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* Immune Regulation Group, Centenary Institute of Cancer Medicine and Cell Biology, Sydney, New South Wales, Australia; and
University of Sydney, Sydney, New South Wales, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The signals required for generating human plasma cells from GC or memory B cells in vitro have been previously investigated (17, 18, 19). In these studies, plasma cells were identified as cells containing intracellular Ig, increased expression of CD38, and concomitant down-regulation of CD20, consistent with the morphology and phenotype of plasma cells present in human bone marrow (20, 21). After initial stimulation of GC or memory B cells with CD40 ligand (CD40L), IL-2, and IL-10, a greater proportion of plasma cells was generated when these activated cells were recultured in the absence of CD40L (17, 18, 19). These findings suggested that CD40L interrupted plasma cell development while concomitantly promoting expansion of nondifferentiated B cell blasts (17, 18, 19). Thus, provision of T cell help, in the form of transiently expressed CD40L, appeared to influence the decision between the generation of plasma cells and memory B cells (22).
During the past 6 years, we and others have shown that numerous lymphocyte differentiation events alter in frequency with consecutive cell divisions (23, 24, 25, 26, 27, 28, 29, 30, 31, 32), leading us to propose that division number may play an important role in key decisions associated with immune regulation (23, 25, 27). The extent to which similar division-linked regulation might apply to lymphocytes that have undergone extensive division and differentiation after Ag stimulation in vivo (i.e., memory cells) is currently unknown. Here, we have applied new quantitative analytical techniques to re-examine the generation of ISCs from human B cells after in vitro stimulation with T cell-derived signals CD40L, IL-2, and IL-10 and to dissect the differential roles played by CD40L and these cytokines in this process. Our data suggest that a division-linked process governs the formation of ISCs. Two types of ISC (CD38- and CD38+) could be generated from proliferating memory B cells, with CD38- ISCs appearing to be precursors of CD38+ ISCs. Strikingly, CD38+ ISCs acquired increased proliferation and survival characteristics that permitted them, but not CD38- B cells, to persist and increase in number in the absence of CD40L. Thus, the primary function of CD40L was to sustain the proliferative potential and survival of CD38- blasts rather than to prevent subsequent generation of effector ISCs. This analysis, therefore, challenges the proposal that removal of CD40L regulates differentiation of memory B cells into ISCs. Rather, our results are consistent with a stochastic division-linked mechanism for controlling the early production of protective Ig while simultaneously preserving, and even expanding, the memory B cell pool, both of which are necessary for sustained humoral immunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PE-conjugated and biotinylated anti-CD38 mAbs were from
Caltag (Burlingame, CA). FITC- and PE-conjugated anti-CD19, CD20,
CD27, anti-5-bromo-2'-deoxyuridine (BrdU), anti-IgM,
anti-IgG, anti-Ig 
L chain mAb, isotype control mAb, and
rabbit polyclonal anti-active caspase-3 antisera were from BD
PharMingen (San Diego, CA). Unconjugated and biotinylated goat
anti-human IgM, IgG, or IgA polyclonal antisera were purchased from
Southern Biotechnology (Birmingham, AL). Recombinant human CD40L
expressed as membranes of the Sf21 insect cell line infected
with baculovirus vector containing human CD40L cDNA was a
generous gift from Dr. M. Kehry (Boehringer Ingleheim, Ridgefield, CT)
(33). IL-2 was purchased from Endogen (Woburn, MA), and
IL-10 was provided by Dr. R. de Waal Malefyt (DNAX Research Institute,
Palo Alto, CA). CFSE was purchased from Molecular Probes (Eugene, OR).
BrdU was purchased from Sigma-Aldrich (St. Louis, MO).
Cells
Normal human spleens were obtained from trauma victims undergoing routine splenectomies (Royal Prince Alfred Hospital, Sydney, Australia) or from organ donors (Australian Red Cross Blood Service, Sydney, Australia). Mononuclear cells were prepared as previously described (28, 34) and cryopreserved in liquid nitrogen until required. Total human B cells (>98% CD19+) were isolated from splenic mononuclear cells using CD19 DYNAbeads (Dynal, Oslo, Norway) (28, 34). These cells were further fractionated into naive (CD27-) and memory (CD27+) (35, 36) populations using CD27 MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany) or by cell sorting using a FACStarPlus (BD Biosciences, San Jose, CA) after labeling with PE-anti-CD27 mAb and collecting CD27- and CD27+ B cells. The recovered naive (CD27-) B cells were uniformly IgM+IgDhigh (>98%) and contained <2% IgG+ and IgA+ cells, whereas memory (CD27+) B cells were heterogeneous for isotype expression, containing IgM-expressing as well as isotype-switched B cells (34, 35, 36).
CFSE labeling and B cell cultures
Purified naive and memory B cells were labeled with CFSE (23, 37) and cultured in 48-well plates (4 x 105/ml; BD Labware, Franklin Lakes, NJ) with CD40L only (final membrane dilution, 1/250) or with IL-2 (50 U/ml) and/or IL-10 (100 U/ml) for 5 days. For reculture experiments, B cells were cultured for 4 days with CD40L, IL-2, and IL-10, harvested, and washed thoroughly with PBS. The cells were recultured with IL-2 and IL-10 in the absence or presence of CD40L for an additional 4 days (17). A known number of CaliBRITE beads (BD Biosciences) were added to culture wells before harvesting, and the number of viable B cells in each culture condition was calculated as a function of the ratio of beads to live cells (24, 26, 27). B cells were cultured in RPMI 1640 containing L-glutamine (Life Technologies, Grand Island, NY), 10% FCS (Life Technologies), 10 mM HEPES, pH 7.4 (Sigma-Aldrich), 0.1 mM nonessential amino acid solution (Sigma-Aldrich), 1 mM sodium pyruvate (Life Technologies), 60 µg/ml penicillin, 100 µg/ml streptomycin, and 40 µg/ml apo-transferrin (Sigma-Aldrich). All cultures were conducted at 37°C in a humidified atmosphere containing 5% CO2.
Immunofluorescent staining
In vitro-activated splenic B cells were harvested from culture
wells and nonspecific binding sites blocked by preincubation with
normal mouse IgG. Cells were then incubated on ice for 20 min with
PE-anti-CD38 mAb (Caltag) and were analyzed on a FACScan flow
cytometer using CellQuest software (BD Biosciences). For determining
expression of active caspase-3, cells were initially labeled with
biotinylated anti-CD38 mAb followed by Streptavidin-Tricolor
(Caltag), fixed for 10 min with 1% formaldehyde, diluted with an equal
volume of PBS containing Tween 20 (final concentration, 0.1%; ICN
Pharmaceuticals, Costa Mesa, CA), and incubated overnight. The cells
were then incubated with PE-anti-caspase-3 Ab.
CD38- and CD38+ B cells in
the same divisions that were present in both the live and dead
populations, as defined by scatter plots, were analyzed. Expression of
intracellular Ig was similarly determined by labeling fixed and
permeabilized cells with anti-IgM, IgG, or anti-Ig L chain
mAb and analyzing CD38- and
CD38+ B cells.
BrdU analysis
Cell cultures were pulsed with 100 µg/ml BrdU for 4–8 h, harvested, labeled with biotinylated anti-CD38 mAb and Streptavidin-Tricolor, and then fixed and permeabilized as described above. DNase I (Roche, Castle Hill, New South Wales, Australia; 50 µg/ml, prepared in 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 100 µg/ml BSA) was added at 37°C for 30 min before incubating with PE-anti-BrdU mAb (24, 25). Cultures that had not been pulsed with BrdU were similarly treated to ascertain the background binding of the specific mAb.
Analysis of Ig secretion
CFSE-labeled memory B cells were cultured with CD40L, IL-2, and IL-10 for 4 days, washed, and then recultured with IL-2 and IL-10 in the presence or absence of CD40L. After 3 days, the cells were harvested and incubated with PE-conjugated anti-CD38 mAb. Activated B cells were sorted into ELISPOT plates (Multiscreen-HA plates; Millipore, Bedford, MA) that had been precoated with goat anti-human IgM, IgG, or IgA polyclonal antisera. B cells were incubated for 4 h at 37°C, after which the wells were washed three times with PBS-Tween 20, before addition of biotinylated anti-human IgG, IgA, or IgM anti-sera followed by streptavidin-conjugated alkaline phosphatase (Amersham Pharmacia, Castle Hill, New South Wales, Australia). Captured cells were visualized and enumerated after the addition of nitroblue tetrazolium/bromochloroindophenol.
After 4 days, supernatants were collected and the levels of Ig were determined. Ninety-six-well microtiter plates (Dynex Technologies, Chantilly, VA) were precoated with goat anti-human IgM, IgG, or IgA polyclonal antisera, and nonspecific binding sites were blocked with 2% FCS prepared in PBS. Culture supernatants and Ig standards were added to the wells and incubated for 2 h at 37° before the addition of biotinylated anti-human IgG, IgA, or IgM antisera. Bound Ab was detected by the addition of streptavidin-conjugated HRP (Amersham Pharmacia) and visualized with ABTS (Sigma-Aldrich; 1 mg/ml) prepared in citrate buffer (pH 4.5) containing 0.03% H2O2.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of continual stimulation with CD40L in inducing B cell
differentiation was examined in cultures of naive and memory B cells
distinguished on the basis of CD27 expression (35, 36).
Commitment to the ISC lineage was initially assessed by acquisition of
CD38, which as a marker for human bone marrow plasma cells (20, 21) has been used extensively to monitor in vitro generation of
plasma cells from human GCs or memory B cells (17, 18, 19, 38, 39, 40, 41). After stimulation with CD40L and IL-10, with or without
IL-2, human CD27+ memory B cells yielded 10–40
times more CD38+ B cells than did
CD27- naive B cells cultured under the same
conditions or than did memory B cells stimulated with CD40L alone
(Table I
). Consistent with this finding
was the demonstration of 5- to 30-fold more IgM, IgG, and IgA secreted
by memory compared with naive B cells under these stimulation
conditions (Table I). The CD38+ cells generated
in these cultures displayed reduced expression of CD20, but maintained
high levels of CD39 (data not shown), thereby distinguishing them from
GC B cells (17, 42).
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, a–d and f–i). IL-10
enhanced proliferation of both naive and memory B cells stimulated with
CD40L, whereas IL-2 selectively increased expansion of memory B cells
(Fig. 1, a and f; Table I
). Consistent with these
findings, the number of cells recovered from cultures of memory B cells
exceeded that of naive B cells irrespective of the culture conditions
(Table I).
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, b–e and g–j). This trend
was potently enhanced by IL-10, as indicated by the fact that 20–50%
of memory B cells that had undergone three or more divisions after
culture with CD40L and IL-10 became CD38+ (Fig. 1, g–j). Although IL-2 caused a 2- to 3-fold
increase in the proportion and absolute number of
CD38+ B cells in memory B cell cultures, as well
as in the amount of secreted Ig (Table I), it did not have a large
effect on the rate of differentiation when measured per division (Fig. 1j). Removing CD40L has distinct effects on the generation and survival of CD38- and CD38+ B cells
Previous studies had suggested that B cells stimulated with CD40L,
IL-2, and IL-10 were maintained in an undifferentiated ("memory")
state until CD40L was removed, when differentiation into
CD38+/+CD20+/- plasma cells occurred
(17, 18, 19). However, according to the data shown in Table I
and Fig. 1, generation of CD38+ B cells, as well
as Ig secretion, clearly occurred in cultures from which CD40L had not
been removed. In an attempt to resolve this apparent discrepancy, the
two-step culture protocol used in previous experiments by other
investigators (17, 18, 19) was combined with the CFSE-based
quantitative technique designed to monitor division-linked
differentiation in terms of both absolute and relative cell numbers.
CFSE-labeled memory B cells were cultured with CD40L, IL-2, and IL-10
for 4 days, when the proportion and absolute number of
CD38- and CD38+ B cells
were determined. Cells were then recultured for 4 days with IL-2 and
IL-10 in the absence or presence of CD40L, and the characteristics of
CD38+ and CD38- B cells
were assessed.
The greatest proportion of CD38+ cells was
generated in secondary cultures containing IL-2 and IL-10 only (Fig. 2a), in agreement with the
previous findings (17, 18, 19). However, when total number
rather than percentage of cells was calculated, 2-fold more
CD38+ B cells was now observed in the presence,
compared with the absence, of CD40L (Fig. 2b). For
CD38- B cells, the situation was different with
the total number, as well as the proportion, of cells being greatly
increased in cultures containing CD40L (Fig. 2, e and
f). By comparing cell numbers at the beginning and end of
secondary cultures, a net increase (2.5- to 5-fold) in
CD38+ B cells was observed irrespective of the
presence of CD40L (Fig. 2b). By contrast, there was a net
loss of CD38- B cells after CD40L withdrawal
(Fig. 2f). This loss was strikingly evident in contour plots
of CFSE vs CD38 expression on B cells obtained from primary and
secondary cultures. After primary culture, 
10% of cells expressed
CD38 (Fig. 3a). CD40L
preserved the population of CD38- B cells
present in the later divisions (Fig. 3b), whereas its
withdrawal resulted in a reduction in the proportion of these cells and
a concomitant increase in the proportion of CD38+
B cells (Fig. 3c). Therefore, CD40L does not inhibit the
generation of CD38+ B cells and, in addition,
plays an important role in maintaining CD38- B
cell blasts.
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The relative increase in CD38+ and loss of
CD38- B cells in the absence of CD40L could
result from one or more mechanisms. First, CD38+
B cells may increase in number by proliferating more than
CD38- B cells. Second,
CD38- B cells may have been more prone to
apoptosis. Third, CD38- B cells may have rapidly
differentiated into CD38+ cells
(17, 18, 19). Each of these possibilities was then examined in
turn. By comparing CFSE intensities of CD38+ B
cells in primary and secondary cultures, it was apparent that
proliferation of these cells during secondary culture was similar
irrespective of the presence of CD40L (Fig. 3). In contrast,
CD38- B cells showed little evidence of
proliferation when recultured without CD40L (compare Fig. 3, b and c). To assess the proliferative
characteristics of CD38- and
CD38+ B cells in more detail, incorporation of
BrdU after a short exposure was measured. Both populations incorporated
BrdU after primary culture, but the percentage of
BrdU+CD38+ B cells was
2-fold greater than that of
BrdU+CD38- B cells (Fig. 2, c and g), confirming
CD38+ B cells to be a more actively dividing
population (mechanism 1). Furthermore, although reculture with CD40L
sustained proliferation of both CD38- and
CD38+ populations, removal of CD40L resulted in a
much greater reduction in the percentage of
BrdU+CD38-, compared with
CD38+ B cells (Fig. 2, c and
g), consistent with the CFSE profiles of the two populations
shown in Fig. 3. To investigate whether the selective loss of
CD38- B cells in the absence of CD40L (Fig. 2f) resulted from cell death (mechanism 2), apoptosis was
quantified by assessing acquisition of active caspase-3
(43). No significant difference in the proportion of
apoptotic CD38- and CD38+
B cells was detected after reculture with CD40L
(p > 0.05; Fig. 2, d and
h). In contrast, removal of CD40L was associated with a
greater increase in apoptotic CD38- vs
CD38+ B cells (p <
0.001; Fig. 2, d and h). Thus, during the
differentiation process, CD38+ B cells acquire
the capacity to both proliferate and survive without continued
stimulation by CD40L, whereas CD38- B cell
blasts require sustained T cell help for their expansion and
survival.
The increase in number of CD38+ B cells after
removal of CD40L (Fig. 2b) may also result from
differentiation of CD38- blasts into
CD38+ B cells (mechanism 3). To investigate this,
memory B cells were cultured with CD40L, IL-2, and IL-10, and
CD38- B cells present in later divisions were
then isolated by cell sorting (Fig. 4, a and b) and
recultured with IL-2 and IL-10 in the absence or presence of CD40L. As
shown in Fig. 4, c and d, after a 2-day
reculture, 38.1% and 17.0% of cells harvested from these cultures
were CD38+ in the absence and presence of CD40L,
respectively, consistent with the notion that the
CD38- B cell population contains precursors of
CD38+ cells. Importantly, the absolute number of
CD38+ B cells generated in these secondary
cultures (Fig. 4e), as well as the extent of their
proliferation (indicated by reduced CFSE intensity; compare Fig. 4, c and d) was similar irrespective of the presence
of CD40L. This reinforces the notion that CD40L does not prevent the
generation of CD38+ cells. Moreover, no loss of
CD38 occurred during secondary culture of sorted
CD38+ cells (data not shown). In contrast, there
were 5-fold fewer CD38- B cells recovered in the
absence, compared with the presence, of CD40L (Fig. 4f),
which was consistent with the data shown in Fig. 2. Furthermore, the
isolated CD38- B cells underwent limited
proliferation in the absence of CD40L, whereas CD40L promoted continued
division of these cells (Fig. 4, b–d). Taken
together, these results confirm that, although the relative proportion
of CD38+ B cells increases when CD40L is removed,
CD40L does not inhibit the generation of these cells, but rather is
required for survival of CD38- B cells.
Consequently, continued proliferation of CD38- B
cells in secondary cultures containing CD40L would also indicate that
it is unlikely that CD38+ B cells are generated
due to CD40L being consumed in these cultures. The preferential
survival of CD38+ B cells in the absence of CD40L
is therefore due to a combination of greater proliferation by the
CD38+ cells per se (mechanism 1), enhanced
apoptosis on the part of the CD38- subset
(mechanism 2), and differentiation of some CD38-
blasts into CD38+ B cells (mechanism 3).
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Traditionally, plasma cells have been identified in vivo by a
CD38++CD20±
phenotype, expression of intracellular Ig, and spontaneous production
of Ig (20, 21, 44, 45, 46). Accordingly, many studies on the
generation of CD38+ B cells from GC or memory B
cells in vitro have led to the suggestion that a direct correlation
exists between increased numbers of CD38+
"plasma" cells and the appearance of increased levels of secreted
Ig (17, 38, 39, 40, 41). Therefore, it has been assumed to be
appropriate to equate in vitro-derived CD38+ B
cells with plasma cells. However, those previous studies did not
compare Ig secretion by in vitro-generated CD38+
B cells with that by CD38- B cells present in
the same cultures to ascertain whether Ig secretion is indeed
restricted to CD38+ B cells. To do so, we
extended the findings shown in Table I and Fig. 1 by examining the
ability of activated B cells defined by division history and CD38
expression to secrete Ig. Memory B cells stimulated in secondary
cultures with CD40L and cytokines were sorted into early divisions
(population 1) and late divisions comprising either
CD38- (population 2) or
CD38+ B cells (population 3; Fig. 5a). Undivided B cells or B
cells that had undergone only a few divisions (population 1) secreted
very little Ig (<5% were ISCs in ELISPOT assays; Fig. 5b).
However, B cells sorted from later divisions produced significantly
more Ig than did B cells in population 1, demonstrating that the
frequency of ISCs increased with division number. Surprisingly, the
overall frequency of ISCs in CD38- (population
2) and CD38+ (population 3) populations of
divided B cells was similar, although the relative proportion of cells
from population 3 secreting IgG and IgA appeared to increase compared
with population 2 (Fig. 5b). A similar trend was observed
when Ig secretion by populations of sorted B cells was measured in the
supernatants of secondary cultures containing CD40L, IL-2 and
IL-10. Thus, although B cells corresponding to populations 2 and 3
secreted comparable amounts of IgM, 2.5-fold more IgG and IgA
were detected in supernatants of CD38+ B cells
(population 3) compared with CD38- B cells
(population 2; Fig. 5c). These results were also obtained
when secondary cultures were performed in the presence of the cytokines
only (data not shown).
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L chain expressed by B cells as they differentiated from population 1
into population 2 and then 3 (Fig. 6)
when measured in terms of mean fluorescence intensity (MFI). Moreover,
a significantly higher proportion of CD38+ B
cells (population 3) expressed intracellular IgG and Ig L chain
than did CD38- B cells in populations 1 and 2
(Table II). The increase in expression of
intracellular Ig by B cells as they underwent division and
differentiation to express CD38 (Fig. 6) paralleled the increase in Ig
secreted by these different populations of B cells (Fig. 5), thereby
confirming the important role of cell division in differentiation of
ISCs from memory B cells and demonstrating that CD38 expression does
not exclusively identify ISCs.
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The experiments described so far indicated that withdrawal of
CD40L is not a critical factor leading to differentiation of
CD38+ "plasma" cells from
CD38- precursors (Figs. 2 and 4). However,
because CD38 expression did not appear to be an absolute marker for
ISCs (Fig. 5), it remained possible that removing CD40L might lead to
an increase in the number of ISCs, if not CD38+ B
cells. To investigate this possibility, the amount of secreted Ig
present in supernatants of 4-day secondary cultures was measured
because such an approach makes no assumptions regarding a phenotype of
ISCs. The prediction was that if CD40L does indeed maintain activated B
cells in an undifferentiated (memory) state (17, 18, 19), a
reduction in total Ig secretion would be observed in the presence of
CD40L. When the cultures were performed without CD40L, large amounts of
IgM, IgG, and IgA were produced (Table III). After addition of CD40L, no
decrease in Ig secretion was observed. On the contrary, an increase in
each isotype was detected (Table III), consistent with our previous
conclusion that withdrawal of CD40L does not trigger differentiation
to ISCs.
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a) and placed in secondary cultures containing IL-2 and
IL-10 with or without CD40L was also determined by ELISPOT. In the
presence of CD40L, 29.9 ± 6.7% of CD38-
population 2 B cells and 45.5 ± 9.5% of
CD38+ population 3 cells were found to secrete Ig
(Fig. 5b). When secondary cultures lacked CD40L, 25.1
± 1.8% and 31.9 ± 2.0% (mean ± SEM, n =
3) of population 2 and 3 B cells, respectively, secreted Ig, thereby
demonstrating that CD40L does not inhibit Ig secretion on a per cell
basis. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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; Fig. 1), which are usually
considered to be synonymous with plasma cells (17, 38, 39, 40, 41). When differentiation of CD40L-activated memory B cells
was studied in relation to cell division, an increasing proportion of
these cells was shown to differentiate at each division to become
rapidly proliferating CD38+ B cells (Fig. 1). By
linking differentiation to cell division, it became feasible to
characterize the distinct effects of IL-10 and IL-2 on CD40L-activated
memory B cells. IL-10 was clearly a differentiation factor for memory B
cells, because the number of CD38+ B cells
generated per division in its absence was low. In contrast, IL-2
appeared to be a growth factor for activated memory cells irrespective
of whether they expressed CD38 or not. Thus, although it did not affect
the division-based rate of differentiation, IL-2 did increase B cell
recovery (Table I; Fig. 1).
The signals required for sustaining division and survival
changed once memory B cells had differentiated into
CD38+ B cells. In particular, removal of CD40L
did not prevent expansion of CD38+ B cells (
Figs. 2–4), although it did arrest proliferation of the
CD38- population. Consequently,
CD38+ B cells rapidly became the dominant ISCs
after withdrawal of CD40L (Figs. 2 and 4). In contrast, <50% of
CD38+ B cells secrete Ig (Fig. 5). When coupled with
their high proliferation rate, it would appear that in vitro-generated
CD38+ cells represent precursors of terminally
differentiated plasma cells. The proportion of cells within this
population that secrete Ig would be expected to increase with further
differentiation, consistent with the recent finding that plasmablasts
must first exit the cell cycle for terminal differentiation into
nondividing high-rate Ig-secreting plasma cells to occur
(47).
At face value, our findings do not support the hypothesis that
removal of CD40L is the molecular trigger for facilitating plasma cell
generation from memory B cells (17, 18, 19, 22).
Theoretically, CD40L could have been consumed in our cultures at a rate
equal to its removal from the cultures reported in earlier studies.
However, such an explanation can be excluded on the grounds that the
absolute number of CD38+ B cells and ISCs
generated in secondary cultures, as well as the level of Ig secreted,
was always greater in the presence of CD40L (Figs. 2 and 4; Table III).
Furthermore, because CD38- B cells require CD40L
to survive, a reduction in the levels of CD40L during in vitro culture
due to consumption would be expected to compromise survival of these
cells. However, because large numbers of viable
CD38- B cells were generated in the presence of
CD40L (Fig. 2, f and h), this did not appear to
occur. Thus, the increased proportion, as opposed to absolute numbers,
of CD38+ B cells observed after removal of CD40L
(Figs. 2 and 4) (17, 18, 19) is likely to have resulted from a
combination of the accelerated cell cycle arrest and death of
CD38- B cell blasts, relatively greater
proliferation of CD38+ vs
CD38- B cells, and differentiation of
CD38- B cells to CD38+ B
cells (
Figs. 2–4). In fact, close examination of the findings
originally presented by Arpin et al. (see Fig. 1c of Ref.
17) reveals data very similar to ours—withdrawal of CD40L
from secondary cultures caused a 10-fold reduction in the number of
CD38-CD20+ B cells, but
had very little effect on the number of CD38+ B
cells present.
The finding here that both CD38- and
CD38+ B cells with the same division history were
ISCs (Fig. 5) also contrasted with previous data indicating that
CD38+ B cells contained more intracellular Ig
than did CD38- B cells (17, 18).
Because withdrawal of CD40L greatly compromised survival of
CD38- B cell blasts (population 2;
Figs. 2–4),
the population of CD38- B cells examined
previously may well have been largely composed of undivided B cells
(population 1), which secrete and contain very little Ig (Figs. 5 and 6). Thus, comparison of secretion and expression of Ig by
differentially divided B cells may explain this discrepancy. Overall,
the major difference between the two models appears to be explicable in
terms of alternative interpretations of similar results, combined with
the use, in our case, of CFSE to resolve B cells into discrete
populations based on division history rather than phenotype alone.
Importantly, our interpretation receives support from other previous
studies showing that B cells produced high levels of Ig in the
continual presence of CD40L (41, 48) and that
proliferation was necessary for generating ISCs from precursor cells
(49, 50).
The detection of CD40L-independent CD38+
and CD40L-dependent CD38- ISCs in vitro is
novel, as is the demonstration that acquisition of CD38 is not an
absolute prerequisite for Ig production. It is likely that the two
populations of cells are related in a linear manner, with
CD38- ISCs subsequently acquiring CD38 together
with altered survival and stimulation requirements. This is consistent
with the observation that isolated CD38- B cell
blasts yielded CD38+ B cells (Fig. 4).
Alternatively, they may represent a different lineage of ISC.
Additional experiments to resolve this question will require the
identification of a reliable marker for the
CD38- ISC population. Nevertheless, if the
changing sensitivity of activated memory B cells to CD40L observed in
vitro can be extrapolated to the in vivo situation, it is possible that
CD38- and CD38+ ISCs
represent short-lived and long-lived ISCs, respectively (14, 50, 51, 52, 53). Thus, acquisition of CD38 expression may correlate with
selection into a population of T cell stimulation-independent, rapidly
proliferating plasma cell precursors, which contribute to an initial
expansion of the selected population of ISCs (9, 10, 47, 49, 54). The rapidly dividing CD38+ ISCs
presumably then acquire altered homing characteristics, resulting in
their migration to sites including bone marrow (55, 56),
where they undergo terminal differentiation to yield long-lived
quiescent CD38+ plasma cells (38, 45, 52, 53, 57, 58). In this way, Ig produced by the selected ISCs will
be sustained for long periods even after Ag clearance (2, 14). In contrast, the CD38- ISCs, being
CD40L dependent, will only survive and contribute Ig as long as Ag and
T cell help are both available. Given that stimulation of activated T
cells requires Ag presentation by memory B cells (1, 42),
successful Ag clearance would act as a feedback mechanism limiting
memory B cell expansion and further development of both short- and
long-lived ISCs, although limited differentiation of the
CD38+ ISCs already formed would continue. In vivo
studies have revealed that the number of Ag-specific B cells present at
the conclusion of a secondary immune response is reduced 10- to 50-fold
compared with the peak of the response, indicative of apoptosis of the
majority of memory blasts after Ag clearance (12). The
surviving memory blasts most likely revert to a quiescent state to
serve as the memory cell pool, which can be reactivated upon subsequent
Ag exposure (12, 15, 16, 59). Our in vitro findings that
CD38- blasts undergo rapid apoptosis after
removal of CD40L (Figs. 2 and 4) are in accord with these in vivo
observations and suggest that a small number of surviving
non-Ig-secreting CD38- blasts may contribute to
this pool of long-lived memory B cells.
The findings here of an increase in differentiation of memory B cells upon consecutive cell divisions to CD38+ B cells with enhanced proliferation and survival characteristics is reminiscent of other important lymphocyte differentiation events, including Ig isotype switching (23, 24, 26, 27, 28), cytokine production (25, 29, 30), and changes in expression of cell surface molecules (23, 30, 31, 32, 34). The transcription factors B lymphocyte-induced maturation protein-1 (60) and X-box-binding protein (61) facilitate development of plasma cells, while B cell-specific activator protein (62) and B cell lymphoma 6 (16, 63) repress this event. An attractive hypothesis for the features of B cell differentiation described here is that cell division alters the level of expression of key transcriptional regulators that affect the rate of differentiation, perhaps by controlling expression of a key master switch protein (11). By regulating the proportion of ISC precursors formed per division, a balance between the numbers of memory cells and plasma cells can be achieved. It is reasonable to conclude that the rate of division-linked differentiation has evolved to deal efficiently with pathogens through rapid development of short- and long-lived ISCs, while ensuring preservation and enhancement of the memory pool for protection against future exposure. Consequently, exploiting mechanisms involved in regulating the generation of ISCs and preserving memory B cells in vivo, for example by modulating IL-10, B lymphocyte-induced maturation protein-1, or bcl-6, may be advantageous for the development of vaccines designed to maximize protective humoral immunity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stuart G. Tangye, Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag #6, Newtown 2042, New South Wales, Australia. E-mail address: s.tangye{at}centenary.usyd.edu.au
3 Current address: Walter & Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia.
4 Abbreviations used in this paper: GC, germinal center; ISC, Ig-secreting cell; CD40L, CD40 ligand; BrdU, 5-bromo-2'-deoxyuridine; MFI, mean fluorescence intensity.
Received for publication July 24, 2002. Accepted for publication October 29, 2002.
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and IL-4 by B cells: positive and negative effects on CD40 ligand-induced proliferation, survival, and division-linked isotype switching to IgG1, IgE, and IgG2a. J. Immunol. 163:4175.This article has been cited by other articles:
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|
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|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
J. R. Darce, B. K. Arendt, S. K. Chang, and D. F. Jelinek Divergent Effects of BAFF on Human Memory B Cell Differentiation into Ig-Secreting Cells J. Immunol., May 1, 2007; 178(9): 5612 - 5622. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. C. Kuo, A. L. Shaffer, J. Haddad Jr., Y. S. Choi, L. M. Staudt, and K. Calame Repression of BCL-6 is required for the formation of human memory B cells in vitro J. Exp. Med., April 16, 2007; 204(4): 819 - 830. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Konforte and C. J. Paige Identification of Cellular Intermediates and Molecular Pathways Induced by IL-21 in Human B Cells J. Immunol., December 15, 2006; 177(12): 8381 - 8392. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. L. Good, V. L. Bryant, and S. G. Tangye Kinetics of Human B Cell Behavior and Amplification of Proliferative Responses following Stimulation with IL-21 J. Immunol., October 15, 2006; 177(8): 5236 - 5247. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
G. R.A. Ehrhardt, J. T. Hsu, L. Gartland, C.-M. Leu, S. Zhang, R. S. Davis, and M. D. Cooper Expression of the immunoregulatory molecule FcRH4 defines a distinctive tissue-based population of memory B cells J. Exp. Med., September 19, 2005; 202(6): 783 - 791. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. C. Macallan, D. L. Wallace, Y. Zhang, H. Ghattas, B. Asquith, C. de Lara, A. Worth, G. Panayiotakopoulos, G. E. Griffin, D. F. Tough, et al. B-cell kinetics in humans: rapid turnover of peripheral blood memory cells Blood, May 1, 2005; 105(9): 3633 - 3640. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. T. Avery, J. I. Ellyard, F. Mackay, L. M. Corcoran, P. D. Hodgkin, and S. G. Tangye Increased Expression of CD27 on Activated Human Memory B Cells Correlates with Their Commitment to the Plasma Cell Lineage J. Immunol., April 1, 2005; 174(7): 4034 - 4042. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Tarlinton and P. D. Hodgkin Targeting Plasma Cells in Autoimmune Diseases J. Exp. Med., June 7, 2004; 199(11): 1451 - 1454. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Arce, E. Luger, G. Muehlinghaus, G. Cassese, A. Hauser, A. Horst, K. Lehnert, M. Odendahl, D. Honemann, K.-D. Heller, et al. CD38 low IgG-secreting cells are precursors of various CD38 high-expressing plasma cell populations J. Leukoc. Biol., June 1, 2004; 75(6): 1022 - 1028. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. I. Ellyard, D. T. Avery, T. G. Phan, N. J. Hare, P. D. Hodgkin, and S. G. Tangye Antigen-selected, immunoglobulin-secreting cells persist in human spleen and bone marrow Blood, May 15, 2004; 103(10): 3805 - 3812. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Nishimura, T. Igarashi, N. L. Haigwood, R. Sadjadpour, O. K. Donau, C. Buckler, R. J. Plishka, A. Buckler-White, and M. A. Martin Transfer of neutralizing IgG to macaques 6 h but not 24 h after SHIV infection confers sterilizing protection: Implications for HIV-1 vaccine development PNAS, December 9, 2003; 100(25): 15131 - 15136. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Deaglio, A. Capobianco, L. Bergui, J. Durig, F. Morabito, U. Duhrsen, and F. Malavasi CD38 is a signaling molecule in B-cell chronic lymphocytic leukemia cells Blood, September 15, 2003; 102(6): 2146 - 2155. [Abstract] [Full Text] [PDF]
|
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), CD40L and IL-10 (
), or CD40L,
IL-2, and IL-10 (•) was determined by division slicing. The
expression of CD38 by CFSE-labeled naive and memory B cells stimulated
with CD40L (b and g), CD40L and IL-10
(c and h), or CD40L, IL-2, and IL-10
(d and i) was determined by labeling with
anti-CD38 mAb, followed by flow cytometric analysis. The contour
plots are representative of at least ten independent experiments. The
percentage of CD38+ B cells in each division of
naïve (e) or memory (j) B cells
after culture with CD40L alone (
