Quantitative Regulation of B Cell Division
Destiny by Signal Strength
Marian L. Turner, Edwin D. Hawkins and Philip D. Hodgkin
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References
J Immunol 2008; 181:374-382; ;
doi: 10.4049/jimmunol.181.1.374
http://www.jimmunol.org/content/181/1/374
The Journal of Immunology
Quantitative Regulation of B Cell Division Destiny by Signal
Strength
Marian L. Turner,*† Edwin D. Hawkins,*† and Philip D. Hodgkin1*
A
ctivation of B cells can occur in a T cell-dependent or
-independent manner. In vitro, both stimulation conditions lead to a proliferative response comprising progression through multiple rounds of cell division and concurrent
differentiation to Ab secreting and isotype-switched effector cells.
The type of stimulatory signal and the features of the signal in
terms of dose, affinity, physical structure, or duration can qualitatively and quantitatively regulate B cell responses in vivo and in
vitro (1–7). Although it is intuitively sensible that a population of
cells will respond better to stronger signals than to weaker ones, a
thorough and quantitative description of how B cell proliferation is
regulated in response to signals of varying strength and quality is
lacking. Understanding the mode of regulation is important not
simply because proliferation determines the overall size of the responding lymphocyte population but also because the differentiation of B cells to Ab secreting cells and the process of isotype
switching are integrally linked to cell division (8 –12). As a consequence of this, qualitative features of the response, such as type
of Ab, will be influenced by the extent of division progression of
the participating cells. Thus, progression through successive divisions and the control of the ultimate number of divisions reached
is an important variable in the generation of an appropriate immune response.
Previous studies exploring the control of lymphocyte division
progression have highlighted two broad classes of behavior.
The first has been called the “autopilot” response (13), in which
a brief encounter with Ag leads to a large number of successive
*Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia;
and †Department of Medical Biology, University of Melbourne, Parkville, Victoria,
Australia
Received for publication January 31, 2008. Accepted for publication April 28, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Philip D. Hodgkin, Walter and
Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050,
Australia. E-mail address: [email protected]
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
www.jimmunol.org
divisions, as exemplified by CD8⫹ T cell behavior. In contrast,
CD4⫹ T and B cell division progression appears to be more
closely dependent on continual stimulation (14, 15). Rush and
Hodgkin (14) showed that B cells are able to undergo only a few
further divisions if the stimulus is removed, before they stop
dividing and die. Clearly this behavior will influence the response mounted, as intrinsic division-linked differentiation
events will only proceed as long as division is maintained. Rush
and Hodgkin (14) used this information to suggest a feedback
mechanism whereby the continued presence of Ag serves to
progress the B cell Ab class through a series of changes to
modify the type of response.
We recently presented a quantitative model of lymphocyte
proliferation (16). In this model, we proposed a control unit in
each cell made up of two independent operators or machines,
one regulating the time for cell division and the other regulating
the time to cell death. The mean values for the time to either
outcome are regulated by internal and external influences and
stochastic variation within the population gives the potential for
many different outcomes. The interaction of these intracellular
“timers” sensitively controls the proliferation dynamics of the
lymphocyte population. A third control mechanism necessary to
complete the model and to fully describe in vitro and in vivo
data was that describing the population “division destiny,” or
the maximum number of divisions each participating cell would
undergo.
In this study, we examine in greater detail the regulation of
division destiny during B cell proliferation. We demonstrate
that in vitro B cell division progression is different for different
stimuli and is further regulated by the concentration of the stimulatory molecule and the timing of exposure to stimulation. Our
results reveal that B cells have an intrinsic division number
restriction, even under the optimal stimulation conditions used
in our experiments, which represents the maximum of the division destiny continuum. We also provide evidence supporting
the hypothesis that the implementation of division destiny is
likely to be achieved via division counting rather than a timing
mechanism.
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Differentiation to Ab secreting and isotype-switched effector cells is tightly linked to cell division and therefore the degree of
proliferation strongly influences the nature of the immune response. The maximum number of divisions reached, termed the
population division destiny, is stochastically distributed in the population and is an important parameter in the quantitative
outcome of lymphocyte responses. In this study, we further assessed the variables that regulate B cell division destiny in vitro in
response to T cell- and TLR-dependent stimuli. Both the concentration and duration of stimulation were able to regulate the
average maximum number of divisions undergone for each stimulus. Notably, a maximum division destiny was reached during
provision of repeated saturating stimulation, revealing that an intrinsic limit to proliferation exists even under these conditions.
This limit was linked directly to division number rather than time of exposure to stimulation and operated independently of the
survival regulation of the cells. These results demonstrate that a B cell population’s division destiny is regulable by the stimulatory
conditions up to an inherent maximum value. Division destiny is a crucial parameter in regulating the extent of B cell responses
and thereby also the nature of the immune response mounted. The Journal of Immunology, 2008, 181: 374 –382.
The Journal of Immunology
375
FIGURE 1. B cell proliferation in
response to varying stimulus concentrations. CFSE-labeled naive resting
B cells were stimulated in vitro with
anti-CD40 and IL-4, LPS, or CpG at
the concentrations shown. Cell numbers (A–C) and the proportion of cells
in each division (D–F) were calculated at representative time points for
each stimulation condition. Each data
point represents the mean of cell numbers or division numbers calculated
from three culture wells. Mean division number (G–I) was calculated at
multiple time points as described in
Materials and Methods.
Flow cytometry and data analysis
Mice
B cells were obtained from mice that were bred and maintained at the
Walter and Eliza Hall Institute (WEHI) animal facilities in specific pathogen-free conditions and in accordance with WEHI animal ethics committee
regulations. Male C57BL/6 mice were used unless specified. Bcl-2 Vav
transgenic mice were provided by Steve Gerondakis (WEHI, Parkville,
Australia). Bim-deficient B cells (17) were provided by Philippe Bouillet
(WEHI). SWHEL mice (18) were provided by Robert Brink (Garvan Institute for Medical Research, Sydney, Australia) and were crossed with
blimp-1GFP/⫹/RAG2⫺/⫺ mice (19) provided by Stephen Nutt (WEHI). B
cells were isolated from mice at age 6 –12 wk.
Flow cytometric data were collected on FACScan or LSR flow cytometers
(BD Biosciences) and analyzed with Flow-Jo software (Tree Star). Propidium iodide was added to cell cultures before harvest to identify dead cells,
which were excluded from analysis. To determine the absolute number of live
cells per culture, a known number of CaliBRITE beads (BD Biosciences) was
added directly to cell cultures before harvesting for flow cytometry. The proportion of cells in each division was calculated from CFSE profiles and bead
counts as previously described (21). Mean division number was calculated by
multiplying the number of cells in each division by the division number (taken
to be 0.5, 1.5, 2.5, etc.). MoFlo or FACSDiva flow cytometers (BD Biosciences) were used for cell sorting.
Reagents and Abs
Results
Anti-CD40 mAb (1C10) was prepared from a hybridoma cell line provided
by the DNAX Research Institute. Recombinant mouse IL-4 was a gift of
Dr. Robert Kastelein (DNAX Research Institute, CA). LPS derived from
Salmonella typhosa was obtained from Sigma-Aldrich. CpG DNA oligonucleotide CpG-1668, fully phosphothioated (sequence TCCATGACGTT
CCTGATGCT) was synthesized by Geneworks (Adelaide, Australia).
Stimuli were used at concentrations of 20 ␮g/ml 1C10, 500 U/ml IL-4, 15
␮g/ml LPS, and 3 ␮M CpG unless otherwise stated. CFSE was purchased
from Molecular Probes and PKH26 was purchased from Sigma-Aldrich.
Hen egg lysozyme (HEL)2 was purchased from Sigma-Aldrich.
B cell isolation and cell culture
Single-cell suspensions were prepared from mouse spleens, subjected to
red cell lysis, and separated on discontinuous Percoll density gradients as
described previously (8). Small dense cells were harvested from the 65:
80% interface and B cells purified via negative selection using MACS
beads (Miltenyi Biotec). B cells were typically ⬎98% B220⫹, CD19⫹ and
⬎95% IgM⫹, IgD⫹ as determined by flow cytometry. Where appropriate,
cells were labeled with CFSE according to the originally published method
(20) or with PKH26 according to the manufacturer’s instructions. B cells
were cultured at 37°C (or 32°C where stated) in B cell medium comprising
RPMI 1640 medium (Life Technologies) with 2 mM L-glutamine, 0.1 mM
non-essential amino acids, 10 mM HEPES (pH 7.4), 100 ␮g/ml of streptomycin, 100 U/ml of penicillin, 50 ␮M 2-ME (all supplements were purchased from Sigma-Aldrich), and 10% heat-inactivated FCS (CSL).
2
Abbreviations used in this paper: HEL, hen egg lysozyme; ASC, Ab-secreting cell.
The stimulus dose determines B cell proliferation capacity by
regulating division number
Naive B cells labeled with CFSE and stimulated in vitro with polyclonal activators can be assessed at various time points during a
stimulation experiment to provide information on the effects of
culture conditions on multiple quantitative parameters of cell proliferation and survival (16). We first sought to assess the effect of
signal strength on the proliferative response of B cells by varying
the concentration of the stimuli. T cell-dependent stimulation was
mimicked using an anti-CD40 mAb (1C10) and IL-4. The TLR
agonists LPS and CpG were used as T cell-independent stimuli. B
cell cultures were treated with varying concentrations of each of
the three stimuli, and proliferation was assessed in terms of cell
number and progression through subsequent divisions. The proliferation profiles followed the same pattern in each condition, showing an increase in cell number to a point at which population
growth ceased and a gradual decline in cell number commenced.
However, a clear effect of stimulus concentration was also observed, with the rate of population growth and the total number of
live cells increasing with an increasing dose (Fig. 1, A–C). Analysis of CFSE profiles revealed that the proportion of cells entering
division was affected by the stimulus dose, such that a larger number of cells remained undivided at low stimulus concentrations
(Fig. 1, D–F). These populations had also progressed through
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Materials and Methods
376
REGULATION OF B CELL DIVISION DESTINY BY SIGNAL STRENGTH
fewer subsequent divisions. Further examination of division progression over the course of the experiment revealed that the mean
population division number increased with increasing concentration of all stimuli (Fig. 1, G–I). Cells stimulated with the highest
concentrations of anti-CD40 and LPS stimulation underwent ⬎8
divisions, such that CFSE autofluorescence was reached and peaks
could no longer be discriminated (data not shown). These cells
were considered as being in division 8⫹ for the purpose of calculating mean division number. In contrast, CFSE profiles of CpGstimulated cells showed maximum division progression within the
range of CFSE resolution, as revealed by the plateau of mean
population division number seen in Fig. 1I.
The duration of stimulation also regulates the division number
reached by the responding population
Another variable of stimulation of a proliferative response that can
be experimentally adjusted is the duration of the stimulus. Division
bursts following transient T-dependent stimulation of B cells have
been observed previously (14). Furthermore, the cessation of proliferation after limited-duration stimulation has been used to verify
the independence of division progression and survival parameters
(16). As in vivo Ag exposure and T cell help are also likely to be
temporally regulated, we studied the effect of the duration of stimulation on B cell proliferation in more detail. B cells activated with
each of the three stimuli tested were capable of entering division
even if the stimulus was removed before the onset of mitosis;
however, the maximum total cell numbers (Fig. 2, A–C) and the
degree of division progression reached (Fig. 2, D–F) were less
than when stimulus was continually present. The extent of prolif-
eration correlated with the duration of stimulus exposure. The proportion of cells entering division was also lower in cultures receiving short stimulation than those in continual stimulation (Fig.
2, D–F). Notably, during the period of population decline, there
was no further division progression, as indicated by the plateau of
mean population division number (Fig. 2, G–I) and by the observation of no change in the proportion of cells in each division class
(Fig. 2, J–L).
Stimulation dose and duration can coordinately regulate B cell
division destiny
We next explored the interaction between stimulus concentration
and duration in regulating proliferation. B cells were subjected to
the same concentration range of stimuli as presented in Fig. 1, but
the stimuli were removed before entry to division. The mean division number achieved was regulated by the dose of stimulus in
all cases (Fig. 3, G–I) in a similar manner to that seen in continual
stimulation. This indicates that the dose of the stimulus is also
capable of regulating the division destiny of a B cell population
that only encounters that stimulus before entry to division. However, the mean division number reached at all concentrations was
considerably less when stimulation was removed early in the culture period (Fig. 3, G–I) than when compared with cell populations
in which the equivalent dose was present throughout the culture
period (Fig. 1, G–I).
A notable observation from this experiment was that while the
mean division number of cells stimulated with anti-CD40 and IL-4
or LPS plateaued at maximum division progression and remained
static during population decline, the mean division number of
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FIGURE 2. B cell proliferation in
response to variable stimulation duration. CFSE-labeled naive resting B
cells were stimulated in vitro with 20
␮g/ml anti-CD40 and 500 U/ml IL-4,
15 ␮g/ml LPS, or 3 ␮M CpG. At the
times indicated (e.g., 30, 40, and 50 h
for anti-CD40 cultures), parallel cultures of cells were harvested, washed
three times in warm medium, and returned to culture in medium alone.
Constant stimulation is represented by
cells washed at the earliest time and
returned to culture with the original
level of stimulation. Cell numbers (A–
C), the proportion of cells in each division (D–F), and the mean division
number (G–I) were determined at various time points as described in Fig. 1.
The proportion of cells in each division at various time points is also
shown for one stimulation duration
condition for each stimulus type (J–
L). Each data point represents the
mean of cell numbers or division
numbers calculated from three culture
wells. We have previously presented a
portion of the data from the
1C10⫹IL-4 stimulation experiment
used in this figure (16).
The Journal of Immunology
377
CpG-stimulated cultures appeared to decrease during this phase
suggesting that, in this system, cells in later divisions may die
more rapidly than those in earlier divisions.
The distribution of cells across divisions at the point of proliferation arrest following stimulus removal has been previously described as the population division destiny (16). The results presented in Figs. 1–3 demonstrate that the division destiny of
responding B cells is able to be regulated in a progressive manner
by both the concentration and the duration of all stimuli tested and
is an important parameter in determining the overall proliferative
outcome.
than their undisturbed counterparts (Fig. 4F). Similar dilution experiments were performed in cultures of LPS or anti-CD40 plus
IL-4 stimulated cells and proliferation was limited in every case
(data not shown). These data indicate that activated B cells are
subject to a limit to proliferation that cannot be overcome by repeated stimulation. For CpG-stimulated B cells, this limit appears
to be exerted at a maximum of six divisions (Fig. 4C). It was not
possible with CFSE techniques to determine the division destiny of
The extent of B cell proliferation depends on the mitogen but is
intrinsically limited in all stimulation conditions
In the experiments presented above, it was observed that responding B cell populations reached a point at which proliferation
ceased even when provision of the highest stimulus concentrations
was maintained. These results suggested that an intrinsic limit to B
cell proliferation capacity exists for a given stimulus, even in maximal stimulation conditions. This appeared to be true for all stimuli
tested, although the onset of population decay occurred at different
population division progression profiles (Fig. 4, A–C) and times
(Fig. 4D) depending on the mitogen used. We hypothesized that
this limit is an extension of the regulable division destiny parameter observed in conditions of restricted stimulus dose or duration.
We first sought to exclude the possibility that the limit to proliferation was imposed by cell overgrowth or insufficient access to
mitogenic signals. Identical cell cultures in CpG stimulation were
grown undisturbed or diluted by a factor of 10 before the point at
which population decay commenced and then were supplemented
with fresh medium and stimulus. Subsequent proliferation was
tracked by cell number and CFSE profile analysis. Despite the
reduction in total cell number, the onset of population decline occurred at a very similar time to undisturbed cultures (Fig. 4E).
Correcting for the dilution factor showed that cells in the diluted
culture were not able to achieve a greater population expansion
FIGURE 4. B cell proliferation in response to repetitive in vitro stimulation. CFSE-labeled naive resting B cells were stimulated in vitro with 20
␮g/ml anti-CD40 and 500 U/ml IL-4, 15 ␮g/ml LPS, or 3 ␮M CpG. Cells
stimulated with CpG were resuspended after 70 h of stimulation and one
tenth of the culture volume was transferred to new culture wells containing
medium and CpG at the required concentration to return the cells to the
original stimulatory conditions. Parallel cultures were maintained undisturbed. CFSE profiles of division progression (A–C) and calculated total
live cell numbers (D, E) are shown at various time points during culture.
Corrected cell numbers were determined by multiplying the actual cell
number by the dilution factor (F). Each data point represents the mean ⫾
SD from three replicate cultures.
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FIGURE 3. The B cell proliferation response to defined stimulation
concentration and duration. CFSE-labeled naive resting B cells were stimulated in vitro with anti-CD40 and
IL-4, LPS, or CpG at the concentrations shown. At 40 (anti-CD40), 30
(LPS), or 27 h (CpG) cells were removed from culture, washed three
times in medium, and returned to culture in medium alone. Cell numbers
(A–C), the proportion of cells in each
division (D–F) and the mean division
number (G–I) were determined at various time points as described in Fig. 1.
Each data point represents the mean
of cell numbers or division numbers
calculated from three culture wells.
378
REGULATION OF B CELL DIVISION DESTINY BY SIGNAL STRENGTH
LPS- or anti-CD40-stimulated B cells but proliferation and decay
profiles (Fig. 4D) suggested that these stimuli may induce a mean
of 10 –12 divisions.
Enforced survival does not extend B cell proliferation
Low temperatures slow proliferation and delay the time of
population contraction
Bcl-2 over-expressing B cells from transgenic mice have been
used to demonstrate that survival and division progression are independently regulated (16). We used these transgenic cells to assess whether enforced survival could overcome the limit to division number observed during continual stimulation. Cultures of
Bcl-2 transgenic cells proliferating in response to CpG stimulation
were left undisturbed or split to reduce cell number and provided
with additional stimuli. Cell number analysis revealed a plateau in
population expansion in each case (Fig. 5A), in contrast with the
cell death seen in cultures of identically stimulated nontransgenic
cells. Our interpretation of this arrest is that the B cells were no
longer cycling, however they remained alive due to the block in
apoptosis caused by over-expression of Bcl-2. Indeed, CFSE profiles revealed that enforced survival did not lead to progression
through ⬎6 divisions (Fig. 5, B and C), the limit for CpG proliferation observed previously (Fig. 4). To ensure that division cessation in the Bcl-2 transgenic B cells was not complicated by dysregulation of c-Myc (22), we repeated the experiment with Bimdeficient B cells, which are also long-lived (17). These B cells also
stopped dividing at a similar time point and division profile to wild
type B cells in CpG stimulation, yet remained alive for considerably longer (data not shown). These results indicate that the division destiny of the responding cells was not influenced by extending their survival.
The conundrum of whether cell fates are determined by counting
division number or time has been addressed in other systems. During the examination of oligodendrocyte precursor cells it was
found that changes in temperature could be used to explore the
relative contribution of time and division based counting mechanisms (23). We adopted this strategy and investigated the effect of
temperature on B cell proliferation. Replicate cultures of B cells
identically stimulated with LPS were incubated at 32°C or 37°C. B
cells cultured at the lower temperature took a significantly longer
time to enter division (Fig. 6B). However, once proliferation commenced, the cells underwent multiple rounds of division in a normal fashion, as assessed by CFSE dilution (data not shown). Similar overall levels of proliferation were achieved by cell
populations cultured at both temperatures, however the time at
which the maximum cell number was reached occurred significantly later in populations of cells grown at the lower temperature
(Fig. 6B). CFSE profiles revealed that cells grown at 32°C lagged
behind in earlier divisions compared with cells cultured at 37°C at
various time points throughout the experiment, but that these cells
had also undergone ⬎8 divisions by the time at which population
decline began (data not shown). These results are consistent with
the existence of a division-based regulator and argue against the
conclusion that cells stop dividing after a given time.
B cells retain their division destiny even when proliferation is
interrupted
B cells sorted from different divisions demonstrate a hierarchy
of subsequent proliferation capacity
The data presented in Figs. 4 and 5 revealed a limit to the in vitro
proliferation of B cells under conditions of saturating stimulation.
In a further experiment designed to distinguish between a time- or
division-imposed limit to proliferation, proliferating B cells were
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FIGURE 5. Proliferation of transgenic B cells with extended survival.
Naive resting B cells over-expressing the anti-apoptotic molecule Bcl-2
were labeled with CFSE and stimulated with 3 ␮M CpG. B cells from wild
type C57BL/6 mice were stimulated in identical conditions. Cells were
resuspended at Day 3 and one half or one tenth of the cultures were transferred to new culture wells containing medium and stimuli at the required
concentration to return the cells to the original conditions. Parallel control
cultures were left undisturbed. Calculated cell numbers corrected for the
appropriate dilution factor are shown (A). CFSE profiles of division progression of undisturbed (B) cultures or cultures split 1/10 (C) are shown
from various time points. Each data point represents the mean ⫾ SD from
three replicate cultures.
This feature of B cell responses operates independently of cell
survival and can be well explained as representing the natural endpoint of a continuous scale of division destiny described in earlier
sections. However, the existing data were insufficient to determine
whether the proliferation limit was imposed by the time in culture
or by the division number of responding cells. This is an important
distinction as it may provide information on the mechanism by
which B cell proliferation is regulated and insight into the control
of contraction of B cell responses in vivo. A series of experiments
was therefore designed to discriminate between these alternatives
(Fig. 6).
Firstly, we used the observation that short stimulation duration
leads to a truncated division destiny (Fig. 2) to conduct an experiment in which progression through divisions was interrupted for
an interval of time. B cells were stimulated with LPS, removed
from culture during the population expansion phase and washed
thoroughly before being returned to culture in either medium alone
or with LPS. As seen in Fig. 2, cells in cultures in which the
stimulus was removed reached a lower total cell number than the
restimulated cells and rapidly commenced dying (Fig. 6A). However, subsequent reprovision of LPS rescued these B cells from
population decay and resulted in resumed proliferation (Fig. 6A).
The second expansion phase also reached a maximal point, but at
a later time point than in cultures stimulated continuously. CFSE
profiles indicated that ⬎8 divisions had been undergone in all cultures by the time at which maximum proliferation was reached
(data not shown). This suggested that the reprovision of stimulus
allowed cells in these cultures to reach a similar division destiny to
those cells in undisturbed cultures, but that this occurred at a later
time due to the proliferation arrest during the period of stimulus
deprivation.
The Journal of Immunology
379
FIGURE 6. Evidence for regulation of B cell proliferation cessation by
division number. Naive resting B cells were stimulated in vitro with 15
␮g/ml LPS and total cell numbers were determined at multiple time points.
Each data point represents the mean ⫾ SD from three replicate cultures. A,
After 40 h in culture, the cells were harvested, washed three times in warm
medium and returned to culture in medium alone. At 80 h, one set of
stimulus-deprived cells was harvested again and returned to culture with
the initial concentration of LPS. B, LPS-stimulated B cells were cultured at
32° or 37°C. C, CFSE-labeled B cells were stimulated with LPS for 3 days
before harvesting. The cells were sorted from individual divisions based on
CFSE peaks and returned to culture in the original stimulation conditions.
D, B cells were labeled with the red division tracking dye PKH26 before
stimulation with 20 ␮g/ml anti-CD40 and 500 U/ml IL-4 for 4 days before
harvesting. The cells were sorted from early or late divisions based on PKH
fluorescence intensity and returned to culture in the original stimulation
conditions. Proliferation profiles after return to culture are shown.
sorted according to the number of completed divisions and returned to separate cultures. Tracking the cells’ subsequent proliferation revealed that cells sorted from later divisions proliferated
less extensively and entered population decay earlier than cells
sorted from earlier divisions (Fig. 6C). All cell populations had
reached CFSE autofluorescence by the time of growth cessation.
This sorting experiment was repeated using B cells stimulated in
T-dependent conditions using anti-CD40 and IL-4. As for LPS
stimulation, cells sorted from early divisions had a greater proliferation capacity on return to culture than those sorted from later
divisions (Fig. 6D).
The results of the experiments presented in Fig. 6 are all consistent with the hypothesis that a division-linked limit is the key
regulator of the extent of B cell proliferation, not the time of response. This implies that populations of naive B cells have an
intrinsic distribution of maximum division number and that this
distribution is a continuation of the division destiny parameter described in earlier sections to be regulated by stimulation dose and
duration.
Division destiny is additionally influenced by B cell
differentiation
The differentiation of B cells to Ab-secreting cells (ASC) has been
shown to be linked to division number (9, 11). Of the three stimulatory conditions used in this study, the number of ASC generated
is most significant for LPS stimulation, reaching nearly 50% of the
population after 4 days of culture (19). Thus, for the LPS stimulation system, it was possible that differentiation was contributing
to the cessation of cell division. We used B cells from blimpGFP/⫹
transgenic mice (19) to assess whether ASC differentiation has an
effect on B cell division destiny. LPS-stimulated cells were sorted
into populations based on their relative division progression and
GFP levels. Of the undifferentiated (GFP negative) cells, those
sorted from earlier divisions exhibited the greater proliferative capacity on return to culture (Fig. 7A), an expected pattern in light of
data presented in Fig. 6C. However, the behavior of the differentiated (GFP positive) cells was strikingly different, as these cells
did not achieve any population expansion on return to culture (Fig.
7A). These results indicated that differentiated and undifferentiated
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FIGURE 7. Differentiation to Ab secreting cells influences B cell division destiny. Naive B cells from blimp-1GFP/⫹ reporter mice were labeled
with PKH26 before culture in LPS stimulation. Cells were sorted based on
ASC differentiation status (GFPhigh, differentiated or GFPlow, undifferentiated) and division progression (PKHhigh, early divisions or PKHlow, late
divisions) before return to culture and tracking of cell number (A). Naive
resting B cells from SWHEL xblimpGFP/⫹ mice were stimulated with 15
␮g/ml LPS only or 15 ␮g/ml LPS supplemented with 100 ng/ml HEL. B
cells in LPS ⫹ HEL cultures were split 1 in 10 at day 2. Differentiated ASC
(GFP positive) cells were identified on day 5 (B) and total live cell numbers
were calculated (C). Each data point represents the mean ⫾ SD from three
replicate cultures.
380
REGULATION OF B CELL DIVISION DESTINY BY SIGNAL STRENGTH
cells have very different division progression competencies and
suggested that the process of differentiation is able to reprogram
the division destiny of individual cells.
To assess whether the process of ASC differentiation has a direct
influence on proliferation limitation, we exploited the ability of
BCR stimulation to potently inhibit differentiation in LPS-stimulated cultures. Specific Ag HEL was added to cultures of HEL
BCR-transgenic (SWHEL) B cells stimulated with LPS. Extensive
proliferation occurs in these cultures but B cells do not progress to
exhibit ASC phenotypic characteristics or secrete Ab (Ref. 24 and
Fig. 7B). However, a limit to proliferation was still observed in
these cultures, even when the cell density was reduced and additional stimulation provided (Fig. 7C). These results are in accordance with a model of B cell proliferation in which division destiny is an independent regulator of B cell division capacity but that
differentiation may further alter the division destiny of a cell.
Discussion
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Lymphocyte differentiation has been shown to be tightly linked to
proliferation and cell division and is therefore a crucial determinant in the nature and effectiveness of the immune responses. The
cyton model of Hawkins et al. (16) introduced a division destiny
parameter to allow for description of lymphocyte proliferation cessation and population contraction. The inclusion of the division
destiny parameter enabled the model to accurately fit data from
both in vitro proliferation experiments and in vivo infection responses highly accurately and is therefore of significant relevance
for a better understanding of lymphocyte behavior in vitro and
physiologically. In this study, we sought to better understand the
regulation of B cell division number and the rules governing the
contraction of B cell responses.
We chose the strength of the activating signal as an appropriate
starting point for investigating the regulation of the extent of immune responses. Naive B cells responded to a variety of activating
signals in vitro in a dose-dependent manner, manifested in the
maximum cell number reached. The effect of stimulus dose on cell
number did not appear to be due to significant differences in the
relative survival rates of cells before first division, consistent with
previous findings (16). However, it was clear that the number of
subsequent divisions undergone was strongly affected by signal
strength.
During these experiments, we noted that responding B cell populations followed a characteristic pattern of population growth,
proliferation cessation, and subsequent cell death, even in conditions of continual stimulation at saturating doses. This pattern resembled a physiological immune response. The onset of population contraction occurred at different times under the various
stimulation conditions and appeared to be an intrinsic feature of
the responses, and not a consequence of restrictive culture conditions (Fig. 4). Furthermore, B cells forced to survive by transgenic
over-expression of Bcl-2 also reached a maximum cell number and
could not be driven to proliferate further (Fig. 5). These results
suggested a limit to B cell proliferation that is imposed by the
stimulation conditions and is independent of the survival capacity
of the cells.
B cells have previously been shown to exhibit “division momentum”: a short period of continued in vitro proliferation following removal of T-dependent stimulation (14). However, these cells
stop dividing earlier than continually stimulated cells. We extended these studies to show that naive B cells were able to undergo multiple rounds of cell division following removal of Tdependent or -independent activating signals (Figs. 2 and 3).
Furthermore, cells could enter and continue proliferation even
when the stimulus was removed before the first mitotic event (Figs.
2 and 3). The striking conclusion for all three stimuli was that the
duration of stimulus exposure was a strong regulator of the number
of subsequent divisions achieved. Thus, both the concentration of
stimulus and the time of exposure are able to regulate division
number, and these variables can work in tandem to yield many
possible quantitative outcomes.
These results can be compared with the reported behavior of T
cells. Ag concentration has been shown to regulate the number of
T cells recruited into the proliferative response and the subsequent
number of divisions they reach (25–27). CD8⫹ T cells can continue to divide in vitro (28) or in vivo (29) even after the activating
signal is removed. In fact, only a very brief pulse of stimulus is
required to initiate multiple rounds of CD8⫹ T cell division and
differentiation to cytotoxic effector and memory cells (30). CD4⫹
T cells that have experienced Ag encounter long enough to enter
the cell cycle are also able to progress through further divisions in
the absence of a continued TCR signal, although their subsequent
proliferative program is not as striking as that of CD8⫹ T cells
(15). Despite this “autopilot” behavior of proliferating T cells (13),
there is ample evidence that the response can be regulated by stimulation conditions (15, 31, 32). We have shown in this study that
the duration and dose of stimulation can operate alone or in tandem
to determine the maximum B cell division number reached. These
mechanisms for regulating division number are also seen in T cells
(25, 33). B cell behavior appears to more closely mimic that of
CD4⫹ than CD8⫹ T cells, in that proliferation can occur without
prolonged or repeated Ag exposure, but that participating cells do
not undergo as many divisions as would be achieved in the presence of continual stimulation.
A proliferation limit could be a cell-intrinsic characteristic or
one imposed by extrinsic factors. Although extrinsic factors are
highly likely to influence the behavior of B cells during physiological responses, the in vitro proliferation system used in these
experiments is not cell density or contact dependent. In mixed
cultures of B cells from late divisions with naive cells, there was
no detrimental effect of the presence of the cycling cells on the
proliferation capacity of the naive cells (data not shown). The data
presented in Fig. 4 show that proliferating B cells reached the point
of maximum population expansion at approximately the same time
regardless of whether they were undisturbed or restimulated. This
implied that either a time- or division-based limit is imposed on
proliferating B cells. A series of experiments (Fig. 6) was designed
to distinguish between the two possible mechanisms: division- or
time-based control of proliferation cessation. Cells subjected to an
interrupted stimulation regime (Fig. 6A) or cultured at low temperatures (Fig. 6B) proliferated to an equivalent degree to cells that
received uninterrupted stimulation but achieved this over an extended time period. Furthermore, cells sorted from late divisions
exhibited a lesser ability to continue proliferating than cells sorted
from earlier divisions (Fig. 6C). These combined results strongly
suggest that the basis for the contraction of B cell populations
responding to continual stimulation is via a division counter. A
plausible mechanistic explanation for this could be the dilution of
an accumulated intracellular factor that occurs with each cell division (14). However, the data still do not formally exclude a timebased counter that operates from the time of entry to first division.
In effect, these two mechanisms would produce very similar outcomes in B cell populations, given the asynchrony of time to enter
first division (10, 16).
There are various examples of intrinsic intracellular timers that
operate during developmental pathways and that may be triggered
by time or division number (reviewed in Ref. 34). Normal somatic
cells undergo only a finite number of cell divisions in vitro before
reaching a state termed replicative senescence (35–37). The onset
The Journal of Immunology
Acknowledgments
We thank Gabrielle Belz for comments on the manuscript.
Disclosures
The authors have no financial conflict of interest.
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