1
FcRIIb controls bone marrow plasma cell persistence and apoptosis.
Zou Xiang1,6, Antony J. Cutler1,4,6, Rebecca J.Brownlie1, Kirsten Fairfax2, Kate E.
Lawlor1, Eva Severinson1,5, Elizabeth U. Walker1, Rudolf A. Manz3, David. M.
Tarlinton2 & Kenneth G. C. Smith1.
1. Cambridge Institute for Medical Research and the Department of Medicine,
University of Cambridge School of Clinical Medicine, Box 139, Addenbrooke’s
Hospital, Cambridge, United Kingdom.
2. The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050,
Australia.
3. German Arthritis Research Center, Berlin, Schumannstrasse 20/21, 10117 Berlin,
Germany.
4. Current address: Institute of Infectious Diseases and Molecular Medicine,
University of Cape Town, Cape Town, Republic of South Africa.
5. Current address: Division of Immunology, Wenner-Gren Institute, Stockholm
University, SE-106 91 Stockholm, Sweden.
6. These authors contributed equally to this work.
7. Correspondence should be addressed to KGCS (kgcs2@cam.ac.uk)
2
Abstract
The survival of long-lived plasma cells, which make most serum immunoglobulin,
plays a central role in humoral immunity. We found that the inhibitory Fc receptor
FcRIIb was expressed on plasma cells and controlled their persistence in the bone
marrow. Cross-linking FcRIIb induced apoptosis of plasma cells, which we propose
contributes to the control of their homeostasis and suggests a method for therapeutic
deletion. Plasma cells from mice prone to systemic lupus erythematosus (SLE) did not
express FcRIIb and were protected from apoptosis. We found that human
plasmablasts expressed FcRIIb and could be killed by cross-linking, as could
FcRIIb-expressing myeloma cells. These results suggest that FcRIIb controls bone
marrow plasma cell persistence, and defects in it may contribute to autoantibody
production.
3
Serum immunoglobulin (Ig) is vital to the maintenance of humoral immunity, with
control of both its amount and antigenic specificity critical for defense against
infection and the prevention of autoimmunity1. After T cell-dependent B cell
activation, two sorts of plasma cells are produced. The first to produce antibody are
short-lived plasmablasts, which proliferate and differentiate in the lymph node and
spleen, rapidly producing low affinity IgM and IgG and then dying by apoptosis2 with
a half-life of approximately three days3. Most serum IgG is, however, made by
plasma cells resident in the bone marrow (BM)4. BM plasma cells are long-lived5,6,
and are predominantly generated in the germinal centers of peripheral lymphoid
organs7, as they show evidence of affinity maturation and selection8,9. Relatively
small numbers are produced in primary immune responses, with more substantial
migration occurring after secondary responses to antigenic rechallenge2,10. Expression
of the chemokine receptor CXCR4 is important for plasma cell homing to the BM11-12.
Once migratory plasma cells have arrived in the BM, they do not divide and their
survival is independent of antigen13. A number of factors contribute to their
longevity. Their survival is critically dependent upon the transcription factor Blimp1. Blimp-1 in turn enhances expression of XBP-1, which induces the unfolded protein
response in plasma cells, controlling the interplasmatic stress caused by antibody
secretion14,15. A number of other survival factors are important, at least as
demonstrated in vitro. These include hyaluronic acid, the chemokine CXCL12,
interleukin (IL)-5, IL-6, tumor necrosis factor (TNF)-, and the TNF family
members BAFF and APRIL16. It is thought that these survival factors are supplied to
the plasma cell in the context of an anatomical ‘niche’. These survival niches are
thought to limit the plasma cell capacity of the BM, which has been shown to be
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approximately 0.1-1% of all BM cells . Thus mice have approximately 1x106 and
17
humans approximately 1x109 BM plasma cells16. Additional niches for plasma cells
can be formed in inflammatory tissue18. Given that niches in the BM appear to limit
the number of plasma cells, mechanisms must exist to maintain long-lived plasma cell
memory for previous antigens, but at the same time allow plasma cells produced in
response to recent antigenic challenge to take up their place in the BM repertoire.
It has been proposed on the basis of work in humans that immunization results in nonantigen-specific activation of memory B cells, which then reinforce the BM plasma
cell population19. Other studies, however, have shown only antigen-specific plasma
cells take up residence in the BM after such immunization20,21. The former model
does not explain how, with limited plasma cell niches, space could be made for
plasma cells of the new antigenic specificity in addition to the new ‘bystander
activation’ plasma cells. That this might occur by ‘competitive dislocation’ has been
proposed22.
We sought to address the issue of BM recruitment and persistence in a mouse model.
We found that, rather than contributing to the BM plasma cell repertoire by ‘bystander
activation’, immunization resulted in a reduction in plasma cells of other specificities.
We then investigated the molecular basis for this reduction. FcRIIb is a low-affinity
inhibitory receptor for the Fc portion of IgG, is expressed as one of a number of Fc
receptors on myeloid cells, but is the only FcR expressed on B cells23. There is scanty
evidence in the literature as to whether long-lived plasma cells express FcRIIb, and
FcRIIb-deficient mice, and autoimmune-prone mice known to have a polymorphism
in FcRIIb promoter which reduces expression on activated B cells, have increased
plasma cell numbers
24,25
5
. Moreover, FcRIIb when cross-linked on naive B cells can
induce apoptosis in a BCR-independent fashion26,27. We therefore hypothesized that
FcRIIb may be expressed on plasma cells and may control their persistence. Here,
we found that Fcgrb was indeed transcribed in plasma cells, and FcRIIb was
expressed at the protein level in both short- and long-lived cells. We further
demonstrated that FcRIIb controlled the persistence of BM plasma cells, and that
cross-linking FcRIIb induced apoptosis in a cell autonomous fashion. These findings
shed light on the control of BM plasma cell homeostasis, and have implications for
autoimmunity and for the therapy of myeloma.
RESULTS
Immunization reduces plasma cells specific for previously encountered antigens
We first determined whether immunization and bystander activation resulted in the
entry of antigen non-specific plasma cells into the long-lived BM pool in mice, as
human studies had provided conflicting evidence19,21. Mice were immunized with the
hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to chicken gamma globulin
(CGG) in alum, and a group analyzed 9 weeks later to determine the number of NPspecific IgG plasma cells in BM and spleen. The remainder were then immunized at
9 and 11 weeks with either normal saline, or with a cocktail of antigens comprising
ovalbumin (OVA), keyhole limpet hemocyanin (KLH), CpG and heat-inactivated
Streptococcus pneumoniae. At 13 and 15 weeks mice were sacrificed from each
group for analysis of anti-NP plasma cell frequency (Fig. 1). The group subject to
non-specific immunization demonstrated an increase in NP-specific plasmablasts in
the spleen at 2 weeks, but a reduction at 4 weeks, when it would be expected that most
residual plasma cells in the spleen would be long-lived. In the BM there was also a
6
consistent decrease by 4 weeks, and consistent with this finding was a reduction in
serum anti-NP IgG after non-specific immunization (Fig. 1). We confirmed that this
reduction was not due to interference in the ELISA by antibody produced by the nonspecific immunization by studies spiking the unimmunized serum with non-specific
IgG (data not shown). The affinity of the anti-NP IgG present in the serum, as
determined by measurement of binding to differentially haptenated NP conjugates,
was not altered (data not shown).
These experiments do not support the concept that bystander activation contributes to
the BM plasma cell population in mice, but rather show that non-specific
immunization may indeed reduce BM plasma cells.
FcRIIb expression in plasma cells
Surface receptors might mediate the reduction in plasma cell number seen after nonspecific immunization, and we considered inhibitory receptors as candidates. Most
such receptors, such as CD22 and CD72, disappear upon plasma cell differentiation2.
The evidence for loss of expression of FcRIIb, an inhibitory Fc receptor, was not as
strong, and in fact occasional reports had suggested FcRIIb was expressed on human
plasmablasts, though it was not clear whether this observation was due to incomplete
differentiation or continued transcription within the plasma cell28. The expression of
FcRIIb on a proportion of myeloma cells29 also raised the possibility that it may be
expressed by normal plasma cells. In addition, FcRIIb-deficient mice have increased
plasma cell numbers, and while this is due at least in part to increased production30, it
is possible that increased persistence of plasma cells could contribute.
7
We therefore measured the expression of FcRIIb on splenic and BM plasma cells,
using two different systems to exclude the risk of observing expression on recently
produced, and thus potentially incompletely differentiated, plasma cells. We first used
heterozygous Prdm1gfp/+ (BLIMP-GFP) mice, in which the gene Prdm1, which
encodes Blimp-1, is replaced by GFP at one allele causing plasma cell-specific
fluorescence, the intensity of which increases with differentiation31. This allowed
confident identification of incompletely (GFP intermediate; GFPint) and fully (GFPhi)
differentiated plasma cells by flow cytometry. Plasma cells from both spleen and bone
marrow expressed FcRIIb, and this expression was higher on fully differentiated
plasma cells than on mature B cells or less differentiated plasma cells (Fig. 2a,b). We
then confirmed expression on antigen-specific plasma cells at known times after
immunization. Mice were immunized with phycoerythrin (PE), and bone marrow
plasma cells identified by confocal microscopy 56 days after immunization. FcRIIb
was expressed on IgG and IgM positive plasma cells in C57BL/6 and BALB/C mice,
but it was not expressed on FcRIIb-deficient mice, nor on various autoimmune-prone
strains (Fig. 2c,d). These findings were extended by examining NP-specific plasma
cells identified using the  light chain as a surrogate marker for NP specificity32.
FcRIIb was expressed on plasmablasts and plasma cells in the spleen 6, 13 and 32
days after immunization, and also in the BM after 32 days (Supplementary Fig. 1a,b
online). There was a trend toward higher expression of FcRIIb on IgM than IgG
positive cells, which reached statistical significance in 3 of 6 comparisons (Fig. 2d
and Supplementary Fig. 1b).
To confirm that this residual expression was associated with transcription we
generated plasmablasts in vitro using LPS, IL-6 and IL-10. Such plasmablasts
8
expressed CD138 (also known as syndecan), and downregulated B220 and CD22 as
expected. They did, however, maintain high expression of FcRIIb (Fig. 3a). We
then sorted these plasmablasts and confirmed their purity (Supplementary Fig. 1c)
before measuring Fcgr2b mRNA using semi-quantitative RT-PCR. Fcgr2b mRNA
could be found in plasma cells, B cells and macrophages. We detected no Cd22 or
Fcgr3 mRNA in plasma cells, excluding contamination by B cells and macrophages
respectively (Fig. 3b). To confirm that transcription was maintained in fully
differentiated plasma cells in vivo, we sorted GFPint and GFPhi plasma cells from the
spleens of BLIMP-GFP mice (see above), and used a sensitive radioactive PCR to
measure Fcgr2b mRNA. Equivalent mRNA expression was seen in both GFPint and
GFPhi populations (Fig. 3c). Thus FcRIIb is expressed at the protein level on both
short- and long-lived plasma cells, and this expression is associated with ongoing
transcription after differentiation.
Increased persistence of BM plasma cells in Fcgr2b-/- mice
Increased plasma cell number and immunoglobin production occurs in response to
immunization in FcRIIb-deficient mice24. We studied the kinetics of the plasma cell
response in both spleen and BM in FcRIIb-deficient mice to determine if FcRIIb
influenced plasma cell persistence. Unexpectedly, there was no significant difference
in the generation of splenic IgM or IgG NP-specific plasmablasts, but what are likely
to be long-lived plasma cells were increased in FcRIIb-deficient mice at D14 (Fig.
4a,b). Consistent with this result, an increase in BM plasma cells was seen in
FcRIIb-deficient mice compared to controls (Fig. 4c). Between days 38 and 78 after
a primary immunization there was a slow but significant fall in NP-specific IgG BM
plasma cells in control mice, but these were maintained in FcRIIb-deficient mice
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(Fig. 4d). Too few NP-specific IgM BM plasma cells were present at these late time
points to allow statistically meaningful analysis. Consistent with this decrease in
plasma cells, there was a decrease in serum anti-NP IgG titers in controls, but not in
FcRIIb-deficient mice (Fig. 4e). To demonstrate that this effect was plasma cell
intrinsic, adoptive transfer studies were performed. Transfer of mature plasma cells in
numbers that allow subsequent detection by ELISPOT was unsuccessful, presumably
because they have down-regulated the chemokine receptors necessary for BM
homing. We therefore transferred splenocytes 6 days after boost immunization, at
which time precursors of BM plasma cells express CXCR4 and home to the BM.
After such cells were transferred from control mice, a similar decrease in both
ELISPOT number and serum levels was found as had been seen in intact mice. This
decrease was not observed when FcRIIb-deficient plasma cells were transferred (Fig.
4f). Consistent with the persistence of FcRIIb-deficient plasma cells, anti-NP IgG
continued to accumulate between days 27 and 76 in mice into which FcRIIbdeficient plasma cells had been transferred, but by this time had reached a plateau
after transfer of control cells (Fig. 4g). It was confirmed that these results were not
due to a de novo primary response, which may occur if antigen or primed T cells were
transferred. IgHb splenocytes were transferred into IgHa recipients after an identical
immunization regimen. No evidence of an IgHa response could be detected after 30
days, and anti-NP IgMa was also not detected after transfer, both consistent with the
absence of a primary response (Supplementary Fig. 2 online). No FcRIIb-deficient
myeloid cells could be detected in the BM after transfer (data not shown).
To determine if increasing FcRIIb expression could decrease plasma cell persistence,
transgenic mice were generated in which FcRIIb was over-expressed on B cells and
10
plasma cells. Primary immune responses were reduced in these mice (RJB, KEL, AJC
and KGCS, in preparation), meaning that it is only possible to assess their decline in
serum IgG relative to controls once the BM plasma cell population has been
established. A marked reduction in persistence of anti-NP IgG was seen, with some
transgenic mice having no detectable antibody 78 days after immunization. This was
never observed in control mice even at later times after immunization, and was
consistent with reduced persistence of BM plasma cells (Fig. 4h). The effect of
FcRIIb deficiency or over-expression on serum IgG was thought to be due to plasma
cell number, as both FcRIIb-deficient and transgenic plasma cells made similar
amounts of IgG as control cells, as assessed by ELISPOT size and intensity
(Supplementary Fig. 3 online).
Thus long-lived BM plasma cells are increased in number in FcRIIb-deficient mice
due to both increased production, but also to increased persistence once their
population has been established. This increased survival is plasma cell intrinsic. In
contrast, absence of FcRIIb has no discernable effect on the generation or persistence
of short-lived splenic plasmablasts.
FcRIIb cross-linking mediates plasma cell apoptosis
FcRIIb expression reduced the persistence of BM plasma cells, so we sought to
identify the mechanism underlying this. Cross-linking of FcRIIb in a BCRindependent fashion can induce the apoptosis of mature B cells26,27. We investigated
whether this also occured in plasma cells. Plasmablasts were generated in vitro from
splenocytes, identified by staining with CD138, sorted and their purity confirmed by
staining for intracytoplasmic immunoglobulin (Supplementary Fig. 1d).
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Lipopolysaccharide (LPS) induced similar numbers of plasmablasts in FcRIIbdeficient, FcRIIb-transgenic and control mice (Supplementary Fig. 1e). In order to
cross-link FcRIIb, plasmablasts were cultured on plates coated with 2.4G2, a
monoclonal antibody recognizing both anti-FcRII and FcRIII (the latter is not
expressed on B cells or plasma cells). This resulted in increased plasmablast apoptosis
as detected by annexin staining of CD138+B220lo cells (Fig. 5a). This was confirmed
by measuring apoptosis by nucleosome release from purified plasmablasts (Fig. 5b),
by using a FcRIIb-specific cross-linking antibody (E16: Fig. 5c), and by using
plasmablasts generated from purified B cells (data not shown). Increased apoptosis
was not observed when plasmablasts were generated from FcRIIb-deficient mice,
confirming the role played by cross-linking of FcRIIb in inducing apoptosis. In fact,
slightly reduced apoptosis was consistently seen after cross-linking in FcRIIbdeficient mice, raising the possibility that activatory signalling might occur in the
absence of FcRIIb (Fig. 5a,b). Thus cross-linking of FcRIIb can induce apoptosis of
plasmablasts in vitro.
Physiological induction of apoptosis by FcRIIb would be achieved by cross-linking
with Fc regions, for example by immune complexes, rather than the high affinity
monoclonal 2.4G2. We sought to determine if apoptosis could be induced by such
cross-linking, and if it could be controlled varying the expression of FcRIIb using
FcRIIb transgenic mice (which express around 30-fold more FcRIIb on their plasma
cells than do controls; Fig. 5d). Cross-linking with mouse IgG1 and a secondary goat
anti-mouse immunoglobulin caused a modest but consistent increase in apoptosis
compared to mouse IgG1 F(ab’)2 or buffer, which was not seen in the absence of
FcRIIb but was substantially increased when FcRIIb was overexpressed (Fig. 5e,f).
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Immune complexes of OVA and OVA antibody caused more marked apoptosis,
which was again increased by FcRIIb overexpression (Fig. 5g). Thus ‘physiological’
cross-linking of FcRIIb by immune complexes can induce plasma cell apoptosis in a
manner dependent on the abundance of FcRIIb.
Lymphocyte apoptosis can be divided into two broad types - that induced by the
cross-linking of death receptors, such as CD95 (Fas) or the TNF receptor, or that
controlled by Bcl-2-related molecules33. As FcRIIb does not contain a death domain
typical of the TNF family of receptors, we hypothesized that plasma cell apoptosis
induced by it would fall into the latter category, and sought to confirm this by
assessing FcRIIb-induced apoptosis in Bim-deficient mice34. Bim is a BH3-only
protein which is pro-apoptotic, mediating this effect by binding to Bcl-2 and its
homologs and counteracting their anti-apoptotic effects33. We found that FcRIIb was
expressed at 3-fold higher levels on plasmablasts from Bim-deficient mice compared
to littermate controls (Fig. 5h) Despite this, cross-linking of FcRIIb failed to induce
apoptosis, as measured by either annexin staining or nucleosome release (Fig. 5i-k).
This result confirms that FcRIIb-induced apoptosis is controlled by Bcl-2 family
members, and suggests failure of this apoptosis in Bim-deficient mice might
contribute to their plasma cell accumulation and autoimmunity (see below).
To determine if FcRIIb could induce apoptosis of plasma cells generated in vivo,
mice were immunized with NP-CGG and 7 days after a secondary immunization
splenocytes were cultured for 4 h with or without cross-linking. Apoptosis was
determined by annexin staining of CD138 positive cells, and cross-linking FcRIIb
induced apoptosis (Fig. 6a). To confirm this, NP-specific plasmablasts were identified
13
by staining for cytoplasmic light chain and apoptosis identified by assessment of
nuclear morphology with fluorescence microscopy (Fig. 6b,c). Finally it was
important to determine if FcRIIb cross-linking could induce apoptosis in long-lived
BM plasma cells, as such cells are particularly refractory to killing by known
methods16. 63 days after immunization BM cells were incubated with or without
cross-linking, antigen-specific plasma cells identified by light chain expression, and
apoptosis determined by fluorescence microscopy for nuclear morphology. As with in
vitro generated plasmablasts, cross-linking induced increased apoptosis which was not
seen in FcRIIb-deficient plasma cells (Fig. 6d). Cross-linking of FcRIIb can thus
induce apoptosis of mature BM plasma cells.
Implications for autoimmunity
Systemic lupus erythematosus (SLE)-prone mouse strains have long been known to
have a markedly increased number of plasma cells35,36. Such strains also have a
Fcgr2b promoter polymorphism which reduces expression of FcRIIb on activated
and germinal center B cells25,37. These strains have no detectable FcRIIb on their
bone marrow plasma cells (Fig. 2c,d and Supplementary Fig. 1a,b). Consistent with
this, plasmablasts derived from SLE-prone NZB or MRL mice could not be killed by
cross-linking FcRIIb (Fig. 7a). That this result was due to reduced expression of
FcRIIb was confirmed by crossing NZB mice with FcRIIb B cell transgenic mice,
using ‘speed congenics’ to maximize the NZB genetic contribution and in particular
to ensure inheritance of the locus containing NZB FcRIIb. This genetic alteration
restored FcRIIb expression on NZB plasma cells (data not shown), and in doing so
restored FcRIIb-mediated apoptosis induced by immune complexes (Fig. 7b). A
failure of FcRIIb-mediated apoptosis could thus contribute to the plasma cell
14
accumulation which occurs in SLE-prone mice and constitute a new ‘checkpoint’
governing peripheral B cell tolerance.
FcRIIb cross-linking kills human plasmablasts and myeloma cells
Myelomas are plasma cell malignancies which, compared to many other B cell
malignancies, are particularly difficult to treat. It is likely that the majority arise from
long-lived plasma cells, as most are somatically mutated and arise in the BM. A
proportion of myelomas express FcRIIb29,38. That FcRIIb cross-linking could kill
BM plasma cells raised the intriguing possibility that it might also kill myeloma cells.
We first confirmed that human plasmablasts generated in vitro could be killed by
cross-linking FcRIIb in a manner analogous to the mouse. An SLE-associated
polymorphism resulting in the replacement of Ile with Thr at position 232 in the
transmembrane domain of FcRIIb has been shown to abolish its SHIP-mediated
inhibitory function39,40 - this polymorphism had no effect on FcRIIb-mediated
plasma cell death (Fig. 7c).
We obtained myeloma cell lines that were either positive (EJM) or negative (LP-1) for
FcRIIb expression41,42. Cross-linking FcRIIb induced apoptosis in EJM, but not LP1 (Fig. 7d-f). We then transfected LP-1 cells with a construct inducing expression of
FcRIIb, and sorted cells into those expressing high or negligible amounts of the
receptor. Only cells with high expression became sensitive to apoptosis induced by
FcRIIb cross-linking (Fig. 7g,h). Thus cross-linking FcRIIb may be of therapeutic
benefit in those myelomas which express it.
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DISCUSSION
Animal studies demonstrating the importance of BM plasma cells in providing
“humoral” memory43 have been reinforced by the observation that humans maintain
normal IgG levels after B cell depletion with rituximab (anti-CD20 monoclonal
antibody), even if peripheral blood B cells are undetectable for months or years44,45. It
is not clear, however, how the persistence in the BM of plasma cells of different
antigenic specificities is controlled. It has been suggested that inflammation or
infection could “reinforce” the BM plasma cell population by bystander activation of
memory B cells19 (though this has not been a consistent finding21). If this was the only
mechanism of recruitment it would result in a BM plasma cell compartment which
does not reflect recent antigenic challenges, but rather maintains potentially irrelevant
specificities. Our data shows that bystander activation may increase non-specific
splenic plasmablast number in the short term, but in fact reduces plasma cells of
specificities previously established in the BM. Even if this reduction was <0.5% in
“physiological” settings (compared to the marked reduction seen with our robust
immunisation schedule) it should still be sufficient to “clear” enough niches to make
way for sufficient new BM plasma cells16.
We sought to explain the mechanism underlying the decline in BM plasma cells after
non-specific immunization. We demonstrated that FcRIIb is expressed on long lived
BM plasma cells, and that in its absence plasma cells show abnormal persistence,
which is reflected in serum IgG titers. We then showed that cross-linking FcRIIb
results in plasma cell apoptosis in an antigen-independent fashion – that this is
independent of the BCR is consistent with the mechanism of FcRIIb-mediated
apoptosis of mature naïve B cells26,27 and with absence of BCR expression on the
16
plasma cell membrane. The amount of FcRIIb-mediated apoptosis seen in vitro - a 20
-30% increase over background levels after 4 h cross-linking - may seem small, but it
is similar to that seen for mature B cells26, and is significantly greater both when
soluble immune complexes are used on plasma cells expressing higher levels of
FcRIIb and on BM plasma cells ex vivo. This apparently modest effect, if applied to
each clone of such a long-lived cell type at each antigenic encounter, would be
expected to be of sufficient magnitude to have a physiological impact.
This allows us to propose a mechanism by which FcRIIb may help control BM
plasma cell persistence. Initial antigenic challenge results in the rapid production of
low affinity antibody by extrafollicular plasmablasts, forming immune complexes
which may circulate and cross-link FcRIIb on BM plasma cells, resulting in the death
of a small proportion of them. Plasmablasts produced by the germinal center could
then migrate to the BM and occupy these newly vacant niches. Such a mechanism is
unlikely to act in a simple fashion in all immune responses. It would be expected that
different antigenic challenges would produce immune complexes of quite different
amount and nature. Whether the death of the plasma cells themselves is a stochastic
event, or if some plasma cells are more vulnerable than others, is not clear. Plasma
cells expressing higher levels of FcRIIb are most susceptible to apoptosis, and thus
control of FcRIIb expression may be important in vivo. If BM plasma cell
recruitment can be shown to occur in FcRIIb-deficient mice once their niches are
saturated, other mechanisms must operate in addition to that proposed here, such as
the direct displacement model postulated by Odendahl and colleagues22.
That FcRIIb-induced plasma cell apoptosis is Bim-dependent indicates that it is
17
mediated through a pathway controlled by Bcl-2 family members (that is, the
“mitochondrial” pathway rather than the “death receptor” one). This is consistent with
the mechanism of FcRIIb-induced apoptosis of mature B cells, which was shown to
involve mitochondrial depolarization and cytochrome C release27. FcRIIb-induced
apoptosis of mature B cells has also been shown to be independent of phosphorylation
of the FcRIIb immunoreceptor tyrosine-based inhibition motifs26 and of the SH2containing 5'-inositol phosphatase (SHIP)27. An Ile for Thr replacement at position
232 in the transmembrane domain of FcRIIb has been shown to abolish its SHIPmediated inhibitory function39,40 - we find that this polymorphism has no effect on
FcRIIb-mediated plasma cell apoptosis, and that SHIP-dependent ERK
phosphorylation is not altered upon cross-linking plasma cell FcRIIb (data not
shown). Thus FcRIIb-mediated plasma cell apoptosis is independent of SHIP and
inhibitory function, and is Bim-dependent.
FcRIIb is important in maintaining tolerance and thus preventing autoimmune
disease. Mice deficient in FcRIIb can develop SLE46 and have increased
susceptibility to inducible immune-mediated disease23. Polymorphisms in the
promoter of FcRIIb are associated with reduced expression of FcRIIb, increased
plasma cell number, and SLE37. Such susceptibility to SLE can be corrected by
increasing FcRIIb expression47, and studies in mice transgenic for anti-DNA
antibodies have suggested that the key immunological defect is an increased number
of autoreactive plasma cells, felt to be due to increased production30. We have
confirmed that increased plasma cell production occurs in FcRIIb-deficient mice, but
in addition have shown that there is an increased persistence of long-lived plasma
cells which are resistant to FcRIIb-mediated apoptosis. Moreover, SLE-prone mice
18
have no measurable expression of FcRIIb on BM plasma cells and are resistant to
FcRIIb-induced apoptosis, which can be reversed by transgenic expression of the
receptor. Thus a failure to control the lifespan of autoreactive plasma cells could
represent a second mechanism by which FcRIIb deficiency contributes to
autoantibody production - accumulation of plasma cells due to defective apoptosis has
been associated with autoimmunity in Bcl-2 transgenic mice48 as well as Bimdeficient ones34. It may act not just in the BM – many or most autoreactive plasma
cells may reside in inflammatory tissue18, where antigen is also present. This would be
expected to result in high local levels of immune complexes, particularly in SLE,
where a failure of normal clearance of immune complexes has long been implicated in
disease pathogenesis49. These immune complexes may be important in promoting
apoptosis and controlling local plasma cell numbers in inflammatory lesions. Failure
of plasma cell apoptosis if FcRIIb expression is reduced could thus result in
persistent autoantibody production, forming more immune complexes, and resulting
in a vicious cycle driving pathological inflammation. Thus FcRIIb-mediated
apoptosis of autoreactive plasma cells may represent a mechanism underlying the
most distal “checkpoint” controlling peripheral B cell tolerance50.
FcRIIb also appears important in human autoimmunity, with both transmembrane
domain and promoter polymorphisms being associated with SLE in various ethnic
groups39,51. The transmembrane domain mutation shown to reduce inhibitory function
has no effect on plasma cell apoptosis, but the less well defined promoter
polymorphisms which control FcRIIb expression52,53 may well influence it.
19
Long-lived plasma cells have proven very resistant to therapeutic deletion16. Plasma
cells killing via FcRIIb thus provides an intriguing option for therapy of autoimmune
disease as specific reagents become available28, perhaps combined with conventional
(e.g. cyclophosphamide, steroids) or more novel (rituximab, anti-BAFF) therapies.
The fact that overexpression of FcRIIb increases death indicates that therapies that
modulate FcRIIb expression on plasma cells may also be worth investigating.
Myelomas are plasma cell malignancies which, compared to other B cell
malignancies, are particularly difficult to treat. The incidence of myeloma is 14,000
per year in the US, and the median survival is 3 years54. It is therefore exciting that
cross-linking FcRIIb has a direct apoptotic effect on myeloma cells, and while killing
is incomplete, it is similar to that induced by other agents found useful in combination
therapy55. This may explain the effect of anti-thymocyte globulin on myeloma cell
lines, in which an anti-CD32 component of this preparation has been implicated42.
METHODS
Mice and immunizations. C57BL/6, BALB/c, 129/Sv, NOD, MRL-Mplpr mice were
obtained from Charles River, and NZB mice from Harlan Olac. FcRIIb-deficient
mice on BALB/c and C57BL/6 backgrounds (backcrossed for at least ten generations)
were provided by J.V. Ravetch and S. Bolland (Rockefeller University)24. Prdm1gfp/+
(BLIMP-GFP) mice and Bim knockout mice have been described31,34. All
experiments were performed under the regulations of the Home Office Scientific
Procedures Act, UK (1986) or the approval of the Melbourne Health Animal Ethics
Committee according to the guidelines of the NHMRC Australia. Chicken -globulin
(Sigma) was coupled to NP-Osu (Biosearch). Mice were immunized intraperitoneally
with 100 g of 4-hydroxy-3nitrophenylacetyl-chicken -globulin (NP-CGG), NP-
20
Keyhole limpet Hemocyanin (NP-KLH) (Biosearch) or Phycoerythrin (PE)
(Molecular Probes) precipitated in alum. Mice were boosted after 3 to 5 weeks with
50 g of soluble antigen i.p. For ‘bystander activation’ experiments, mice previously
immunized with NP-CGG in alum were inoculated i.p. with 100 g of OVA and KLH
(Sigma) in alum, 10 g CpG (Sigma Genosys) and 1x105 heat inactivated
Streptococcus pneumoniae at week 9 post-immunization. Mice were boosted i.p. 2
weeks later with 100 g soluble OVA and KLH, 10 g CpG and 1x105 heat
inactivated S. pneumoniae.
Transgenic overexpression of FcRIIb specifically on B cells. A construct
containing a VH promoter, the Igh intron enhancer and the Ig 3’ enhancer had been
previously reported to direct transgenic expression in B cells56. We introduced the
mouse FcRIIb.1 (Ly17.1 allotype) tagged with both V5 and His6’ epitopes into this
construct. The B cell transgenic mice (TG) were established by injecting the DNA
fragment containing the tagged Fcgr2b cDNA, VH promoter and the enhancers into
CBA fertilized C57BL/6 eggs. Transgenic offspring were backcrossed onto C57BL/6
mice for at least 5 generations. The presence of transgene was identified by tail DNA
PCR assays. B-cell TG mice were also backcrossed for 2 generations to NZB mice to
generate (B-TG x NZB)N1 mice. Mice were screened by tail DNA PCR assays for
NZB homozygosity at chromosome 1 using agarose resolvable mapping markers
(D1Mit132, D1Mit308, D1Mit111) and primers recognising the polymorphism in the
Fcgr2b promoter region of autoimmune prone NZB mice57. F2 mice used in plasma
cell cross-linking experiments were homozygous for the NZB Fcgr2b promoter
polymorphism.
Antibodies and flow cytometry analysis. Anti-mouse B220-allophycocyanin (APC;
RA3-6B2), CD19-phycoerythrin (PE; 1D3), CD16/32-fluoroscein isothiocyanate
21
(FITC; 2.4G2), CD138-PE and biotin (281-2), Annexin V-FITC, anti-human CD138PE and CD38-APC (all BD Biosciences), anti-human FcRIIB (Macrogenics) and
anti-CD22-FITC (2D6) (Southern Biotech Associates) were all used to phenotype
and/or identify plasmablasts/plasma cells. 7-aminoactinomycin D (7-AAD)
(Molecular Probes) was used to exclude dead cells. Cells were analyzed using a
FACSCaliburTM flow cytometer (BD) and FCS Press software (R. Hicks, University
of Cambridge).
Cell lines. Human myeloma cell lines EJM and LP-1 were from H. Wiklund
(Uppsala). In some experiments LP-1 was transfected with a construct expressing
human FcRIIb (R A. Floto) - low and high expressing populations were sorted by
Dakocytomation MoFlo.
Microscopy. Cells were adhered to coverslip by poly-L-Lysine (Sigma), fixed and
permeabilized with acetone and methanol at a 1:1 ratio at –20 oC. Cells were stained
with combinations of anti-mouse IgG-Cy5, IgM-Cy5, IgG-Texas Red, -Texas red
and -Texas red (Jackson Immunoresearch) and in some cases, the nuclear dye
Hoechst 33342 (Molecular Probes) to reveal nuclear morphology. Fluorescence was
analyzed by immunofluorescence confocal microscopy (Leica) or wide-field
fluorescence microscopy (Zeiss AxioSkop 2 plus). Fluorescence was quantitated
using Leica software. Four intersects were placed through the acquired image. The
fluorescence signals gained at the rim of the cell were expressed as relative
fluorescence intensity.
ELISPOT and ELISA. Anti-NP secreting plasma cells were detected using the
ELISPOT assay as described previously2. Briefly, 96-well plates (Millipore) were
coated with NP18-BSA and blocked with 10% FCS in PBS prior to addition of BM or
splenic cells. Cells were incubated overnight at 37 oC and 5% CO2 in a humidified
22
incubator in complete medium: RPMI-1640, 10% FCS, penicillin (100 units/ml),
streptomycin (100 g/ml; all Gibco) and 2-mercaptoethanol (50 M; Sigma). AntiNP secreting cells were revealed using anti-mouse IgM, IgG or IgG1-HRPO
(Southern Biotech) and AEC (Sigma). Anti-NP antibodies were detected by ELISA
as described8.
In vitro generation and purification of mouse plasmablasts and enrichment of
plasma cells from the BM. Plasmablasts were prepared in a number of ways; Total
spleen cells were cultured in the presence of 10 g/ml LPS (Salmonella typhimurium;
Sigma) for 3 to 4 days and plasmablasts were analyzed by flow cytometry gated on
B220lo CD138+ population directly or further sorted by flowcytometry (MoFlo) based
on B220lo CD138+. Spleen cells were negatively selected using CD43-specific
microbeads (Miltenyi Biotech) and CD43- cells were incubated with LPS (10 g/ml)
and IL-10 (10 ng/ml) (Peprotech) for 3 days. Cultured cells were washed and
restimulated with LPS (10 g/ml), IL-10 and IL-6 (each 10 ng/ml) for a further 3
days. Splenic B cells were positively selected using CD19 specific microbeads
(Miltenyi Biotech) to a purity of >98%. CD19+ cells were incubated with LPS at 10
g/ml and IL-10 at 10 ng/ml for 3 days. Cultured cells were washed and restimulated
with LPS (10 g/ml), IL-10 and IL-6 (10 ng/ml) for a further 3 days. Plasmablasts
were sorted on the basis of CD138hi, B220lo expression and exclusion of the vital dye,
7-AAD by flow cytometry and then further purified using anti-CD43 microbeads
(Miltenyi Biotech) collecting the positive fraction. The purity of the plasmablasts thus
obtained was higher than 99.6%. Purity of plasmablasts was assessed by ELISPOT
assay or staining of intracellular light chains. Plasma cells were enriched from PE
immunized mice by staining with anti-CD138-biotin and subsequent selection by antibiotin microbeads (Miltenyi Biotech).
23
In vitro generation of human plasmablasts. Human peripheral blood was obtained
from healthy donors with informed consent, under a protocol approved by the
Cambridge Research Ethics Committee. Mononuclear cells were isolated by density
gradient using Ficoll-Paque and cultured in the presence of recombinant human
sCD40L (1 g/ml) and human IL-4 (10 ng/ml) (Peprotech) for 10 days.
In vitro cross-linking on FcRIIb. Mouse plasmablasts or plasma cells were plated
out onto 96-well plates (Nunc) coated with anti-FcRII/III (2.4G2) or anti-FcRIIb (E16) (Santa Cruz Biotechnology), Rat IgG F(ab’)2 (Cortex Biochem) at 20g/ml, or
coating buffer. The cells were incubated for 4 h. Alternatively, FcRIIb was crosslinked either by using monoclonal mouse IgG1 intact (5 g/ml, MOPC 31C, Ancell)
together with polyclonal goat anti-mouse Ig(H+L) (10g/ml)(Southern Biotechnology
Associates), or F(ab’)2 fragments (5 g/ml) of IgG1 (MOPC 31C), or by OVA
immune complexes (OVA-IC). OVA-IC were made by adding rabbit anti-OVA serum
(Sigma) to OVA (Fluka BioChemika) at 1:5 ratio and incubated at 37 C for 30 min
before adding to cultures for 4 h. In some experiments normal rabbit serum was
incubated with OVA prior to the addition to culture, which did not induce apoptosis
above background, or OVA alone was used as a control. Human myeloma cells and
plasmablasts were incubated with mouse anti-human CD32 (clone FL-18.26, 5 g/ml)
and polyclonal goat anti-mouse Ig(H+L) (5 g/ml) (Southern Biotechnology
Associates) in suspension for 8 h.
Apoptosis assays. Cells were analyzed by flow cytometry measuring Annexin V and
7-AAD, or by light microscopic analysis of nuclear fragmentation. Mono- and oligonucleosome release in supernatants from purified plasmablasts was measured by Cell
Death Detection ELISA (Roche Diagnostics).
24
Real time semiquantitative reverse transcription-PCR. RNA was prepared and
mRNA expression was quantitated as previously described58. Briefly, RNA was
extracted from equal numbers of plasmablasts using TRIzol® Reagent (GIBCO BRL),
and reverse transcribed using Super RT (HT Biotechnology). Fcgr2b, Cd22 and Fcgr3
mRNA abundance was assessed relative to Gapdh using real-time semiquantitative
reverse transcription (RT)-PCR (ABI Prism 7700 Sequence Detection System;
Applied Biosystems). The Gapdh control primers and probe were Taqman Rodent
control reagents (Applied Biosystems). All primers and probes were designed using
Primer Express software (Applied Biosystems) and manufactured by Sigma-Genosys.
Radioactive RT-PCR. Resting B cells (B220+) and plasma cells (GFP+CD138+)
were isolated from Prdm1gfp/+ (BLIMP-GFP) mice and RNA extracted using an
RNeasy kit (Qiagen). First strand cDNA synthesis was performed using random
hexameric primers and Superscript II RT (GIBCO), following the manufacturer’s
protocol. Fcgr2 mRNA was quantified using plasma cell cDNA as a template for a 32P
PCR carried out as described59. Essentially, dATP was substituted with 32-P-alphadATP (Amersham) such that products of the PCR reaction incorporated the
radioactive nucleotide during extension. PCR products were resolved on an 8%
polyacrylamide gel, which was subsequently dried and exposed to a PhosphorImager
cassette (Molecular Dynamics) for 12 h at room temperature. Signal intensity was
quantified using ImageQuant v3.3 software (Molecular Dynamics) and Hprt1 for
comparison. The primers for Fcgr2b used were as reported58.
Adoptive transfers. Cells were isolated from spleens 4 days after boost immunization
with NP-CGG. Cells were washed and pooled, before injecting i.v. into C57BL/6
recipient mice. Recipient mice were sacrificed 6 days and 76 days as well as bled on
25
day 27, respectively, after transfer. BM was analyzed by ELISPOT for the frequency
of NP-specific plasma cells. Ratios were corrected relative to the number of NPspecific donor cells for each genotype.
Statistical analysis. Two-tailed unpaired t-tests were used to determine statistical
significance except for Fig. 4f where one-tailed tests were used, and Fig. 7c, where
two-tailed paired t-tests were used.
ACKNOWLEDGEMENTS
This work was supported by a Wellcome Research Leave Award for Clinical
Academics (Grant 067543AIA). RAM was supported by DFG grant MA 2273/2-4; 42. DMT, KF and KEL were supported by the National Health and Medical Research
Council of Australia. We would like to thank H. Wiklund for providing myeloma cell
lines; T. Tsubata and R. Floto for constructs; S. Koenig (Macrogenics) for antibodies;
C. Watson for Bim-/- mice; S. Bolland and J. Ravetch for Fcgr2b-/- mice; L. Willcocks,
A. Rankin, W. Ouwehand and N. Watkins and the staff and donors of the National
Blood Service Cambridge Apheresis Clinic for human primary lymphocyte
preparation; P. Lyons and A. Strasser for helpful advice.
Figure legends
Figure 1 Non-specific immunization (NSI) reduces antigen-specific BM plasma cells
and serum IgG. (a) Experimental protocol. C57BL/6 mice were immunized with 100
g NP-CGG in alum i.p. At week 9 post-immunisation mice were either (A) analyzed
for anti-NP responses (n=5) or immunized and boosted with either saline (filled
circle) or a cocktail of OVA, KLH, 10 g CpG and 1x105 heat inactivated S.
26
pneumoniae (open circle) at weeks 9 and 11 (NSI; n=10 per group). Anti-NP
responses were measured at week 13 (B) or week 15 (C). (b-c) Anti-NP IgG plasma
cells in the spleen (b) and BM (c) of untreated (filled circles) or NSI (open diamonds)
mice were enumerated by ELISPOT. Horizontal lines represent the mean value. (d)
Anti-NP IgG in the serum of untreated (filled bars) or NSI (open bars) mice was
measured by ELISA.
Figure 2 Plasma cells express FcRIIb. (a) Plasma cells in BLIMP-GFP mice were
identified on the basis of CD138 staining and GFP expression. The intensity of FcRII
staining on these gated plasma cells from spleen and bone marrow was compared to
the staining of an isotype control antibody. The percentage of cells in each of the
gated regions is shown. (b) Histograms showing the relative intensity of FcRII
staining on B cells, plasma cells of different maturation stages defined by Blimp-1
expression and T cells. Staining of a control antibody on BM plasma cells is also
shown. The mean GFP fluorescence on each population is given within each
histogram. Results in (a) and (b) are representative of three independent experiments.
(c,d) Mice were immunized with PE in alum. 56 days post-immunization BM cells
were pooled from 3 mice, enriched for plasma cells by MACS sorting on CD138, and
stained for intracellular PE-binding (red), IgG (blue) and surface FcRIIb expression
(green), before analysis by confocal microscopy. (d) FcRIIb staining was determined
on individual IgG+ or IgM+ intracellular PE-binding plasma cells by confocal
microscopy and levels expressed as relative fluorescence intensity. Each dot
represents measurement of an individual cell.
27
Figure 3 Plasma cells continue to express Fcgr2b mRNA. (a) Surface expression of
FcRIIb and CD22 on CD138+ plasmablasts generated in vitro by stimulation of
splenic B cells with LPS, IL-10 and IL-6. (b) Plasmablasts (PC), B cells (B), T cells
(T) and macrophages (M) were analyzed for expression of Fcgr2b, Cd22 and Fcgr3
mRNA by semi-quantitative real-time PCR. Data is expressed relative to Gapdh. (c)
Radioactive PCR was conducted on cDNA that had been synthesized from the mRNA
extracted from B cells or plasma cells that had been sorted as immature (GFPint) and
mature (GFPhi) from BLIMP-GFP mice. Hprt and Fcgr2 primers were added
simultaneously, allowing Fcgr2 expression to be compared to the Hprt loading. Both
Fcgr2 cDNA and contaminating genomic DNA (gDNA) are amplified in this reaction,
but could be clearly resolved. For b and c, 1 of 2 representative experiments using
independent mRNA preparations is shown.
Figure 4 FcRIIb and the persistence of splenic plasmablasts and long-lived BM
plasma cells. Mice were immunized with NP-KLH in alum and antibody-forming
cells secreting anti-NP antibodies were enumerated by ELISPOT; (a) IgM spleen, (b)
IgG spleen, (c, d, f) IgG BM. (e,g,h) Anti-NP IgG in serum measured by ELISA. (ae) show comparisons between NP responses in control (filled circles) and FcRIIbdeficient mice (open circles). (f,g) show NP-specific plasma cells in recipient mice 6,
27 and 76 days after adoptive transfer of 3.3x107 control (filled circles) or 1.8x107
FcRIIb-deficient (open circles) splenocytes from immunized donor mice 6 days after
boosting with NP-CGG. Numbers of cells transferred were adjusted to achieve
transfer of similar numbers of NP-specific plasma cell precursors from FcRIIbdeficient and control mice. (h) shows a comparison between NP responses in control
(filled circles) and FcRIIb-transgenic mice (open triangles). Each experiment shown
28
is representative of either 2 or 3 independent experiments. Each dot shows data from a
single mouse and horizontal lines represent the mean value.
Figure 5 FcRIIb cross-linking induces apoptosis of in vitro generated plasmablasts.
Plasmablasts were generated from splenocytes by culture with LPS and FcRIIb was
cross-linked by culturing in wells pre-coated with antibodies, or by soluble immune
complexes. (a) B cells were isolated from splenocytes by negative selection with
CD43 specific microbeads and cultured with LPS and IL-10 for 3 days and then with
LPS, IL-10 and IL-6 for another 3 days. Cells were then incubated for 4 h in wells
pre-coated with or without 2.4G2. Cells were analyzed by flow cytometry gating on
the plasmablast population (see Supplementary Figure 1d), and percentage of
apoptotic cells (annexin V+/7-AAD–) were detected (mean  s.e.m. of 3 experiments).
(b) Plasmablasts generated after 4 days culture with LPS were sorted (see gates in
Supplementary Figure 1d), cultured for 4 h in wells precoated with 2.4G2, rat
F(ab’)2, or PBS, and apoptosis determined by measurement of nucleosome release by
ELISA (mean  s.e.m. of triplicates of 2 experiments). (c to j) Splenocytes cultured
with LPS for 3 to 4 days were analyzed by flow cytometry (gates as in
Supplementary Figure 1d). (c) Cells from Fcgr2b+/+ mice were incubated in wells
pre-coated with or without anti-FcRIIb (E-16) (mean  s.e.m of 4 experiments). (d)
Cells from Fcgr2b-/- (red), Fcgr2b+/+ (blue) and B-cell transgenic (TG) (green) mice
were analyzed by flow cytometry for surface expression of FcRIIb using 2.4G2.
Isotype control curves were identical to those of Fcgr2b-/- shown and were omitted for
clarity. The geometric mean fluorescence of each population is shown. (e)
Plasmablasts were incubated for 4 h in culture medium containing mouse IgG1 and
goat anti-mouse Ig(H+L), mouse IgG1 F(ab’)2, or buffer alone (mean  s.e.m in
29
duplicate of one representative experiment of 3). (f) Data from all 3 experiments (see
e) were pooled and shown as percentage increase in apoptosis of intact IgG1 treatment
over F(ab’)2. (g) Plasmablasts were incubated for 4 h in culture medium containing
OVA which was previously incubated with rabbit anti-OVA polyclonal antibody for
30 min, or containing OVA alone (mean  s.e.m in duplicate of one representative
experiment of 2). (h) Plasmablasts (gated as in Supplementary Figure 1d) from
Bim+/+ and Bim-/- mice were stained with 2.4G2 antibody (Bim+/+ green, Bim-/- black)
or isotype control (Bim+/+ red, Bim-/- blue). The geometric mean fluorescence is
shown. (i) Plasmablasts from Bim+/+ and Bim-/- mice were incubated for 4 h in wells
pre-coated with or without 2.4G2. Cells were analyzed after 4 h of culture (mean 
s.e.m in duplicate of one representative experiment of 3). (j) Data from all 3
experiments (see i) were pooled and shown as percentage of increase in apoptosis of
24G2 treatment over buffer control. (k) Plasmablasts cultured from Bim+/+ and Bim-/mice were sorted and analyzed as in b.
Figure 6 FcRIIb cross-linking induces apoptosis of ex vivo plasma cells. C57BL/6
mice were immunized and boosted with NP-CGG. (a to c) 7 days after boost
splenocytes were cultured for 4 h in wells precoated with 2.4G2, rat F(ab’)2, or PBS.
(a) Antibody-forming cells were gated as B220lo CD138+ (as in Fig. 5a) and the
percentages of Annexin V+ 7-AAD– cells determined. (b) Cells were permeabilized
and stained with anti-immunoglobulin light chains to allow detection of plasma cells,
and counterstained with the Hoechst dye to identify apoptotic nuclear morphology.
Representative fields (of over 30 per group examined at each time point) indicating
apoptotic plasma cells (arrows) are shown. (c) Number of apoptotic plasma cells
30
identified (see b) was quantified. (d) 63 days after boost immunization, BM cells from
both Fcgr2b+/+ and Fcgr2b-/- mice were removed and analyzed as in c.
Figure 7 FcRIIb cross-linking and apoptosis in plasma cells from autoimmune-prone
mice, human plasmablasts and myeloma cells. (a) Plasmablasts were generated from
mice of the strains indicated by culture with LPS for 4 to 6 days, then FcRIIb was
cross-linked by incubation in wells pre-coated with the 2.4G2 antibody for 4 h.
Apoptosis was assessed by annexin staining (see Fig. 5a). (b) Mouse splenocytes
from the B-cell specific FcRIIb transgenic mice backcrossed to NZB (TG-1 and -2
are two individual transgenic mice and NTG is a nontransgenic littermate control)
were differentiated into plasmablasts and analyzed as in Fig. 5g (mean  s.e.m in
duplicate of one representative experiment of 3). (c) Human primary cultured
plasmablasts from donors with different FcRIIb transmembrane polymorphism
genotypes (Ile or Thr at position 232) were incubated with (XL) or without (C)
FL18.26, together with polyclonal goat anti-mouse Ig(H+L), for 8 h. Annexin V+ 7AAD- apoptotic cells were determined by flow cytometry gating on CD38+ CD138+
(mean  s.e.m of 9 experiments for I/I, 10 for I/T and 7 for T/T). (d,e) FcRIIb
expression on myeloma cell lines EJM and LP-1 analyzed by flow cytometry with a
mouse anti-human FcRIIb (2B6) (unshaded) or isotype control (shaded). (f) EJM and
LP-1 cells were cross-linked by incubation with (XL) or without (C) FL18.26,
together with polyclonal goat anti-mouse Ig(H+L), for 8 h, and annexin V+/7-AAD–
cells were determined by flow cytometry. (g) LP-1 cells were transfected with a
construct expressing human FcRIIb (LP-1-FcRIIb) and sorted into populations
expressing high or low amounts of FcRIIb. (h) Apoptosis was assessed in these two
31
populations as in g. In each panel mean  s.e.m of at least 3 experiments is shown
unless otherwise indicated.
Author contributions
A.J.C. mainly contributed Figures 1-4e in collaboration with E.U.W., K.F., R.A.M.
and D.M.T; Z.X. mainly contributed Figures 4f-7 with contributions from R.J.B.,
K.E.L. and E.S.; A.J.C. and R.J.B. generated the transgenic mice; K.G.C.S. conceived
and directed the experiments; and Z.X. and K.G.C.S. wrote the paper, with input from
all authors.
32
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