to guidelines of the University of California (Los Angeles, CA) Animal Research Committee. Mice used in all experiments were between 6 and 12 wk old.
Cells and reagents
Single cell suspensions were prepared from splenocytes depleted of erythrocytes by lysis with ammonium chloride solution. Murine splenocytes were cultured in RPMI 1640 with 5% FCS plus supplement (glutamine, 2-ME, penicillin, streptomycin, 10 mM HEPES). Cells were stimulated with 10 µg/ml polyclonal goat F(ab')2 anti-mouse IgM Ab (The Jackson Laboratory, Bar Harbor, ME) or 1 µg/ml each of PMA plus ionomycin (Calbiochem, La Jolla, CA).
Flow cytometry
For cell surface staining, 5 x 105 cells per sample were incubated with appropriate Abs. Data were collected on a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA) and analyzed using CellQuest software (BD Biosciences). Anti-CD21-FITC (7G6), anti-CD24(HSA)-PE (M1/69), anti-CD24(HSA)-biotin (M1/69), anti-CD23-biotin (B3B4), anti-B220-PE (RA3-6B2), anti-CD69-FITC (H1.2F3), and streptavidin-allophycocyanin were purchased from BD PharMingen (San Diego, CA). Anti-IgM-Cy5 Abs were purchased from The Jackson Laboratory, and anti-IgD-PE (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) were from Southern Biotechnology Associates (Birmingham, AL). All staining profiles were based on live-gated cells, as determined by forward and side scatter. For cell sorting, 5 x 107 cells per sample were incubated in 500 µl of staining medium (RPMI 1640 with 2.5% FCS plus supplement) with various Abs. Cells were sorted on a FACSVantage cell sorter (BD Biosciences) into 1 ml of collection medium (RPMI 1640 with 20% FCS plus supplement).
[3H]Thymidine uptake proliferation assay
Purified cells were incubated at 5 x 104/well in RPMI 1640 with 5% FCS plus supplement. Unless otherwise stated, cells were pulsed with 1 µCi [3H]thymidine for 12 h prior to harvesting. Cells were harvested and [3H]thymidine uptake was analyzed using a scintillation counter.
Immunoblotting
Total cell lysates were prepared by boiling in SDS-containing sample buffer for 10 min. Samples were resolved by 12% SDS-PAGE and transferred to a nitrocellulose membrane. Western blot analysis was performed using standard procedures. Lanes were loaded based on equal cell numbers. However, because certain cell populations (e.g., T2 cells) become more activated than others, we adjusted the loading volumes to approximate equivalent actin levels when necessary. Immunoblotting Abs used included: anti-cyclin D2 (M-20), anti-Bcl-xL (S-18; each from Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho Ser473-Akt, anti-Akt (New England Biolabs, Beverly, MA); anti-actin (A-2066; Sigma-Aldrich, St. Louis, MO). Normalized densitometry readings were generated by subtracting the background and dividing by the respective control (actin) band intensities. Densitometry readings are presented as ratios relative to the normalized readings of the first lane of each blot.
A1 RT-PCR
Total RNA was extracted from FACS-purified B cell populations using TRIzol reagent (Life Technologies, Rockville, MD), and single-stranded cDNA was synthesized using Powerscript RT (Clontech Laboratories, Palo Alto, CA) according to the manufacturer’s instructions. PCR was performed using Titanium Taq polymerase (Clontech Laboratories) using A1-specific oligonucleotides (sense, CCT GGC TGA GCA CTA CCT TCA; antisense, CTG CAT GCT TGG CTT GGA). PCR conditions were as follows: a hot start at 95°C for 10 min followed by 24–36 cycles of denaturing at 95°C for 30 s, annealing at 60°C for 30 s, and synthesis for 1 min at 72°C. PCR products were analyzed on a 1.2% agarose gel. Normalized densitometry readings were performed as described above. Experiments were initially done to identify the PCR cycle range reflecting a linear increase in the A1 PCR product band intensity. Samples were analyzed at every three cycles (24–36 cycles), and all the experiments shown used PCR cycle numbers within the linear range.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The initial characterization of splenic T1 and T2 cells
distinguished these two populations using the CD21 and CD24 (HSA) cell
surface markers (22). T1 (CD21low)
and T2 (CD21high) cells subdivide the immature
(HSAhigh) splenic B lymphocytes and are
distinguished from mature splenic B cells, which are
HSAlow.Surface IgM and IgD expression also
distinguishes T1
(IgMhighIgDlow), T2
(IgMhighIgDhigh), and M
(IgMlowIgDhigh) cells (data
not shown) (22). For cell sorting, previous studies have
isolated these splenic populations primarily using the CD21 and HSA
markers to avoid potential preactivation with the anti-IgM staining
Abs (22, 23). Following this same rationale, we stained
primary murine splenocytes for CD21 and HSA and used FACS to isolate
T1, T2, and M cells. The percentage of T1
(CD21lowHSAhigh), T2
(CD21highHSAhigh), and M
(CD21lowHSAlow) cells
falling within their respective gates was similar to that reported in
earlier studies (Fig. 1
A)
(22). After FACS isolation of these cells, reanalysis of
the sorted samples consistently revealed a sorting purity of between 84
and 98% for each population (Fig. 1A). We further
characterized the sorted cells by evaluating their B220, IgM, and IgD
surface expression levels (Fig. 1B). These results confirm
the phenotypes of the FACS-isolated T1, T2, and M cells and are
consistent with previous reports of these cell populations
(22).
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A). The immature T1 subset
did not proliferate after BCR stimulation (Fig. 2A). In
striking contrast to T1 cells, the T2 subset of immature B cells
proliferated robustly in response to BCR engagement. T2 cells
proliferated to a similar extent as M cells over a series of time
points (24, 36, and 48 h) (Fig. 2A). Of note, the
FACS-isolated T1 and T2 cells expressed equivalent levels of surface
IgM (Fig. 1
B), excluding the possibility that differences in
BCR responsiveness were secondary to differences in Ag receptor
density.
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60% of
CD21highHSAhigh cells are
CD23+ (T2), whereas 40% are
CD23- (MZ) (Fig. 1C). After FACS
isolation using all three surface markers, reanalysis of the sorted
populations revealed a purity of 88% for T2 cells and 95% for MZ
cells (Fig. 1C).
Equal numbers of purified T2 and MZ cells, along with T1 and M cells,
were assessed for BCR-mediated proliferation. Strikingly,
CD23+ T2 cells proliferated robustly, whereas
CD23- MZ cells responded minimally to BCR
activation (Fig. 2B). These results are consistent with
previous reports showing that MZ cells do not proliferate in response
to BCR cross-linking and instead undergo apoptotic cell death
(24). Using this more stringent purification strategy, T2
cells exhibited greater proliferation than M cells (Fig. 2B), indicating that the presence of contaminating MZ cells
in the CD21highHSAhigh
population led to an underestimation of the T2 proliferative response
in Fig. 2A.
Earlier reports indicated that T2 cells, compared with T1 or M cells,
contain a higher percentage of cells basally in the
G2/M phase of cell cycle (22). Cell
cycle analysis using propidium iodide revealed a similar increase in
cycling T2 cells (data not shown). We further investigated this early
activation of T2 cells using a proliferation assay at very early time
points (6–24 h) after BCR engagement. Interestingly, T2 cells entered
cell cycle much more rapidly than either T1 or M cells (Fig. 2C). Though the significance of this early BCR
responsiveness is unclear, these observations highlight the unique BCR
response of T2 cells and distinguish them from both T1 and M cells.
We also evaluated the capacity of T1, T2, and M cells to up-regulate
surface expression of the early activation marker CD69. BCR stimulation
led to an equivalent up-regulation of CD69 expression on the viable
cells in all three subpopulations (Fig. 2D). Of note, the
capacity of splenic T1 cells to up-regulate CD69 is distinct from a
previous report indicating that phenotypically similar immature B cells
in the BM fail to up-regulate this marker (25). Thus, the
T1, T2, and M splenic B cell populations each exhibit the ability to
generate at least a subset of early BCR-dependent signals, despite
having distinct proliferative responses.
T2, but not T1, B cells up-regulate cyclin D2 after BCR engagement
Cyclin D2 is a critical cell cycle regulator that is expressed in
splenic B cells after BCR engagement (26). Therefore, we
tested the ability of splenic T1, T2, and M cell populations to
up-regulate cyclin D2. Strikingly, after 12 h of BCR stimulation,
cyclin D2 expression was predominantly induced in
CD21highHSAhigh T2 cells
(Fig. 3A). In contrast, T1
cells only minimally induced cyclin D2 (Fig. 3, A and
B). Interestingly, the cyclin D2 up-regulation in M cells
was also significantly less robust than in T2 cells at all time points
evaluated (Fig. 3, A and B, and data not shown).
This difference was consistent with the early proliferative response of
T2 cells (Fig. 2C). The marked differences in cyclin D2
up-regulation between T1, T2, and M cells persisted for at least
48 h post-BCR engagement (data not shown).
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B. BCR cross-linking led
to no significant induction of cyclin D2 expression in
CD23- MZ cells (Fig. 3B). In
contrast, BCR-activated
CD23+CD21highHSAhigh
T2 cells strongly up-regulated cyclin D2 expression (Fig. 3B). In summary, the inability to fully up-regulate cyclin
D2 in T1 and MZ cells correlates with their inability to proliferate in
response to BCR cross-linking. The rapid and robust cyclin D2 induction
in T2 cells further correlates with their early proliferative
capacity. A BCR-dependent survival signal is generated in T2 but not T1 B cells
In addition to proliferative signals, BCR engagement
leads to antiapoptotic signals in splenic B cells
(27, 28, 29). After 48 h of in vitro culture in the
absence of any stimulation, the majority of primary murine splenocytes
undergo cell death (<30% viable) as assessed by forward and side
scatter (Fig. 4A). However, in
the presence of BCR cross-linking Abs, splenocyte viability remained
nearly as high (75–80%) as that observed in freshly isolated cells
(80% viable; Fig. 4A). These results suggest that a
BCR-mediated signal can protect at least some splenic B cells from
death.
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B). However, in the
presence of BCR stimulation, T2 (78% viable) and M (85% viable) cells
were rescued from death (Fig. 4B). In contrast, BCR
stimulation did not rescue T1 cells from death and, instead,
reproducibly enhanced cell death in this developmental subset (<10%
viable).
A1/Bfl-1 is an antiapoptotic factor in mature B lymphocytes
(30, 31, 32). A1 is also highly expressed in long-lived mature
splenic B cells (33). Expression array analysis indicates
that A1 is a rapidly induced, BCR-dependent, splenic B cell gene
product (T. T. Su and D. J. Rawlings, manuscript in
preparation). To assess the ability of T1, T2, and M cells to
up-regulate A1, we used RT-PCR to quantitate the levels of A1 mRNA
after BCR engagement. After 12 h of BCR stimulation, A1 was
markedly induced in T2 and M cells (Fig. 4C). In contrast,
A1 was only minimally induced in T1 cells (Fig. 4C).
Like A1, Bcl-xL is an additional, inducible B
cell survival factor (27, 28, 31). Furthermore,
Bcl-xL-/- mice exhibit a
dramatic loss of the peripheral lymphoid system (34).
Bcl-xL induction in T1, T2, and M cells was
assessed by Western blot analysis. Similar to the results with A1,
Bcl-xL expression was consistently (50%)
higher in T2 cells than in T1 cells (Fig. 4D).
Akt/PKB is a proto-oncogene important for a range of cellular growth
and survival signals (35, 36). Akt is a serine/threonine
kinase known to phosphorylate and inactivate the proapoptotic protein
Bad (37, 38). Recent evidence has also implicated Akt in
NF
B-dependent survival signaling (39, 40, 41). Both A1 and
Bcl-xL are NFB-dependent genes (30, 31, 32, 42), and Akt activity is induced upon Ag receptor engagement in
total splenic B cells and mature B cell lines (data not shown)
(43). Therefore, we investigated the ability of T1, T2,
and M cells to activate Akt. Akt activity, as measured by site-specific
phosphorylation at serine 473, peaks at 2 min after BCR stimulation and
gradually decreases after 5, 10, and 20 min (data not shown). At the
peak time point, Akt activity was predominantly induced in T2 cells
after BCR activation (Fig. 4E). In contrast, little or no
Akt activation was observed in T1 cells (Fig. 4E). In
addition, M cells exhibited a significantly lower level of Akt
activation than T2 cells, suggesting that Akt-independent mechanisms of
NFB activation may exist in long-lived mature B cells. The
difference in Akt activation may also be exaggerated, in part, by the
early BCR responsiveness of T2 cells. Together, these data demonstrate
that the immature T1 and T2 subsets clearly differ in their ability to
generate a BCR-dependent survival signal as revealed by both cellular
and molecular analysis.
BCR engagement drives the differentiation of T2 B cells into a mature B cell phenotype
The data presented demonstrate clear differences in BCR responsiveness between the T1 and T2 immature B cell subsets. T2 cells uniquely proliferate and survive in response to BCR cross-linking, consistent with a potential role in positive selection. However, in addition to these molecular events, the process of positive selection requires that the selected subset also be capable of differentiating into the subsequent developmental stage.
To begin to address the developmental capacity of these B lineage
populations, the ability of T2 cells to differentiate in vitro into M
cells was evaluated. For these studies, we used an in vitro assay
similar to that previously described in studies of the B cell growth
factor, BAFF (23). T1, T2, and M cells were FACS isolated
using CD21/HSA surface markers as above (Figs. 1A and
5A). The cell populations were
cultured in the presence or absence of BCR stimulation and stained
again for CD21/HSA after 48 or 96 h. Consistent with our earlier
results, the majority of T1 cells died within 48 h of receptor
engagement (Fig. 4B). However, the CD21/HSA profile (gated
on the live population) remained essentially unchanged in both T1 and M
populations (data not shown). In contrast, BCR stimulation of purified
T2 cells led to a progressive decrease in
CD21highHSAhigh T2 cells
(Fig. 5, A and B) and a time-dependent increase
in CD21lowHSAlow M cells
(Fig. 5, A and C). Ag receptor activation of T2
cells led to the generation of 34 and 54% of cells exhibiting a mature
cell phenotype by 48 and 96 h, respectively. This increase in M
cells is not likely a result of preferential outgrowth or selective
survival of a small fraction of contaminating M cells, because T2 cells
proliferate and survive similarly to M cells (Figs. 2 and 4B). In addition, because MZ cells do not proliferate in
response to BCR activation, contaminating MZ cells within the sorted
CD21highHSAhigh T2 gate are
also unlikely to contribute to the production of M cells in this assay.
Consistent with this prediction, CD21/CD23 staining in the T2 cell
differentiation assay revealed a specific loss of
CD21highCD23- MZ cells
upon BCR stimulation (Fig. 5D).
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E). Thus, the ability of purified T2 cells
to differentiate into an M cell phenotype appears to be mediated
uniquely through the B cell Ag receptor. BCR-dependent responses in T2 cells require Btk
To begin to address the molecular requirements for T2 cell
signaling, we used Xid mice, mutated in the Btk gene (16).
Btk is a critical component of the BCR signalosome required for the
generation of both 1,2-diacylglycerol and calcium-dependent
signals (44). Xid mice have reduced numbers of M cells but
an intact T1 and T2 pool (22). We purified T1, T2, and M
cells from BALB/c (wild-type (WT)) or BALB/xid (Xid) mice using
CD21/HSA cell sorting, as in Fig. 1 (data not shown). In contrast to WT
cells, T2 and M cells from Xid mice failed to proliferate after 48
h of BCR activation (Fig. 6A).
In addition, after 12 and 36 h of BCR stimulation, Xid T1, T2, and
M cells exhibited significantly enhanced cell death compared with WT
controls (Fig. 6B). Notably, Xid T2 cells were only 15%
viable, compared with 69% in WT T2 cells after 36 h. At the
molecular level, both total and purified T2 splenocytes from Xid mice
exhibited only minimal BCR-induced cyclin D2 up-regulation (Fig. 6C). However, stimulation with PMA (a 1,2-diacylglycerol
analog) plus ionomycin (a calcium ionophore) rescued cyclin D2
expression, indicating that signaling components downstream of Btk are
unaltered in Xid cells (Fig. 6C). Furthermore, live-sorted
(by propidium iodide and annexin V FACS sorting just before
immunoblotting) Xid splenocytes still exhibited reduced cyclin D2
levels (data not shown), suggesting that the Xid cyclin D2 defect was
independent of the survival defect. Together, these data demonstrate a
direct requirement for Btk in the BCR responsiveness of T2 cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2, or VAV1 and
VAV2 each have reduced numbers of mature B cells but an intact T2 cell
pool (data not shown) (17, 18, 19, 20). Consistent with this
hypothesis, T2 cells purified from Btk-deficient Xid mice fail to
generate proliferative and survival signals (Fig. 6). Together, these
data suggest that BCR engagement and intact BCR signaling at the T2
cell stage are imperative for the development of mature B cells. Differences in BCR responsiveness suggest distinct roles for the T1 and T2 immature B cell subsets
Of the 2 x 107
IgM+ B cells that are generated from the BM
daily, 10% enter the spleen and only 1–3% enter the mature B cell
pool (49, 50). Studies done in 
-HEL BCR transgenic mice
indicate negative selection is responsible for at least part of this
process (51). Several lines of evidence demonstrate that B
lymphocyte negative selection occurs at the splenic immature B cell
stage (21). Our data demonstrate that the T1 immature
subset does not proliferate in response to BCR cross-linking, but
rather dies (Figs. 2, A and B, and
4B). Furthermore, T1 cells do not up-regulate genes required
for cell proliferation and survival (Figs. 3 and 4, C and
D). Together, these data are consistent with the T1 subset
of immature B cells being the target for BCR-induced negative selection
in the periphery.
In addition to negative selection, mounting evidence suggests that positive selection also plays a role in peripheral B cell development. Whereas MHC molecules are the selecting ligands for T cell positive selection, analogous ligands for the BCR remain largely unknown. This has made it difficult to directly evaluate positive selection in B lymphocytes. Recent work using transgenic BCR models, however, has provided clear evidence for positive selection for two unique B cell lineages, peritoneal B1 cells and MZ cells (52, 53). In addition, analysis of Ag receptor diversity revealed that, in comparison to HSAhigh immature B cells, HSAlow mature B cells have a much more restricted repertoire of expressed H-L chain pairs (10, 11). The generation of mature cells expressing only a very limited subset of H-L chain pairs (in several independent transgene models) is most consistent with the positive selection of a minority of immature B cells (11, 14, 15). Notably, a subsequent study using a combination of transgenic H and dysfunctional L chain pairs clearly indicates that positive selection operates in this oligoclonal receptor model (54). In that model, the level of H chain expression and the nature of the L chain expressed coordinately controlled generation of the mature peripheral B cell pool. These combined observations suggest that the repertoire restriction occurring at the immature to mature splenic B cell transition is primarily due to positive selection. Despite these supportive data, however, an appropriate target population for positive selection in the periphery has not yet been described.
The immature T2 subset is an immediate precursor to mature B cells in
vivo, presenting T2 cells as a potential target population for
selection into the mature B cell pool (22). The current
report presents significant further data supporting this hypothesis. In
response to BCR stimulation, T2 cells enter cell cycle rapidly and
proliferate (Fig. 2, A–C), correlating with
early and robust cyclin D2 induction (Fig. 3). BCR engagement
of T2 cells also activates survival signals (Fig. 4) and leads to a
change in surface phenotype consistent with a mature follicular B cell
(Fig. 5). Taken together, these results strongly support the hypothesis
that T2 cells are a likely target for B lineage positive selection into
long-lived mature B cells.
Together, our findings suggest that T1 and T2 cells may act as
distinct checkpoints for selection into the mature B cell pool. The T1
subset of immature B cells transits from the BM, through the
bloodstream, to the splenic PALS (22). In contrast, the T2
subset is primarily located in the splenic follicle, adjacent to mature
B cells (22). Their distinct anatomic locations and
distinct responses to BCR engagement suggest a model whereby T1 cells
with receptor specificities for blood-borne self-Ags are deleted by
negative selection, whereas T2 cells with specificities for alternative
follicular Ags become positively selected into the mature B cell pool
(Fig. 7). This model, however, does not
exclude the possibility that additional selection steps may also occur
within the mature B cell pool itself.
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Consistent with the short lifespan (3–4 days) of immature splenic
B cells, in vivo transfer studies indicate that T1 cells differentiate
into T2 cells within 48 h (22). The clear differences
in BCR responsiveness between T1 and T2 cells suggest that during this
relatively brief time period, important changes rapidly occur to
transform T1 cells, which die upon BCR activation, into T2 cells, which
proliferate, survive, and mature upon receptor engagement (Fig. 7, lower panel). It is unclear what drives this T1 to T2
transition. However, recent evidence indicates BAFF-deficient
mice have a developmental block at the T1 stage, suggesting that this
novel B cell activator may play a key role in the T1 to T2 transition
(55). In addition, signal transduction through Syk may
also be required for this developmental step.
Syk-/- B cells do not enter the splenic
follicle, the precise anatomic restriction separating T1 from T2 cells
(56).
In addition to identifying the signals driving this T1 to T2 step, it will be equally important to identify the molecular changes induced at this transition. What is it about the BCR signaling machinery of T2 cells that allows this population to respond so differently to T1 cells? The very similar surface phenotype and limited time required to form T2 from T1 cells suggests that a relatively restricted set of expression differences may distinguish these developmental subsets. Gene expression profiling and biochemical analysis of the BCR signalosome within purified subpopulations should help identify these key molecular differences.
Recent studies indicate that BCR responsiveness in splenic B cells is
correlated with the capacity to colocalize the BCR with lipid rafts
(57, 59, 60). Upon BCR activation, association of the BCR
with lipid rafts was observed in >75% of HSAlow
mature B cells vs only 30% of HSAhigh
immature splenic B cells (57). We are currently evaluating
the hypothesis that the subfraction of HSAhigh
immature B cells capable of colocalizing the BCR to lipid rafts is the
BCR-responsive T2 population. Further studies comparing the specific
signaling components present in the lipid rafts of T1 vs T2 cells will
be important in confirming our predictions and further understanding
the molecular differences between these two closely related, yet
functionally distinct, B lymphocyte populations.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David J. Rawlings, Department of Pediatrics, University of Washington, School of Medicine, Box 356320, Seattle, WA 98195-6320. E-mail address: drawling{at}u.washington.edu
3 Abbreviations used in this paper: BM, bone marrow; M, mature follicular; HSA, heat-stable Ag; BCR, B cell receptor; Btk, Bruton’s tyrosine kinase; T1, transitional 1; T2, transitional 2; PALS, periarteriolar lymphoid sheath; MZ, marginal zone; WT, wild type.
Received for publication September 12, 2001. Accepted for publication December 19, 2001.
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References |
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B-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes. Proc. Natl. Acad. Sci. USA 96:9136.B that blocks TNF-induced apoptosis. Genes Dev. 13:382.B activation, and Bcl-xL levels in vivo. J. Exp. Med. 191:1721.B activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 401:82.[Medline]
B is a target of AKT in anti-apoptotic PDGF signalling. Nature 401:86.[Medline]
B family directly activates expression of the apoptosis inhibitor Bcl-xL. Mol. Cell. Biol. 20:2687.This article has been cited by other articles:
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  I. Castro, J. A. Wright, B. Damdinsuren, K. L. Hoek, G. Carlesso, N. P. Shinners, R. M. Gerstein, R. T. Woodland, R. Sen, and W. N. Khan B Cell Receptor-Mediated Sustained c-Rel Activation Facilitates Late Transitional B Cell Survival through Control of B Cell Activating Factor Receptor and NF-{kappa}B2 J. Immunol., June 15, 2009; 182(12): 7729 - 7737. [Abstract] [Full Text] [PDF]
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A. Palanichamy, J. Barnard, B. Zheng, T. Owen, T. Quach, C. Wei, R. J. Looney, I. Sanz, and J. H. Anolik Novel Human Transitional B Cell Populations Revealed by B Cell Depletion Therapy J. Immunol., May 15, 2009; 182(10): 5982 - 5993. [Abstract] [Full Text] [PDF]
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A. J. Gross, J. R. Lyandres, A. K. Panigrahi, E. T. L. Prak, and A. L. DeFranco Developmental Acquisition of the Lyn-CD22-SHP-1 Inhibitory Pathway Promotes B Cell Tolerance J. Immunol., May 1, 2009; 182(9): 5382 - 5392. [Abstract] [Full Text] [PDF]
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J. Lee, S. Kuchen, R. Fischer, S. Chang, and P. E. Lipsky Identification and Characterization of a Human CD5+ Pre-Naive B Cell Population J. Immunol., April 1, 2009; 182(7): 4116 - 4126. [Abstract] [Full Text] [PDF]
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P. A. Blair, K. A. Chavez-Rueda, J. G. Evans, M. J. Shlomchik, A. Eddaoudi, D. A. Isenberg, M. R. Ehrenstein, and C. Mauri Selective Targeting of B Cells with Agonistic Anti-CD40 Is an Efficacious Strategy for the Generation of Induced Regulatory T2-Like B Cells and for the Suppression of Lupus in MRL/lpr Mice J. Immunol., March 15, 2009; 182(6): 3492 - 3502. [Abstract] [Full Text] [PDF]
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L. Le Pottier, V. Devauchelle, A. Fautrel, C. Daridon, A. Saraux, P. Youinou, and J.-O. Pers Ectopic Germinal Centers Are Rare in Sjogren's Syndrome Salivary Glands and Do Not Exclude Autoreactive B Cells J. Immunol., March 15, 2009; 182(6): 3540 - 3547. [Abstract] [Full Text] [PDF]
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S. F. Andrews and D. J. Rawlings Transitional B Cells Exhibit a B Cell Receptor-Specific Nuclear Defect in Gene Transcription J. Immunol., March 1, 2009; 182(5): 2868 - 2878. [Abstract] [Full Text] [PDF]
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 S. Matsuda, Y. Mikami, M. Ohtani, M. Fujiwara, Y. Hirata, A. Minowa, Y. Terauchi, T. Kadowaki, and S. Koyasu Critical role of class IA PI3K for c-Rel expression in B lymphocytes Blood, January 29, 2009; 113(5): 1037 - 1044. [Abstract] [Full Text] [PDF]
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J. C. Nixon, S. Ferrell, C. Miner, A. L. Oldham, U. Hochgeschwender, and C. F. Webb Transgenic Mice Expressing Dominant-Negative Bright Exhibit Defects in B1 B Cells J. Immunol., November 15, 2008; 181(10): 6913 - 6922. [Abstract] [Full Text] [PDF]
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J. C. Rodriguez-Alba, M. E. Moreno-Garcia, C. Sandoval-Montes, V. H. Rosales-Garcia, and L. Santos-Argumedo CD38 induces differentiation of immature transitional 2 B lymphocytes in the spleen Blood, April 1, 2008; 111(7): 3644 - 3652. [Abstract] [Full Text] [PDF]
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 A. Meyer-Bahlburg, S. F. Andrews, K. O.A. Yu, S. A. Porcelli, and D. J. Rawlings Characterization of a late transitional B cell population highly sensitive to BAFF-mediated homeostatic proliferation J. Exp. Med., January 21, 2008; 205(1): 155 - 168. [Abstract] [Full Text] [PDF]
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G. Hart, T. Avin-Wittenberg, and I. Shachar IL-15 regulates immature B-cell homing in an Ly49D-, IL-12 , and IL-18 dependent manner Blood, January 1, 2008; 111(1): 50 - 59. [Abstract] [Full Text] [PDF]
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J. L. Lamoureux, L. C. Watson, M. Cherrier, P. Skog, D. Nemazee, and A. J. Feeney Reduced receptor editing in lupus-prone MRL/lpr mice J. Exp. Med., November 26, 2007; 204(12): 2853 - 2864. [Abstract] [Full Text] [PDF]
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F. J. X. Spillmann, G. Beck-Engeser, and M. Wabl Differentiation and Ig-Allele Switch in Cell Line WEHI-231 J. Immunol., November 15, 2007; 179(10): 6395 - 6402. [Abstract] [Full Text] [PDF]
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B. N. Teague, Y. Pan, P. A. Mudd, B. Nakken, Q. Zhang, P. Szodoray, X. Kim-Howard, P. C. Wilson, and A. D. Farris Cutting Edge: Transitional T3 B Cells Do Not Give Rise to Mature B Cells, Have Undergone Selection, and Are Reduced in Murine Lupus J. Immunol., June 15, 2007; 178(12): 7511 - 7515. [Abstract] [Full Text] [PDF]
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M. Shankar, J. C. Nixon, S. Maier, J. Workman, A. D. Farris, and C. F. Webb Anti-Nuclear Antibody Production and Autoimmunity in Transgenic Mice That Overexpress the Transcription Factor Bright J. Immunol., March 1, 2007; 178(5): 2996 - 3006. [Abstract] [Full Text] [PDF]
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K. L. Hoek, P. Antony, J. Lowe, N. Shinners, B. Sarmah, S. R. Wente, D. Wang, R. M. Gerstein, and W. N. Khan Transitional B Cell Fate Is Associated with Developmental Stage-Specific Regulation of Diacylglycerol and Calcium Signaling upon B Cell Receptor Engagement J. Immunol., October 15, 2006; 177(8): 5405 - 5413. [Abstract] [Full Text] [PDF]
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R. A. Barrington, M. Borde, A. Rao, and M. C. Carroll Involvement of NFAT1 in B Cell Self-Tolerance J. Immunol., August 1, 2006; 177(3): 1510 - 1515. [Abstract] [Full Text] [PDF]
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B. R. Herrin and L. B. Justement Expression of the Adaptor Protein Hematopoietic Src Homology 2 is Up-Regulated in Response to Stimuli That Promote Survival and Differentiation of B Cells J. Immunol., April 1, 2006; 176(7): 4163 - 4172. [Abstract] [Full Text] [PDF]
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C. M. Grimaldi, V. Jeganathan, and B. Diamond Hormonal Regulation of B Cell Development: 17beta-Estradiol Impairs Negative Selection of High-Affinity DNA-Reactive B Cells at More Than One Developmental Checkpoint. J. Immunol., March 1, 2006; 176(5): 2703 - 2710. [Abstract] [Full Text] [PDF]
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A. K. Cuss, D. T. Avery, J. L. Cannons, L. J. Yu, K. E. Nichols, P. J. Shaw, and S. G. Tangye Expansion of Functionally Immature Transitional B Cells Is Associated with Human-Immunodeficient States Characterized by Impaired Humoral Immunity J. Immunol., February 1, 2006; 176(3): 1506 - 1516. [Abstract] [Full Text] [PDF]
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L. L. Dragone, M. D. Myers, C. White, T. Sosinowski, and A. Weiss Src-Like Adaptor Protein Regulates B Cell Development and Function J. Immunol., January 1, 2006; 176(1): 335 - 345. [Abstract] [Full Text] [PDF]
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M. Viau, N. S. Longo, P. E. Lipsky, and M. Zouali Staphylococcal Protein A Deletes B-1a and Marginal Zone B Lymphocytes Expressing Human Immunoglobulins: An Immune Evasion Mechanism J. Immunol., December 1, 2005; 175(11): 7719 - 7727. [Abstract] [Full Text] [PDF]
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A. Craxton, K. E. Draves, A. Gruppi, and E. A. Clark BAFF regulates B cell survival by downregulating the BH3-only family member Bim via the ERK pathway J. Exp. Med., November 21, 2005; 202(10): 1363 - 1374. [Abstract] [Full Text] [PDF]
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B. Srivastava, W. J. Quinn III, K. Hazard, J. Erikson, and D. Allman Characterization of marginal zone B cell precursors J. Exp. Med., November 7, 2005; 202(9): 1225 - 1234. [Abstract] [Full Text] [PDF]
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E. Vigorito, L. Gambardella, F. Colucci, S. McAdam, and M. Turner Vav proteins regulate peripheral B-cell survival Blood, October 1, 2005; 106(7): 2391 - 2398. [Abstract] [Full Text] [PDF]
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E. Peeva, J. Venkatesh, and B. Diamond Tamoxifen Blocks Estrogen-Induced B Cell Maturation but Not Survival J. Immunol., August 1, 2005; 175(3): 1415 - 1423. [Abstract] [Full Text] [PDF]
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G. P. Sims, R. Ettinger, Y. Shirota, C. H. Yarboro, G. G. Illei, and P. E. Lipsky Identification and characterization of circulating human transitional B cells Blood, June 1, 2005; 105(11): 4390 - 4398. [Abstract] [Full Text] [PDF]
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J. Rolf, V. Motta, N. Duarte, M. Lundholm, E. Berntman, M.-L. Bergman, L. Sorokin, S. L. Cardell, and D. Holmberg The Enlarged Population of Marginal Zone/CD1dhigh B Lymphocytes in Nonobese Diabetic Mice Maps to Diabetes Susceptibility Region Idd11 J. Immunol., April 15, 2005; 174(8): 4821 - 4827. [Abstract] [Full Text] [PDF]
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 J. Rajaiya, M. Hatfield, J. C. Nixon, D. J. Rawlings, and C. F. Webb Bruton's Tyrosine Kinase Regulates Immunoglobulin Promoter Activation in Association with the Transcription Factor Bright Mol. Cell. Biol., March 15, 2005; 25(6): 2073 - 2084. [Abstract] [Full Text] [PDF]
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M. E. Moreno-Garcia, L. N. Lopez-Bojorques, A. Zentella, L. A. Humphries, D. J. Rawlings, and L. Santos-Argumedo CD38 Signaling Regulates B Lymphocyte Activation via a Phospholipase C (PLC)-{gamma}2-Independent, Protein Kinase C, Phosphatidylcholine-PLC, and Phospholipase D-Dependent Signaling Cascade J. Immunol., March 1, 2005; 174(5): 2687 - 2695. [Abstract] [Full Text] [PDF]
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C. M. Grimaldi, R. Hicks, and B. Diamond B Cell Selection and Susceptibility to Autoimmunity J. Immunol., February 15, 2005; 174(4): 1775 - 1781. [Abstract] [Full Text] [PDF]
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X. Zhu, R. Hart, M. S. Chang, J.-W. Kim, S. Y. Lee, Y. A. Cao, D. Mock, E. Ke, B. Saunders, A. Alexander, et al. Analysis of the Major Patterns of B Cell Gene Expression Changes in Response to Short-Term Stimulation with 33 Single Ligands J. Immunol., December 15, 2004; 173(12): 7141 - 7149. [Abstract] [Full Text] [PDF]
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 A. C. Donahue, K. L. Hess, K. L. Ng, and D. A. Fruman Altered splenic B cell subset development in mice lacking phosphoinositide 3-kinase p85{alpha} Int. Immunol., December 1, 2004; 16(12): 1789 - 1798. [Abstract] [Full Text] [PDF]
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Z. S. M. Rahman and T. Manser B Cells Expressing Bcl-2 and a Signaling-Impaired BAFF-Specific Receptor Fail to Mature and Are Deficient in the Formation of Lymphoid Follicles and Germinal Centers J. Immunol., November 15, 2004; 173(10): 6179 - 6188. [Abstract] [Full Text] [PDF]
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 L. A. Humphries, C. Dangelmaier, K. Sommer, K. Kipp, R. M. Kato, N. Griffith, I. Bakman, C. W. Turk, J. L. Daniel, and D. J. Rawlings Tec Kinases Mediate Sustained Calcium Influx via Site-specific Tyrosine Phosphorylation of the Phospholipase C{gamma} Src Homology 2-Src Homology 3 Linker J. Biol. Chem., September 3, 2004; 279(36): 37651 - 37661. [Abstract] [Full Text] [PDF]
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T. C. Chiles Regulation and Function of Cyclin D2 in B Lymphocyte Subsets J. Immunol., September 1, 2004; 173(5): 2901 - 2907. [Abstract] [Full Text] [PDF]
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P. W. Yu, R. S. Tabuchi, R. M. Kato, A. Astrakhan, S. Humblet-Baron, K. Kipp, K. Chae, W. Ellmeier, O. N. Witte, and D. J. Rawlings Sustained correction of B-cell development and function in a murine model of X-linked agammaglobulinemia (XLA) using retroviral-mediated gene transfer Blood, September 1, 2004; 104(5): 1281 - 1290. [Abstract] [Full Text] [PDF]
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L. Flaishon, S. Becker-Herman, G. Hart, Y. Levo, W. A. Kuziel, and I. Shachar Expression of the chemokine receptor CCR2 on immature B cells negatively regulates their cytoskeletal rearrangement and migration Blood, August 15, 2004; 104(4): 933 - 941. [Abstract] [Full Text] [PDF]
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E. M. Fuentes-Panana, G. Bannish, N. Shah, and J. G. Monroe Basal Ig{alpha}/Ig{beta} Signals Trigger the Coordinated Initiation of Pre-B Cell Antigen Receptor-Dependent Processes J. Immunol., July 15, 2004; 173(2): 1000 - 1011. [Abstract] [Full Text] [PDF]
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P. A. Silveira, J. Dombrowsky, E. Johnson, H. D. Chapman, D. Nemazee, and D. V. Serreze B Cell Selection Defects Underlie the Development of Diabetogenic APCs in Nonobese Diabetic Mice J. Immunol., April 15, 2004; 172(8): 5086 - 5094. [Abstract] [Full Text] [PDF]
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C. E. Blanco-Betancourt, A. Moncla, M. Milili, Y. L. Jiang, E. M. Viegas-Pequignot, B. Roquelaure, I. Thuret, and C. Schiff Defective B-cell-negative selection and terminal differentiation in the ICF syndrome Blood, April 1, 2004; 103(7): 2683 - 2690. [Abstract] [Full Text] [PDF]
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C. S. Goodyear, M. Narita, and G. J. Silverman In Vivo VL-Targeted Activation-Induced Apoptotic Supraclonal Deletion by a Microbial B Cell Toxin J. Immunol., March 1, 2004; 172(5): 2870 - 2877. [Abstract] [Full Text] [PDF]
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S. Middendorp and R. W. Hendriks Cellular Maturation Defects in Bruton's Tyrosine Kinase-Deficient Immature B Cells Are Amplified by Premature B Cell Receptor Expression and Reduced by Receptor Editing J. Immunol., February 1, 2004; 172(3): 1371 - 1379. [Abstract] [Full Text] [PDF]
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H. Wang and S. H. Clarke Evidence for a Ligand-Mediated Positive Selection Signal in Differentiation to a Mature B Cell J. Immunol., December 15, 2003; 171(12): 6381 - 6388. [Abstract] [Full Text] [PDF]
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S. Middendorp, G. M. Dingjan, A. Maas, K. Dahlenborg, and R. W. Hendriks Function of Bruton's Tyrosine Kinase during B Cell Development Is Partially Independent of Its Catalytic Activity J. Immunol., December 1, 2003; 171(11): 5988 - 5996. [Abstract] [Full Text] [PDF]
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R. Wen, Y. Chen, L. Xue, J. Schuman, S. Yang, S. W. Morris, and D. Wang Phospholipase C{gamma}2 Provides Survival Signals via Bcl2 and A1 in Different Subpopulations of B Cells J. Biol. Chem., October 31, 2003; 278(44): 43654 - 43662. [Abstract] [Full Text] [PDF]
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 M. J. Walmsley, S. K. T. Ooi, L. F. Reynolds, S. H. Smith, S. Ruf, A. Mathiot, L. Vanes, D. A. Williams, M. P. Cancro, and V. L. J. Tybulewicz Critical Roles for Rac1 and Rac2 GTPases in B Cell Development and Signaling Science, October 17, 2003; 302(5644): 459 - 462. [Abstract] [Full Text] [PDF]
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C.-P. Danzer, B. E. Collins, O. Blixt, J. C. Paulson, and L. Nitschke Transitional and marginal zone B cells have a high proportion of unmasked CD22: implications for BCR signaling Int. Immunol., October 1, 2003; 15(10): 1137 - 1147. [Abstract] [Full Text] [PDF]
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M. Hikida, S. Johmura, A. Hashimoto, M. Takezaki, and T. Kurosaki Coupling Between B Cell Receptor and Phospholipase C-{gamma}2 Is Essential for Mature B Cell Development J. Exp. Med., August 18, 2003; 198(4): 581 - 589. [Abstract] [Full Text] [PDF]
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S. Ogilvy, C. Louis-Dit-Sully, J. Cooper, R. L. Cassady, D. R. Alexander, and N. Holmes Either of the CD45RB and CD45RO Isoforms Are Effective in Restoring T Cell, But Not B Cell, Development and Function in CD45-Null Mice J. Immunol., August 15, 2003; 171(4): 1792 - 1800. [Abstract] [Full Text] [PDF]
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L. R. Whyburn, K. E. Halcomb, C. M. Contreras, C. A. Lowell, O. N. Witte, and A. B. Satterthwaite Reduced Dosage of Bruton's Tyrosine Kinase Uncouples B Cell Hyperresponsiveness from Autoimmunity in lyn-/- Mice J. Immunol., August 15, 2003; 171(4): 1850 - 1858. [Abstract] [Full Text] [PDF]
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C. Wang, M. Khalil, J. Ravetch, and B. Diamond The Naive B Cell Repertoire Predisposes to Antigen-Induced Systemic Lupus Erythematosus J. Immunol., May 1, 2003; 170(9): 4826 - 4832. [Abstract] [Full Text] [PDF]
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H. Dalwadi, B. Wei, M. Schrage, T. T. Su, D. J. Rawlings, and J. Braun B Cell Developmental Requirement for the G{alpha}i2 Gene J. Immunol., February 15, 2003; 170(4): 1707 - 1715. [Abstract] [Full Text] [PDF]
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T. M. Yankee, S. A. Solow, K. D. Draves, and E. A. Clark Expression of the Grb2-Related Protein of the Lymphoid System in B Cell Subsets Enhances B Cell Antigen Receptor Signaling Through Mitogen-Activated Protein Kinase Pathways J. Immunol., January 1, 2003; 170(1): 349 - 355. [Abstract] [Full Text] [PDF]
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J. B. Petro, R. M. Gerstein, J. Lowe, R. S. Carter, N. Shinners, and W. N. Khan Transitional Type 1 and 2 B Lymphocyte Subsets Are Differentially Responsive to Antigen Receptor Signaling J. Biol. Chem., December 6, 2002; 277(50): 48009 - 48019. [Abstract] [Full Text] [PDF]
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S. Xu and K.-P. Lam Delayed Cellular Maturation and Decreased Immunoglobulin {kappa} Light Chain Production In Immature B Lymphocytes Lacking B Cell Linker Protein J. Exp. Med., July 15, 2002; 196(2): 197 - 206. [Abstract] [Full Text] [PDF]
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 T. T. Su, B. Guo, and D. J. Rawlings Emerging Roles for PKC Isoforms in Immune Cell Function Mol. Interv., June 1, 2002; 2(3): 141 - 144. [Abstract] [Full Text]
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-actin as a control for relative protein loading. Data are
representative of more than three experiments.
