Regulation of Memory Antibody Levels: The
Role of Persisting Antigen versus Plasma Cell
Life Span
This information is current as
of June 17, 2017.
Dominique Gatto, Stephen W. Martin, Juliana Bessa, Erica
Pellicioli, Philippe Saudan, Heather J. Hinton and Martin F.
Bachmann
J Immunol 2007; 178:67-76; ;
doi: 10.4049/jimmunol.178.1.67
http://www.jimmunol.org/content/178/1/67
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References
The Journal of Immunology
Regulation of Memory Antibody Levels: The Role of Persisting
Antigen versus Plasma Cell Life Span
Dominique Gatto,1 Stephen W. Martin,1 Juliana Bessa, Erica Pellicioli, Philippe Saudan,
Heather J. Hinton, and Martin F. Bachmann2
Protective Ab levels can be maintained for years upon infection or vaccination. In this study, we studied the duration of Ab
responses as a function of the life span of plasma cells and tested the role of persisting Ag in maintaining B cell memory. Our
analysis of B cell responses induced in mice immunized with virus-like particles demonstrates the following: 1) Ab titers are
long-lived, but decline continuously with a t1/2 of ⬃80 days, which corresponds to the life span of plasma cells; 2) the germinal
center (GC) reaction, which lasts for up to 100 days, is dependent on Ag associated with follicular dendritic cells; and 3) early GCs
produce massive numbers of plasma and memory B cell precursors, whereas the late Ag-dependent GCs are dispensable for the
maintenance of Ab levels and B cell memory. The Journal of Immunology, 2007, 178: 67–76.
Cytos Biotechnology, Zurich-Schlieren, Switzerland
Received for publication April 26, 2006. Accepted for publication October 4, 2006.
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
D.G. and S.W.M. contributed equally to this work.
2
Address correspondence and reprint requests to Dr. Martin F. Bachmann, Cytos
Biotechnology AG, Wagistrasse 25, CH-8952 Zurich-Schlieren, Switzerland. E-mail
address: [email protected]
3
Abbreviations used in this paper: FDC, follicular dendritic cell; AFC, Ab-forming
cell; GC, germinal center; PNA, peanut agglutinin; VLP, virus-like particle; LT, lymphotoxin; LT␤R, LT ␤ receptor.
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$2.00
www.jimmunol.org
sist on FDCs (13, 14). Thus, VLPs are able to induce strong B cell
responses in the absence of infection or long-term Ag deposits,
other than those persisting on FDCs. BrdU-labeling experiments
were performed to study the life span of memory B cells and
plasma cells and relate their turnover with the maintenance of specific IgG Ab titers. To modulate Ag persistence, we depleted FDCs
at various time points after immunization using a lymphotoxin
(LT) ␤ receptor (LT␤R)-Ig fusion protein. LT␣␤ signaling is
required for the maintenance of mature FDC networks via
LT␤R expressed by FDCs and LT␣1/␤2 expressed by B cells.
Inhibition of LT␣1/␤2 signaling by the injection of LT␤R-Ig
fusion proteins has been shown to cause a rapid disappearance
of functional FDCs and the markers specific of this population,
such as FDC-M1, FDC-M2, and CR1 (15, 16). Furthermore,
LT␤R-Ig treatment has been demonstrated to prevent the trapping of newly formed immune complexes, as well as to eliminate previously sequestered Ags (15). Earlier studies have
reported that injection of LT␤R-Ig before immunization abolished GC formation and led to impaired Ab responses to SRBC
(16). However, the effect of LT␤R-Ig treatment after the B cell
response has been induced and, in particular, at late time points
after immunization has not yet been addressed.
In this study, we report that the maintenance of the GC reaction,
early B cell proliferation, and the development of B cell memory
were highly Ag dependent, whereas persisting Ag was not essential for the maintenance of B cell and Ab memory in the late phase
of the response. GCs had a high output of memory B cells and
plasma cells within the first month after immunization. At later
time points, the contribution of the ongoing GC reaction to the
pool of long-lived memory B cells and bone marrow plasma cells
became negligible. In this late phase of the response, Ab titers
declined with a t1/2 of ⬃3 mo. The kinetics of this decline was
dictated by the t1/2 of the plasma cell population, which was found
to be in a similar range. These findings suggest a major role for
long-lived plasma cells residing in the bone marrow in maintaining
memory Ab levels and attribute an important role to persisting Ag
in the early, but not late phase of the B cell response.
Materials and Methods
Mice and Ags
C57BL/6 mice (Harlan) were immunized i.v. with 10 ␮g of Q␤. Animal
protocols used were reviewed and approved by the Swiss Federal Veterinary Office.
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T
he success of current vaccines is based on the induction of
long-lived Ab responses. Despite this, relatively little is
known about how the duration of Ab responses is regulated. It has been shown that plasma cells can be very long-lived
in experimental animals (1, 2). In light of this, it seems plausible
that such long-lived plasma cells may maintain Ab titers for long
periods of time in an Ag-independent fashion, as has been observed in individuals exposed to vaccinia virus (3). In contrast, Ag
can persist on follicular dendritic cells (FDCs)3 for long periods of
time, and potentially contribute to the maintenance of Ab responses in an Ag-dependent fashion (4, 5). In support of this, it has
been shown that increasing levels of persisting Ag can result in
increased Ab titers, and long-lived germinal centers (GC) associated with Ag-carrying FDCs have been observed (6, 7). Ag may
also persist in the absence of FDCs, for example during latent viral
infections or after vaccination, in which the use of adjuvants results in local Ag depots (8). Thus, it is difficult to create experimental conditions that exclude Ag persistence. A further mechanism that may be used to maintain Ab memory is the nonspecific
stimulation of memory B cells by TLR ligands, which activates
memory B cells to differentiate into Ab-producing plasma cells (9).
Hence, the relative contribution of long-lived bone marrow plasma
cells and continually differentiating memory B cells to the maintenance of humoral memory remains controversial (8, 10, 11).
In the present study, we used virus-like particles (VLPs) derived
from the bacteriophage Q␤ (12) to study the regulation of B cell
and Ab memory. We have shown previously that these VLPs are
highly immunogenic in the absence of adjuvant and efficiently per-
68
REGULATION OF MEMORY Ab LEVELS
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FIGURE 1. Kinetics of Q␤-specific B cell response. Mice were immunized i.v. with 10 ␮g of Q␤, and the B cell response was followed over time. A,
Kinetics of the IgG immune response directed against Q␤ Ag. Anti-Q␤ IgG titers are given as serum dilutions reaching half-maximal absorbance at 450
nm. B, Number of Q␤-specific IgG-producing AFCs was enumerated by ELISPOT in the spleen and bone marrow in response to Q␤ immunization.
The Journal of Immunology
Q␤ capsids were expressed using the vector pQ␤10 and purified, as
described (12).
Depletion of FDCs and retained Ag
LT␤R-Ig was prepared by fusing the coding region of the extracellular
domain of the LT␤R to the C region of human IgG1. The construct was
transfected into EBV-encoded nuclear Ag cells (Invitrogen Life Technologies) using Lipofectamine Plus (Invitrogen Life Technologies) and cultured in serum-free medium. The LT␤R-Ig fusion protein was purified
using protein G-Sepharose columns (Pharmacia).
Mice were injected i.p. with 300 ␮g of LT␤R-Ig on days 9 and 11 (early
FDC depletion), 39 and 41 (late FDC depletion), and 100 and 102 (very
late FDC depletion) after immunization with Q␤ particles.
BrdU labeling
For BrdU labeling, either BrdU (Sigma-Aldrich) was administered as a 0.8
mg/ml solution in the drinking water (light protected and changed every
second day), or 1 mg of BrdU was injected in saline solution i.p.
ELISAs
ELISAs were performed, as described (13). Titers represent log2 dilutions
of 40-fold prediluted sera at half-maximal OD.
Q␤-specific Ab-forming cell (AFC) frequencies were determined, as described (14). Briefly, 24-well plates were coated with 10 ␮g/ml Q␤. Spleen
or bone marrow cells were added in MEM containing 2% FCS and
incubated for 5 h at 37°C. Cells were washed off and plates were incubated successively with goat anti-mouse IgG (EY Laboratories) and
alkaline phosphatase-conjugated donkey anti-goat IgG Abs (Jackson
ImmunoResearch Laboratories) before development of alkaline phosphatase color reactions.
Flow cytometry
In all cases, Fc receptors were blocked with anti-mouse CD16/32
(2.4G2). Abs were purchased from BD Biosciences, unless otherwise
specified.
Detection of B cells expressing Q␤-specific surface Ig was performed by
incubation with Q␤, followed by a polyclonal rabbit anti-Q␤ serum (produced by RCC) and Cy5-conjugated donkey anti-rabbit IgG serum (Jackson
ImmunoResearch Laboratories), as previously described (13). Isotypeswitched B cells were detected in two ways. 1) cells were stained with
the following FITC-conjugated Abs: anti-IgD (11-26c.2a); goat antiIgM serum (Jackson ImmunoResearch Laboratories); anti-CD4
(GK1.5); anti-CD8 (53-6.7); anti-CD11b (M1/70); anti-Gr-1 (RB68C5); and PerCP-Cy5.5-conjugated anti-CD19 (1D3) Abs. Biotinylated
peanut agglutinin (PNA; Vector Laboratories) and streptavidin PE were
used to assess PNA binding. Dead cells were excluded by staining with
0.005 ␮g/ml YO-PRO-1 (Molecular Probes). 2) Cells were stained with
the following CyChrome-conjugated Abs: anti-IgD (11-26c.2a); antiCD4 (GK1.5); anti-CD8 (53-6.7); anti-CD11b (M1/70); anti-Gr-1 (RB68C5); and PE-conjugated anti-CD19 (ID3). In this case, BrdU incorporation
was detected by intracellular staining using a FITC-conjugated anti-BrdU Ab
(B44) after cell permeabilization, as described below.
To detect intracellular Q␤-specific Ig-positive bone marrow cells, surface Q␤-specific Ig was blocked with excess unlabeled Q␤ VLP. Cells
were permeabilized with 2⫻ FACS lysing solution (BD Biosciences;
349202) containing 0.06% (v/v) Tween 20. Intracellular Q␤-specific Ig
was detected by staining with Q␤ particles labeled with Alexa 647 (Molecular Probes), prepared according to the manufacturer’s instructions. Simultaneous detection of BrdU incorporation was performed using a FITCconjugated anti-BrdU Ab (B44), as described below.
Detection of incorporated BrdU
BrdU incorporation was measured by the method of Tough and Sprent
(17). Briefly, surface-labeled and/or intracellular Ig-labeled spleen and
bone marrow cells were fixed in ice-cold 95% (v/v) ethanol for 30 min,
followed by permeabilization in 1% (w/v) paraformaldehyde containing
0.01% (v/v) Tween 20. Cells were treated with 50 Kunitz U/ml bovine
pancreatic DNase I and stained subsequently with FITC-labeled anti-BrdU
(B44; BD Biosciences).
Immunohistochemistry
Freshly removed organs were snap frozen in liquid nitrogen. Tissue
sections of 5- to 7-␮m thickness were cut in a cryostat and fixed with
acetone. Q␤ Ag was detected by incubating sections with rabbit anti-Q␤
serum (RCC), followed by biotinylated sheep anti-rabbit Igs (The Binding Site) and alkaline phosphatase-labeled streptavidin (Roche). Alternatively, Alexa 488-labeled secondary Abs were used for detections.
FDCs were visualized using the rat FDC-M1 (BD Biosciences) Abs,
followed by biotinylated rabbit anti-rat Abs (Jackson ImmunoResearch
Laboratories) and alkaline phosphatase-labeled streptavidin (Roche).
IgD-expressing B cells were stained with sheep anti-mouse IgD Abs
(The Binding Site) and HRP-labeled rabbit anti-sheep Abs (Jackson
ImmunoResearch Laboratories). Alkaline phosphatase was visualized
using the Vector Blue substrate (Vector Laboratories) and HRP with the
substrate diaminobenzidine. GC B cells were stained with PNA biotin,
followed by Alexa 546-labeled streptavidin (red). Q␤-specific B cells
were stained with Alexa 488-labeled Q␤ (green).
Statistical analysis
Levels of statistical significance between means were determined using
Student’s t test. Average life spans of cell populations were calculated from
multiple experiments. Results are indicated as average ⫾ SEM. Only time
points after day 45 were used for the analysis.
Results
Characterization of the B cell response induced by immunization
with VLPs derived from Q␤
We have shown previously that a single injection of VLPs derived
from the bacteriophage Q␤ elicits strong and long-lasting IgG responses, which are strictly dependent on T cell help (13) and are
mainly of the IgG2a isotype (our unpublished observation). As described before, mice immunized i.v. with 10 ␮g of Q␤ mounted a
strong VLP-specific IgG response that peaked ⬃3– 4 wk later and
declined thereafter (Fig. 1A). The decay of the response at later time
points occurred with a t1/2 of 80 days (⫾7 days). Immunization resulted in high numbers of VLP-specific IgG-secreting AFCs in the
spleen and bone marrow (Fig. 1B). After the peak of the response,
numbers of AFCs declined over time both in spleen and bone marrow.
To quantify isotype-switched Q␤-specific B cells, we used a previously described flow cytometry-based detection system (Fig. 1C) (13,
14); the specificity of the staining was controlled using VLPs from
bacteriophage AP205 (data not shown). As observed for AFCs, numbers of Q␤-specific isotype-switched B cells in the spleen gradually
decayed with time (Fig. 1D). Immunization with Q␤ induced efficient
GC reactions (Fig. 1, E and F): up to 80% of all Q␤-specific isotypeswitched B cells were PNAhigh 3 wk after immunization, and PNAhigh
cells were maintained at low levels at least up to day 100 (Fig. 1E)
(13). Histological analysis revealed large GCs containing Q␤ Ag (Fig.
1F), which colocalized with FDCs (data not shown), as well as
with Q␤-specific B cells (Fig. 1G). Moreover, large numbers of
plasmablasts were observed outside GCs (Fig. 1G). Because Q␤
particles cannot replicate in the host and administration in adjuvant is
not required, they are only expected to persist as immune complexes
on the surface of FDCs.
C, Representative staining showing Q␤-binding isotype-switched B cells in spleen of immunized and naive mice. D, The number of Q␤-binding
CD19⫹IgM⫺IgD⫺ cells in the spleen was assessed by flow cytometry. The calculated limit of detection was 2 ⫻ 104 cells. E, The percentage of GC B cells
within the pool of Q␤-specific GC B cells was assessed by staining Q␤-binding CD19⫹IgM⫺IgD⫺ B cells additionally using PNA. F and G, Histological
sections were stained 12 days after s.c. immunization for PNA (red), identifying GC, Q␤-specific B cells (blue), as well as persisting Q␤ Ag (green). F,
An overlay for PNA and persisting Ags is shown. G, An overlay for Q␤-specific B cells and persisting Ags is shown. Note that Q␤-specific B cells are
found in either GCs or large aggregates as plasmablasts outside B cell follicles.
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ELISPOT assay
69
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REGULATION OF MEMORY Ab LEVELS
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FIGURE 2. Rapid turnover of splenic B cells vs slow turnover of bone marrow plasma cells: BrdU labeling from day 0 until day 20. A, Mice were
immunized i.v. with 10 ␮g of Q␤, and BrdU was administered in the drinking water for the first 20 days until the peak of the response. Mice were
sacrificed subsequently at the indicated time points, and B cells were assessed for incorporation of BrdU. B, Representative staining showing labeling
of BM Q␤-specific B cells in immunized and naive mice. C, Representative staining showing BrdU labeling of splenic Q␤-specific B cells. Splenic
Q␤-specific CD19⫹IgM⫺IgD⫺ B cells were stained for BrdU. As a control, spleen cells from immunized mice, but not pulsed with BrdU were
stained. D, The percentage of Q␤-specific B cells that were BrdU⫹ is shown. E, The frequency of total Q␤-specific B cells and the frequency of
BrdU⫹ Q␤-specific cells are shown. Note that a fraction of the spleens was used for histology, and absolute numbers of Q␤-specific B cells therefore
cannot be given. The detection limit was assessed as being 0.04% for total Q␤-specific B cells and 7 ⫻ 10⫺3% for BrdU⫹ Q␤-specific B cells. F
and G, Q␤-specific plasma cells of the bone marrow were stained for BrdU. F, The percentage of Q␤-specific plasma cells that were BrdU⫹ is shown.
G, The number of total Q␤-specific plasma cells and the number of BrdU⫹ Q␤-specific plasma cells are shown. The detection limit was 7 ⫻ 102
for total Q␤-specific plasma cells and 100 for BrdU⫹ Q␤-specific plasma cells.
Rapid turnover of splenic Q␤-specific B cells vs slow turnover
of bone marrow plasma cells
To characterize the life span and turnover of splenic Q␤-specific B
cells and bone marrow plasma cells, BrdU-labeling experiments
were performed. BrdU, which is a DNA base analog, is incorpo-
rated into the DNA of proliferating cells, marking them at a given
time point. The life span of a cell as measured in such experiments
is defined as the time it takes for a cell to either divide, which leads
to loss of BrdU, or die. For an optimal estimation of life span, two
types of experiments should be performed, as follows: 1) all cells
The Journal of Immunology
71
FIGURE 3. Rapid turnover of splenic B
cells vs slow turnover of bone marrow plasma
cells: BrdU labeling from day 20 until day 110.
A, Mice were immunized i.v. with 10 ␮g of
Q␤, and BrdU was administered in the drinking water from day 20 until day 110. Mice
were sacrificed subsequently at the indicated
time points, and B cells and plasma cells were
assessed for incorporation of BrdU. B, Splenic
Q␤-specific CD19⫹IgM⫺IgD⫺ B cells were
stained for BrdU. The percentage of Q␤specific B cells that were BrdU⫹ is shown.
C, Q␤-specific plasma cells of the bone
marrow were stained for BrdU. The percentage of Q␤-specific plasma cells that
were BrdU⫹ is shown.
corporation followed over 100 days (Fig. 3A). In the spleen, 80%
of the specific B cells incorporated BrdU within a few days,
whereas 20% remained unlabeled for the rest of the experiment
(Fig. 3B), most probably corresponding to the population of B cells
that did not lose the BrdU label in Fig. 2D. BrdU labeling in the
pool of bone marrow plasma cells was less pronounced and only
reached ⬃40% of the cells at the end of the experiment. Thus, a
substantial population of nondividing plasma cells was already
present in the bone marrow at day 20 after immunization (Fig. 3C).
A sizeable population of B cells shows high turnover late after
immunization
To determine the proportion of B cells that were actively dividing
at different time points after immunization, BrdU was added to the
drinking water for 10 days preceding analysis (Fig. 4A). In the
spleen, a surprisingly large fraction of B cells was still actively
dividing at later time points; indeed, ⬃30% of Q␤-specific B cells
proliferated between days 90 and 100 after immunization (Fig. 4,
B and C). This observation was consistent with the ongoing GC
reaction at these late time points. In contrast, Q␤-specific BrdU⫹
bone marrow plasma cells were found in large numbers only early
after immunization, and by day 100 only a small fraction of plasma
cells was positive for BrdU (Fig. 4, D and E). Thus, there is ongoing proliferation in the splenic B cell population, whereas there
is almost no turnover in the bone marrow plasma cell population.
These data indicate that the two populations are rather independent, and that, therefore, few proliferating splenic B cells enter the
bone marrow plasma cell pool at later time points.
Depletion of FDCs and associated Ag by treatment with
LT␤R-Ig
In a next set of experiments, we tested the importance of Ag in
driving B cell responses and memory by depleting FDCs at various
time points after immunization using a LT␤R-Ig fusion molecule.
It has been shown previously that LT␤ is continuously required for
survival of FDCs and that these cells rapidly die if the action of
LT␤ is blocked by LT␤R-Ig (15, 16). To evaluate the efficacy of
this treatment in depleting FDCs and the associated Ag, mice were
immunized with Q␤-derived VLPs, and LT␤R-Ig fusion protein
was injected on days 9 and 11 after immunization. Disappearance
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are labeled initially, the treatment is stopped, and the number of
BrdU⫹ cells followed over time; 2) cells are exposed to BrdU at
later time points, and rate of accumulation of BrdU⫹ cells is measured. The combination of these two methods allowed us to distinguish between the loss of BrdU due to proliferation vs cell
death. For this purpose, we immunized mice with Q␤ and administered BrdU into the drinking water during the first 20 days of the
response (Fig. 2A). Q␤-specific splenic B cells that had incorporated BrdU were clearly detectable by flow cytometry, even at late
time points when present at low frequencies (Fig. 2, B and C).
Numbers of Q␤-specific plasma cells in the bone marrow were
assessed similarly by flow cytometry, and were in the same range
as those obtained by ELISPOT assay (Fig. 1B). The specificity of
this intracellular staining has been demonstrated previously (14).
As expected, virtually all VLP-specific splenic B cells and plasma
cells in the bone marrow were BrdU⫹ on days 10 –20 (Fig. 2, D
and F). However, the frequency of BrdU⫹ Q␤-specific B cells in
the spleen rapidly declined by ⬃80% within a few days, but remained stable thereafter (Fig. 2D). Note, however, that the overall
frequency of Q␤-specific B cells also declined over time (t1/2 of
the population is 20 ⫾ 4 days) and, at later time points, the frequency of BrdU⫹ cells within the overall declining population of
Q␤-specific B cells remained constant (Fig. 2E). This indicates
that the BrdU⫹ B cells were lost with the same kinetics as the total
population of specific B cells and, consequently, that the life span
of BrdU⫹ B cells was the same as the life span of the specific B
cell population as a whole. In contrast to Q␤-specific B cells in the
spleen, the loss of BrdU⫹ bone marrow plasma cells specific for
Q␤ occurred more slowly and the frequency of BrdU⫹ plasma
cells declined over more than 1 mo (Fig. 2F). This indicates that
bone marrow plasma cells were generated at a lower rate than
splenic B cells. At later time points, the overall number of specific
BrdU⫹ plasma cells declined slowly, but the frequency of BrdU⫹
plasma cells remained constant, indicating that the life span of
BrdU⫹ plasma cells was the same as the life span of the population
(Fig. 2G). The decay of plasma cells was biphasic, the later phase
being characterized by a t1/2 of 80 ⫾ 21 days, roughly corresponding to the decay of the IgG titers (Fig. 1A).
These results were complemented by a second experiment, in
which BrdU labeling was performed from day 20 and BrdU in-
72
REGULATION OF MEMORY Ab LEVELS
of FDCs and trapped Q␤ particles was assessed by immunohistochemistry 1 wk after LT␤R-Ig treatment. As shown in Fig. 5A,
injection of LT␤R-Ig led to the loss of FDCs, as revealed by an
absence of staining for the FDC-specific marker FDC-M1. Consistent with the disappearance of FDCs, no Q␤ Ag could be detected in mice treated with LT␤R-Ig, in contrast to the readily
visible deposits present in untreated control mice (Fig. 5B). The
activity of the LT␤R-Ig fusion protein was further confirmed by
the reduction in frequencies of marginal zone B cells observed in
treated mice (data not shown), which is in agreement with the
reported alteration of the marginal zone organization caused by
inhibition of LT␤ signaling (16, 18). Thus, a substantial depletion
of FDC-associated Ag was achieved by treatment with LT␤R-Ig.
LT␤ inhibition and disappearance of FDCs have been shown to
prevent the formation of GC in the spleen (16). We therefore expected that treatment with LT␤R-Ig fusion protein would lead to perturbed GC reactions also when administered after their formation at
later stages of the response, thus emphasizing their output at several
stages after immunization. To confirm the longevity of GCs induced
by immunization with Q␤ particles, we assessed the number of VLPspecific PNA⫹ GCs by histology (Fig. 5C). GC numbers were highest
3 wk after immunization and declined thereafter. Few GCs could still
be observed ⬎100 days after immunization, indicating that GC reactions were still ongoing, as observed by flow cytometry. No Q␤specific GCs were detected in spleens of LT␤R-Ig-treated mice, indicating that they had been disrupted by the treatment (Fig. 5C).
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FIGURE 4. Presence of a proliferating population of B cells up to 100 days after immunization. A, Mice were immunized i.v. with 10 ␮g of Q␤. BrdU
was administered in the drinking water for periods of 10 days, and mice were sacrificed at the indicated time points. B cells and plasma cells were assessed
for incorporation of BrdU. B and C, Splenic Q␤-specific CD19⫹IgM⫺IgD⫺ B cells were stained for BrdU. B, The percentage of Q␤-specific B cells
that were BrdU⫹ is shown. C, The number of total Q␤-specific B cells and the number of BrdU⫹ Q␤-specific cells are shown. Calculated detection
limits were 5 ⫻ 104 for total Q␤-specific B cells and 5000 for BrdU⫹ Q␤-specific B cells. D and E, Q␤-specific plasma cells of the bone marrow
were stained for BrdU. D, The percentage of Q␤-specific plasma cells that were BrdU⫹ is shown. E, The number of total Q␤-specific plasma cells
and the number of BrdU⫹ Q␤-specific plasma cells are shown. The limit of detection was 7 ⫻ 102 for total Q␤-specific plasma cells and 100 for
BrdU⫹ Q␤-specific plasma cells.
The Journal of Immunology
73
the slower turnover of bone marrow plasma cells (Fig. 1B). However,
no short-term effect of the treatment on specific serum Ab titers was
observed. The analysis of mice 45 days after FDC depletion highlighted a major impact of an early LT␤R-Ig treatment on the longterm Ab response induced by Q␤. At this later analysis time point, the
role of FDCs and the associated Ag in establishing the slowly turning
over bone marrow plasma cell pool and memory Ab titers becomes
evident. Indeed, treated mice displayed a 3-fold reduction in anti-Q␤
serum Ab levels (Fig. 6A), which was the result of a corresponding
reduction of Q␤-specific AFCs in both the spleen (data not shown)
and bone marrow (Fig. 6A). Similarly to what was observed at the
earlier time point of analysis, frequencies of Q␤-specific isotypeswitched B cells were also lower in spleens of mice that had been
depleted of FDC-associated Ag (Fig. 6A). Thus, in the early stages of
B cell responses, FDCs and the Ag retained on their processes play a
crucial role in the maintenance of GCs and in establishing long-term
B cell and Ab memory.
Role of FDCs in maintaining the late B cell responses
Role of FDCs in maintaining the early B cell responses
We studied the effect of depleting FDCs and the related role of Ag
persisting on this cell population at three different stages of the B cell
response, as follows: early (⬃day 10), late (⬃day 40), and very late
(⬃day 100) after immunization. For these analyses, the frequencies of
Q␤-specific isotype-switched B cells and GC B cells in the spleen,
numbers of Q␤-specific IgG AFC in the bone marrow, and anti-Q␤
serum IgG titers were determined. In the first set of experiments,
FDCs and retained Q␤ Ag were depleted by injections of LT␤R-Ig on
days 9 and 11 after immunization, i.e., shortly before GC reactions
reach their peak, and the impact on the B cell response was assessed
10 and 45 days after the last LT␤R-Ig treatment (corresponding to
days 21 and 56 after immunization). As shown in Fig. 6A, isotypeswitched B cells specific for Q␤ were 10-fold reduced in spleens of
treated animals compared with controls 10 days after depletion of
FDCs and the associated Ag. Q␤-specific GC B cells were also drastically (13-fold) reduced, and they accounted for most of the loss of
specific cells, because 80 –90% of Q␤-binding B cells were PNAhigh
at this time point (Fig. 6A). Ten days after depletion, frequencies of
anti-Q␤ IgG AFCs in the spleen (data not shown), but not those in the
bone marrow, were reduced (Fig. 6A). This is consistent with the very
short life span of splenic AFCs at this early time point compared with
Role of persisting Ag in driving B cell proliferation
The impact of FDC depletion on B cell proliferation was assessed
next. Accordingly, mice were immunized with Q␤, and FDCs were
depleted 10 days later by LT␤R-Ig treatment. Ten days after FDC
depletion, BrdU was injected, and the frequencies of Q␤-specific
BrdU⫹ B cells and plasma cells were determined 24 h later (Fig. 7A).
This early depletion of FDCs and associated Ag had a strong impact
on the frequency of splenic Q␤-specific BrdU⫹ B cells (Fig. 7A), as
well as on PNAhigh B cells (data not shown). Thus, FDC-associated
Ag was required for proliferation of B cells in the spleen early after
immunization. As expected, the treatment had less short-term influence on the frequency of Q␤-specific BrdU⫹ plasma cells accumulating in the bone marrow (Fig. 7B).
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FIGURE 5. Depletion of FDCs and associated persisting Ag by LT␤R-Ig
treatment. Mice were immunized i.v. with 30 ␮g of Q␤. LT␤R-Ig injections
were performed on days 9 and 11 after immunization. A, Immunohistochemical staining of spleen sections with FDC-specific Abs FDC-M1 (blue) and
anti-IgD (brown) 1 wk after treatment with LT␤R-Ig (right panels) and in
control untreated mice. B, Histological analysis of Q␤ Ag (stained in blue) on
spleen sections from LT␤R-Ig-treated mice and control untreated mice. Note
that injection of control IgG preparations did not affect Ag persistence (data not
shown). C, Quantification of GC reactions by histological analysis. Spleen
sections were stained 22, 56, and 122 days after immunization for PNA and
Q␤-specific B cells in untreated and LT␤R-treated mice. On the left side, an
overlay for PNA (red) and Q␤-specific B cells (green) is shown for a spleen
122 days after immunization.
We next addressed the importance of Ag retained on FDCs at later
time points. For this purpose, mice were treated with LT␤R-Ig on
days 39 and 41 after immunization with Q␤; the effect of the treatment was analyzed subsequently 21, 50, and 106 days after the last
injection of the fusion protein (corresponding to days 60, 89, and
145 after immunization). Depletion of FDCs and the associated Ag
resulted in a short-term reduction of isotype-switched Q␤-specific
B cells (Fig. 6B). As expected from the fact that GC reactions were
still ongoing, the largest impact was seen on the frequency of
PNAhigh GC B cells. However, generation and/or maintenance of
memory B cells were not significantly affected by this late injection of
the LT␤R-Ig, as demonstrated by comparable frequencies of Q␤binding B cells present in spleens of treated and control mice 145 days
after immunization (Fig. 6B). The same was true for the number of
plasma cells in the bone marrow and long-term Ab titers, which were
not affected by the treatment. This suggests that most Q␤-specific
memory B cells and plasma cells had been generated within the first
month after immunization. At later time points, the GC reaction may
still be ongoing; however, the net output for the pool of memory B
and plasma cells was negligible after day 40, a result consistent with
the BrdU-labeling experiments shown above.
In accordance with these results, depletion of persisting Ag very
late after immunization (by LT␤R-Ig treatment on days 100 and
102) had no noticeable effect on the frequency of Q␤-specific
memory B cells or on the number of bone marrow plasma cells
secreting anti-Q␤ Abs (Fig. 6C). The number of PNAhigh GC B
cells was slightly, but not significantly reduced by the treatment,
indicating that the late presence of PNAhigh cells was largely Ag
independent. Thus, the maintenance of Q␤-specific long-term B
cell effector populations, bone marrow plasma cells, and memory
B cells did not require the presence of the Ag persisting on FDCs.
74
REGULATION OF MEMORY Ab LEVELS
Surprisingly, when FDCs were depleted 100 days after immunization, proliferation of Q␤-specific B cells in the spleen was hardly
affected (Fig. 7C), indicating that the low turnover seen at this late
time point occurs Ag independently (note that the fraction of Agdependent PNAhigh GC B cells is low at this late time point; Fig. 6C).
Potential mechanisms for this proliferation, including the role of nonspecific stimulation by TLR ligands or cytokines, remain to be established.
Discussion
In this study, we determined the relation between maintenance of Ab
titers, plasma cell life span, and turnover of B cells. In addition, the
role of persisting Ag in driving B cell proliferation, GC reactions, and
plasma cell differentiation was analyzed.
The life span of plasma cells in the bone marrow has been the
subject of intensive discussion. Although some authors suggested the
plasma cells to be short-lived, others concluded that they may live for
up to several years (8, 10, 11). Most experimental systems used to
date exhibit some technical limitations, rendering the assessment of
plasma cell life span, under physiological conditions in the absence of
exogenous Ag, difficult. On the one hand, mice have been immunized
with proteins in depot-forming adjuvants, leading to persistence of Ag
in an unphysiological manner, different from the natural Ag persistence on FDCs. Live lymphocytic choriomeningitis virus was also
used to measure the life span of plasma cells (1). However, lymphocytic choriomeningitis virus tends to remain in the host as infectious
virus (19). If high amounts of Ag persist in a depot or as infectious
virus, B cell responses may remain ongoing through continuous
antigenic stimulation, potentially leading to an underestimation of the
plasma cell life span. In contrast, low level persistence of Ag may
stimulate differentiation of memory B cells into plasma cells in the
absence of proliferation, leading to an overestimation of the plasma
cell life span in BrdU-labeling experiments.
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FIGURE 6. Distinct effects of FDC depletion on GC formation vs plasma cell induction. Mice were immunized with Q␤ particles and treated subsequently with LT␤R-Ig at day 10 (A), day 40 (B), or day 100 (C) postimmunization. The frequency of isotype-switched Q␤-specific B cells and of
Q␤-specific PNAhigh GC B cells as well as the frequency of Q␤-specific AFCs in the bone marrow and Q␤-specific IgG Abs in the serum are shown. Note
that no reduction in Q␤-specific total or GC B cells was observed in mice injected with control Ig protein (data not shown). All data represent the mean ⫾
SEM; mean values statistically different in treated mice are indicated by asterisks (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001).
The Journal of Immunology
To resolve this issue, we immunized mice with noninfectious VLPs
derived from bacteriophage Q␤ (20) in the absence of adjuvant. These
VLPs are rapidly transported into B cell follicles and efficiently retained on FDCs (13, 14), but are not expected to persist in any other
form in the host. This efficient trapping of VLPs on FDCs and their
ability to activate Th cell and induce T cell-dependent isotype switching lead to the generation of long-lasting GC reactions and strong
long-lived IgG responses. Recent findings have revealed that the presence of contaminating TLR ligands in preparations of pneumococcal
polysaccharides or hepatitis B nucleocapsids is required for induction
of IgG Ab responses by these Ags (21, 22). Although endotoxin levels
are low in our VLP preparations (1–10 EU LPS/␮g capsid), we cannot exclude that contaminating bacterial LPS might enhance the immunogenicity of the VLPs and contribute to the activation of B cells.
In addition, the VLPs contain bacterial RNA, a potential ligand for
TLR3 and TLR7. We are currently studying the role of this RNA in
driving B cell responses in detail. Nevertheless, the VLP preparations
fail to induce isoytpe switching in the absence of T cells (13, 23) or
CD40-CD40L interactions (our unpublished observations), indicating
that Th cells, more than TLR-mediated costimulatory signals, are critical for the induction of IgG to Q␤. The requirement for T cell help
for the generation of IgG responses strongly suggests that GC reactions induced by immunization with VLPs are T cell dependent and
hence support affinity maturation. This notion is supported by reduced
GCs in absence of costimulation as well as the presence of somatic
hypermutations in Ab sequences from VLP-specific B cells (our unpublished data).
Using Q␤ particles and an Ag-specific B cell detection system,
we measured turnover and life span of B cells and plasma cells. As
expected, we observed a relatively high turnover of splenic B cells
in contrast to a lower turnover of bone marrow plasma cells. Moreover, two populations of splenic B cells were identified, a short-
lived population with a turnover of a few days (5 days; all cells of
the early phase of Fig. 4C and a subpopulation in the later phase)
and a population of long-lived splenic B cells with a life span of
⬃43 days (late phase of Fig. 2E). In contrast, the life span of bone
marrow plasma cells was found to be longer, in the range of 3 mo
(late phase of Fig. 2G). The observation that in the late phase of the
response the number of plasma cells declines exponentially indicates that at any given point in time, the cells have the same probability to die. Hence, plasma cells do not become old, and their
expected future life span is independent of their age. The simplest
explanation for such behavior is a competition model, whereby cells
compete for niches within the bone marrow. Importantly, the calculated plasma cell life span of 80 days closely paralleled the decline of
memory IgG titers, which also exhibited a t1/2 of ⬃80 days. Thus, the
life span of plasma cells dictates the lifetime of the Ab response.
These data also show that plasma cells may live for several months,
but eventually decline as a population with a given t1/2 of ⬃80 days.
Ag may persist on FDCs for months or even years, and has been
postulated to be important for driving B cell proliferation, plasma
cell differentiation, and Ab production (4, 5, 24). FDCs are particularly important for the GC reaction, because the native Ags
retained on their surface via Fc and complement receptors maintain the specificity of the GC and allow for selection of highaffinity B cell clones, leading to affinity maturation. Recently, the
importance of Ag associated with FDCs has been questioned, and
the idea has been put forward that FDCs maintain the GC reaction
nonspecifically (25, 26), because Ag-specific GCs could be observed in the absence of detectable persisting Ag. Two explanations may account for this surprising observation. On the one hand,
the detection methods used may simply not be sensitive enough or,
in contrast, Ag leaking from the adjuvant depot may have allowed
to artificially keep the GC reaction ongoing. Thus, the bulk of the
data still indicates that the key role of FDCs is to expose native Ag
to GC B cells (4, 5, 7, 27). By depleting FDCs and associated Ag,
we found that B cell proliferation at early time points after immunization was strongly FDC dependent; in addition, the GC reaction
remained FDC dependent, for most of the observed time span. B
cell turnover at very late time points (⬎day 100) occurred slowly,
and only ⬃10% of splenic B cells incorporated BrdU within a 10-day
labeling period. Surprisingly, this late B cell proliferation occurred in
the absence of FDCs and retained Ag, indicating that the population
of memory B cells may be maintained by slow proliferation by cytokines, as has been observed for IL-15-dependent proliferation of
CD8⫹ memory T cells (28 –30). Alternatively, memory B cell proliferation may be due to environmental exposure to TLR ligands (9).
Despite a long-lived GC reaction, B cell proliferation, and
plasma cell production, maintenance of IgG Ab titers was not dependent on these active B cell responses after day 30. Indeed,
plasma cell numbers and Ab titers were independent of persisting
Ag and FDCs at later time points. This observation may be explained by overall low numbers of plasma cells produced by the
GC at later time points. In addition, it is possible that the generation of precursors for long-lived plasma cells is restricted to the
first 30 days, and that the late GC reaction fails to generate these
cells. The late GC reaction may therefore be more important to
maintain a flexible, hypermutated B cell repertoire in case of reemergence of the infection rather than for keeping high Ab levels.
Taken together, our results demonstrate that Ag deposits on FDCs
are required in the early phase of B cell responses to Q␤, when GCs
afford a significant output of cells destined to differentiate into memory B cells and long-term Ab-secreting plasma cells. At later time
points after immunization, although some B cells continue to proliferate in GCs, their overall contribution to the pool of memory B cells
and bone marrow plasma cells is limited. These experiments indicate
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FIGURE 7. FDC depletion blocks B cell proliferation. Mice were immunized with Q␤ particles and treated with LT␤R-Ig 10 days (A and B) or
100 days (C) postimmunization. A and B, Ten days after FDC depletion,
BrdU was injected, and the analysis was performed 24 h later (i.e., on day 11
after depletion). The percentage of BrdU⫹ Q␤-specific splenic B cells (A) and
bone marrow plasma cells (B) was determined by flow cytometry. C, BrdU
was added to the drinking water either 12 or 46 days after depletion for a
period of 10 days. The percentage of Q␤-specific splenic B cells that were
BrdU⫹ was determined 22 and 56 days after depletion. Values are expressed
as the mean ⫾ SEM, with significant differences between means indicated by
asterisks (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01).
75
76
that Ab production late after immunization with Q␤ could be maintained in the absence of Ag retained on FDCs, and suggest a major
role for long-lived plasma cells residing in the bone marrow in preserving long-term elevated serum Abs. However, the presented results
establish that the pool of bone marrow plasma cells does not survive
for the lifetime of a mouse, but declines with a t1/2 of ⬃3 mo.
REGULATION OF MEMORY Ab LEVELS
15.
16.
17.
Disclosures
18.
The authors have no financial conflict of interest.
19.
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