International Immunology, Vol. 11, No. 10, pp. 1693–1700
Multivalent cross-linking of membrane
Ig sensitizes murine B cells to a broader
spectrum of CpG-containing
oligodeoxynucleotide motifs, including their
methylated counterparts, for stimulation of
proliferation and Ig secretion
Bruce E. Goeckeritz1,4, Michael Flora3, Kim Witherspoon1, Quirijn Vos1,
Andrew Lees1, Gregory J. Dennis4, David S. Pisetsky5, Dennis M. Klinman6,
Clifford M. Snapper2 and James J. Mond1
Departments of 1Medicine and 2Pathology, and 3Biomedical Instrumentation Center, Uniformed Services
University of the Health Sciences, Bethesda, MD 20814-4799, USA
4Rheumatology Service, Department of Medicine, Walter Reed Army Medical Center, Washington,
DC 20307, USA
5Medical Research Service, Durham Veterans Administration Hospital, Division of Immunology, Division of
Rheumatology and Immunology, Duke University Medical Center, Durham, NC 27710, USA
6Retroviral Immunology Section, Center for Biologics Evaluation, Food and Drug Administration,
Bethesda, MD 20892, USA
Keywords: antibodies, B lymphocytes, cellular activation, CpG, oligodeoxynucleotides, rodent, spleen
Abstract
We have previously reported that B cells that are activated by multivalent but not bivalent
membrane Ig cross-linking ligands synergize with various B cell activators culminating in
enhanced B cell proliferation. In this study we asked whether B cells that are activated by a
multivalent mIg cross-linking agonist could respond to oligodeoxynucleotides (ODN) containing
non-stimulatory motifs. Earlier reports have shown that ODN containing a CpG motif in which the
cytosine is unmethylated and is flanked by two 59 purines and two 39 pyrimidines induce high
levels of B cell activation, while ODN whose CpG are methylated or flanked by sequences other
than the optimal two 59 purines and two 39 pyrimidines were non-stimulatory. In this manuscript we
show that when B cells are stimulated in vitro with dextran-conjugated anti-IgD antibodies
(anti-IgD–dex), as the multivalent mIg ligand, their proliferation is enhanced and they can be
induced to secrete Ig in response to ODN containing various non-optimal motifs, both methylated
and non-methylated. Furthermore we could induce synergistic levels of proliferation with
concentrations of anti-IgD–dex that were in the picomolar concentration range and with
concentrations of ODN that were 10- to 100-fold less than previously reported to be necessary for
mitogenic activity. These data provided a model to explain how low concentrations of a multiepitope-expressing microorganism in the context of mammalian (methylated) or microorganism
(non-methylated) DNA can lead to dysregulated B cell proliferation and Ig secretion.
Correspondence to: B. E. Goeckeritz
Transmitting editor: Z. Ovary
Received 25 May 1999, accepted 8 July 1999
1694 B cell stimulating activities of ODN of various motifs
Introduction
Over the last several years many investigators have studied
the stimulatory effects of DNA on the immune system. They
have demonstrated that microorganism DNA and synthetic
oligodeoxynucleotides (ODN) containing a specific dinucleotide motif (CpG), in which the cytosine is unmethylated and
which is 16 times more frequent than in mammalian DNA, is
extremely immunostimulatory (1,2). DNA and synthetic ODN
containing this immunostimulatory motif have been shown to
activate murine and human B cells for proliferation and Ig
secretion (1,3), to enhance murine T, B and NK cell cytokine
production, to increase NK cell lytic activity, and to activate
macrophages for tumor necrosis factor-α production (2–8).
In addition, ODN containing this immunostimulatory motif are
being investigated for their role as vaccine adjuvant (9).
Of particular interest is the observation that not all ODN
containing an unmethylated CpG dinucleotide are equally
stimulatory. The sequences flanking the CpG dinucleotide
appear to play an important role in determining the immunostimulatory effects of a given ODN (1,10). ODN containing a
CpG flanked on the 59 end by two purines and on the 39 end
by two pyrimidines are maximally stimulatory, whereas ODN
containing a CpG dinucleotide flanked by any other combination of sequences are much less so. Also of interest is
the observation that methylating the cytosine of the CpG
dinucleotide motif significantly decreases its immunostimulatory ability (1). While the specific mechanism of action of
these compounds is unknown, they appear to bind intracellular
components and induce NF-κB-dependent pathways of
activation in a T cell-independent manner.
The fact that unmethylated motifs are a characteristic of
microorganism DNA suggests that microbial products, either
alone or in synergy with the intact organism, may be able to
adjuvant humoral antibody responses to their own determinants which may be poorly immunogenic in the absence of
adjuvanting signals. We have shown earlier that high levels
of Ig secretion can be induced in a T cell-independent manner
when mIg on B cells is cross-linked in a multivalent manner
in the presence of low concentrations of polyclonal B cell
activators (13–15). This mechanism may underlie the ability
of ODN as polyclonal B cell activators to synergize with a
multivalent mIg cross-linking signal induced by an infecting
organism, whose antigenic epitopes are frequently multivalent,
and thereby to initiate either antigen-specific responses even
at low concentrations of a particular epitope.
The ability of antigen-initiated B cell activation to synergize
with ODN-mediated B cell signaling has been reported by
Krieg et al. (1), who demonstrated that B cells stimulated by
unconjugated anti-IgM antibody showed enhanced proliferative responses when cultured in vitro in the presence of
stimulatory ODN. For effective synergy high molar concentrations of anti-IgM were used. Since anti-Ig in vitro is used to
model the effects of ligation of the antigen receptor by antigen
this suggests that high molar concentrations of antigens might
be necessary to synergize with ODN. Such high concentrations of soluble antigens may never be attained and/or
maintained under physiologic conditions of infection. Furthermore, many of the antigenic epitopes that are displayed on
the viral or bacterial surface appear in a repetitive array, thus
enabling them to ligate their specific antigen receptor in a
multivalent manner. Since soluble anti-Ig is bivalent it might
not provide a suitable model to mimic the effect of these
multivalent epitopes.
We have previously shown that mIg-mediated activation of
B cells by a multivalent cross-linking agonist, e.g. anti-IgD
conjugated to high mol. wt dextran (anti-IgD–dex), induces a
.100- to 1000-fold higher B cell proliferative response at
concentrations that are 1000-fold less than unconjugated
anti-IgD (16). Furthermore, unlike unconjugated anti-Ig, antiIgD–dex stimulates very high levels of Ig secretion at nanomolar concentrations in the presence of cytokines (17). For these
and other reasons, which are discussed in earlier publications,
we believe that this agonist provides a useful model for
studying T cell-independent B cell activation. It provides a
tool to study the effect of membrane Ig cross-linking induced
by low concentrations of multivalent antigens as might be
displayed by viral or bacterial organisms. We therefore
employed anti-IgD–dex to study whether under conditions of
multivalent mIg signaling poorly stimulatory ODN, including
those with a less than optimally stimulatory motif or those
stimulatory ODN whose motif is methylated, could be shown
to be stimulatory to B cells. In this paper we show that unlike
bivalent unconjugated anti-Ig, anti-IgD–dex synergizes at
ng/ml concentrations with both stimulatory and poorly stimulatory ODN, including those that are methylated, to enhance
B cell proliferation. These data suggest that microorganisms
that express their B cell-specific epitopes in a multivalent array
may be very effective in synergizing with both mammalian and
microorganism DNA fragments of diverse motifs. This provides
an interesting model to study the parameters that regulate
the steps culminating in dysregulated cell growth and lead
to the development of autoimmunity.
Methods
Mice
Female DBA/2 and C3H/HeN mice, 6–8 weeks old, were
obtained from The National Cancer Institute (Bethesda, MD)
and female C3H/HeJ and (CBA3C57B16) mice, 6–8 weeks
old, were obtained from the Jackson Laboratories (Bar
Harbor, ME).
ODN and other reagents
ODN 74, 1082 and 2105 are phosphorothioated, and were
synthesized using standard phosphoramidite chemistry
methods by Midland Certified Reagent (Midland TX). Their
sequences are as follows: 74 (59-AAAAAAAAAAAAAACGTTAAAAAAAAAAA-39), 1082 (59 GCCGAGGTCCATGTCGTACGC-39) and 2105 (59-TTGCTTCCATCTTCCTCGTC-39). 74m
is the ODN 74 with the only difference being that the
cytosine is methylated. 1082m is the ODN 1082 except that
each cytosine preceding a guanine is methylated
(59-GCXGAGGTCCATGTXGTAXGC-39, where X indicates a
methylated cytosine). 74m and 1082m are phosphorothioated
and were synthesized at the USUHS (Bethesda, MD) as
follows: methylation of the desired base at a given position
B cell stimulating activities of ODN of various motifs 1695
in the oligonucleotide was accomplished using 5-Me-dC-β
cyanoethyl-diisopropyl phosphoramidite (Cruachem, Dulles,
VA). This phosphoramidite delivers a methylated dC at any
site (other that the terminal 39 base) as directed using a
standard synthesis protocol. The company reports a coupling
efficiency of at least 97% for the Me-dC phosphoramidite.
These particular ODN were produced on an Applied
Biosystems Model 394 DNA/RNA Synthesizer. Thioation of the
oligonucleotide was accomplished with Beaucage Reagent
(Cruachem, Dulles, VA) using the manufacturer’s basic instructions for incorporation of the sulfur. In addition, 74 and
74m were synthesized as phosphodiesters at USUHS using
phosphoramidite technology similar to that used with the
phosphorothioates except that oxygen is substituted for sulfur.
The monoclonal anti-T cell reagents, anti-Thy-1.2 (clone 30H12) (18) and anti-CD4 (clone GK 1.5) (19), were grown as
ascitic fluid in nude mice. The mouse mAb against rat IgG κ
chain, MAR 18.5 (20), was produced by cells grown in tissue
culture. The mAb Hδa/1 (21) with specificity for the heavy
chain of IgD was grown in nude mice. Antibodies were purified
as described previously (22).
Conjugation of the Hδa/1 mAb to dextran was done as
described (23) and the Hδa/1–dextran conjugate (anti-IgD–
dex) that was used in these experiments had on average six
molecules of anti-IgD per dextran molecule. Concentrations
of dextran-conjugated antibodies that are noted in the text
reflect only the anti-Ig antibody concentration and not that of
the entire dextran conjugates.
Murine B cell purification
Suspensions of single spleen cells were washed with a mixture
of RPMI (M. A. Bioproducts, Walkersville, MD), 10% FCS
(Intergen, Purchase, NY) and 25 mM HEPES buffer solution
(Life Technologies, Grand Island, NY), and then treated with
monoclonal T cell antibodies anti-Thy 1.2 and anti-CD4 per
107 spleen cells for 30 min on ice. This was followed by
45 min incubation with 10% newborn rabbit complement
(PelFreez, Rogers, AR) in the presence of a 1/10 dilution of
tissue culture fluid containing an anti-rat κ chain mAb MAR
18.5 at 37°C. Small resting B cells were separated from this
T cell-depleted spleen cell suspension by Percoll density
gradient centrifugation by removing cells that banded
between 65 and 70% Percoll.
Measurement of murine B cell [3H]thymidine incorporation
B cells were cultured for 24 h in a final volume of 0.2 ml in
modified Mishell–Dutton medium in flat-bottom 96-well trays
(Costar, Cambridge, MA). [3H]Thymidine (1.0 µCi)
(Amersham, Arlington Heights, IL), with a sp. act. of
20 Ci/mmol, was added to the cultures for a final 18 h and
cultures were harvested with a PHD cell harvester (Cambridge
Technology, Watertown, MA) onto glass fiber filters. Specific
incorporation of thymidine was analyzed by liquid scintillation
spectroscopy and results are expressed as the arithmetic
mean of triplicate cultures.
Quantification of secreted Ig in culture supernatant (SN)
For purposes of Ig quantification B cell cultures were established as for proliferation studies and allowed to incubate for
6 days at which time SN were harvested. IgM concentrations
were measured by an ELISA assay. For determination of
concentrations of secreted IgM in culture SN, Immulon 2,
96-well flat-bottom ELISA plates (Dynatech, Alexandria, VA)
were coated with unlabeled affinity-purified polyclonal goat
anti-mouse IgM (Southern Biotechnology Associates,
Birmingham, AL). Plates were then washed, blocked with
FBS-containing buffer, and incubated with various dilutions
of culture SN and standard. After washing, plates were
incubated with alkaline phosphatase-conjugated affinity-purified, polyclonal goat anti-mouse IgM (Southern Biotechnology
Associates), as indicated, and washed again, and a fluorescent product was generated by cleavage of exogenous 4
methylumbilliferyl phosphate (Sigma, St Louis, MO) by the
plated-bound alkaline phosphatase-conjugated antibodies.
Fluorescence was quantified on a 3M FluoroFAST 96 Fluorometer (Mountain View, CA) and fluorescence units were
converted to Ig concentrations by interpolation from standard
curves that were determined with known concentrations of
purified mouse IgM (Southern Biotechnology Associates).
ELISA for IgG was as described above for IgM with the
exception that alkaline phosphatase-conjugated anti-IgG was
used in place of anti-IgM.
Quantification of IgM-secreting cells in the cultures of stimulated B cells
IgM secreting cells were detected using the spot-ELISA
according to a modified version of a previously described
protocol (24). The individual wells of cellulose ester membrane
96-well plates (Millipore Multiscreen; Millipore, Molsheim,
France) were coated with 50 µl of a 5 mg/ml solution of goatanti-mouse κ light chain antiserum in PBS by incubation
overnight at 4°C. After discarding the coating solution, the
plates were post-coated by adding 50 µl of culture medium
to each well followed by a 30 min incubation at 37°C. After
removal of the SN, the cell suspensions from the stimulated
B cell cultures were added. To this end the suspensions
from the triplicate cultures were pooled, centrifuged and
resuspended in 500 µl culture medium. Next, three sequential
10-fold dilutions of the suspensions were made, and 100 µl
of each of the dilutions was added in triplicate, followed by
a 6 h incubation in a humidified atmosphere containing 5%
CO2 at 37°C. Next, SN were discarded, the plates were
washed and dried, and 50 µl of a 1 mg/ml solution of goat
anti-mouse IgM alkaline phosphatase in FBS-containing buffer
was added to each well, followed by overnight incubation at
4°C. Subsequently, SN were discarded and the plates were
washed. After drying the plates, 50 µl of 5-bromo-4-chloroindolyl-phosphatase and nitro-blue tetrazolium substrate solution (Kirkegaard & Perry, Gaithersburg, MD) were added to
each well and allowed to react for 5 min at room temperature,
followed by two washes with demineralized water and drying.
Spots representing individual IgM-secreting cells were enumerated using an inverting microscope.
Results
Comparison of multivalent versus bivalent mIg activation on
B cell responsiveness to CpG-containing ODN
Since many microbial antigens express their B cell epitopes
in a multivalent array, we wished to study the role of multivalent
1696 B cell stimulating activities of ODN of various motifs
Fig. 1. Anti-IgD–dex at concentrations as low as 0.1 ng/ml synergize
with the CpG-containing ODN 74 for B cell proliferation. B cells
obtained from DBA/2 mouse spleen were stimulated with varying
concentrations of anti-IgD–dex in the presence or absence of the
CpG-containing ODN 74 (1.0 µg/ml). [3H]Thymidine was added 24 h
after initiation of culture for an 18 h period and cultures were then
harvested for scintillation spectroscopy. Data are expressed as the
mean 6 SE of triplicate cultures and are representative of three
similar experiments. Similar results were obtained when using B cells
from C3H/HeJ spleen (data not shown).
mIg ligation on B cell responses to CpG-containing ODN
which in this model represent microbial derived fragments of
DNA. In view of our past findings that anti-IgD–dex, a model
for multivalent mIg ligation, stimulates B cell activation at
picomolar concentrations we wished to determine whether it
could induce synergistic proliferation of B cells with stimulatory
ODN even at these low concentrations (Fig. 1). Concentrations
of anti-IgD–dex as low as 0.1 ng/ml synergized with the CpG
ODN 74 to stimulate enhanced B cell proliferation. Dextran
by itself had no stimulatory effect on ODN-activated B cells
(data not shown). To study whether this reflected the multivalency of the agonist we compared its effect with that of
anti-IgD and anti-IgM, bivalent mIg cross-linking agonists that
were not conjugated to high mol. wt dextran (Fig. 2). B cells
were cultured with the CpG ODN 74 together with either
anti-IgD–dex at 1.0 ng/ml, anti-IgD at 1.0 µg/ml or anti-IgM
at 10 µg/ml, the concentrations of anti-IgD and anti-IgM being
1000- to 10,000-fold greater than that of anti-IgD–dex. While
low-level synergistic stimulation was seen when anti-IgD and
74 were added together, it was inconsistent. This was in
contrast to anti-IgD–dex, where consistent and significant
synergy was seen whenever anti-IgD–dex and the CpG ODN
74 were co-cultured. When anti-IgM was co-cultured with 74
a synergistic response of comparable magnitude to that
previously published (1) was seen. However, with anti-IgD,
the response was inconsistent and significantly less than
that observed with anti-IgD–dex, and was seen only with a
concentration of 10.0 µg/ml, 10,000-fold greater than required
when using dextran conjugated to anti-IgD. In view of studies
suggesting that phosphorothioation may in and of itself
enhance the immunostimulatory ability of a given ODN we
conducted B cell proliferation studies using the ODN 74 as
a phosphodiester and a phosphorothioate. Phosphodiesters
by themselves even at 30 µg/ml did not induce proliferation
above background nor did they synergize with anti-IgD–dex
(data not shown). To determine the role of other B cell
Fig. 2. Multivalent mIg cross-linking agonist (anti-IgD–dex) is superior
to bivalent mIg cross-linking agonists (anti-IgD and anti-IgM) for
synergizing with the CpG-containing ODN 74 for B cell proliferation.
B cells from DBA/2 mouse spleen were stimulated with either antiIgD–dex at 1.0 ng/ml, anti-IgD at 1.0 µg/ml or anti-IgM at 10 µg/ml
and then co-cultured with 10 µg/ml of the CpG-containing ODN 74.
[3H]Thymidine was added 24 h after initiation of culture for an 18 h
period and cultures were then harvested for scintillation spectroscopy.
Data are expressed as the mean 6 SE of triplicate cultures and are
representative of three similar experiments. Similar results were
obtained when using B cells from C3H/HeJ and C3H/HeN spleen
(data not shown).
activators in enhancing B cell proliferation in the presence of
ODN we studied the effects of lipopolysaccharide (LPS), Lipo
Osp A and trivalent membrane CD40 ligand (CD40L). While
membrane CD40L and Lipo Osp A failed to induce any
synergistic stimulatory responses with CpG ODN, LPS
induced low-level and variable enhancements (data not
shown). These results suggest that the mode of B cell
activation is critical in determining the B cell response to
ODN and that activation via the antigen receptor with a
multivalent agonist is the most effective mode of stimulation
in this context.
Anti-IgD–dex synergizes both with ODN containing stimulatory
CpG motifs and those containing stimulatory CpG motifs
which have been methylated
In view of published reports demonstrating that ODN that
contain the stimulatory CpG motif lose their stimulatory activity
when the motifs’ cytosine is methylated, we investigated
whether the inhibitory effects of methylation could be overcome by stimulating the B cells using a multivalent Ig crosslinking stimulus. B cells were cultured with the CpG-containing
ODN 74 or with its methylated counterpart 74m. As previously
shown the stimulatory CpG motif, when acting alone, lost its
stimulatory activity when it was methylated (Fig. 3). However,
when B cells were activated with anti-IgD–dex both the
methylated and the non-methylated CpG motif induced synergistic proliferative responses. The response induced by the
non-methylated ODN in concert with anti-IgD–dex was greater
in magnitude and was observed over a wider concentration
range than that induced by its methylated counterpart. No
synergy was observed when the methylated ODN was cultured with unconjugated anti-IgD and only low-level synergy
was seen when cells were cultured with unconjugated antiIgM at concentrations 1000-fold greater than was required
with anti-IgD–dex (data not shown). These results demonstrate
B cell stimulating activities of ODN of various motifs 1697
CpG motifs, many motifs that have been described as nonstimulatory can in fact synergize to induce B cell proliferation.
Anti-IgD–dex enhances the Ig-secretory ability of both
unmethylated and methylated ODN
Fig. 3. Anti-IgD–dex synergizes with ODN containing stimulatory
CpG motifs and those containing stimulatory motifs that have been
methylated. B cells from DBA/2 mouse spleen were stimulated with
varying concentrations of the CpG-containing ODN 74 and 74m (in
which the cytosine of the CpG dinucleotide is methylated) in the
presence or absence of anti-IgD–dex (1.0 ng/ml). [3H]Thymidine was
added 24 h after initiation of culture for an 18 h period and cultures
were then harvested for scintillation spectroscopy. Data are expressed
as the mean 6 SE of triplicate cultures and are representative of
three similar experiments.
that the methylation status of the ODN is not a critical factor
in determining whether the ODN can enhance proliferation of
B cells that have been activated by a multivalent mIg crosslinking ligand. However, methylation does influence the magnitude of the response that is observed and the concentrations
range of the ODN that is required to induce optimal stimulation.
Anti-IgD–dex synergizes with both stimulatory and poorly
stimulatory non-methylated CpG-containing ODN
In view of the ability of anti-IgD–dex to enhance proliferation
of B cells stimulated by the methylated CpG ODN 74m we
investigated whether it could synergize with CpG-containing
ODN with poorly stimulatory motifs. B cells were cultured with
the stimulatory CpG-containing ODN 74 or with the poorly
stimulatory CpG-containing ODN, 1082 or 2105 (neither of
which contain the optimally stimulatory bases flanking the
CpG dinucleotide). In concert with anti-IgD–dex significant
synergistic proliferative responses were noted with both the
stimulatory and poorly stimulatory CpG ODN with greater
levels of stimulation observed with the stimulatory CpG ODN
74 (Fig. 4a). With lower concentrations of anti-IgD–dex significant synergy was seen only with the stimulatory but not with
the non-stimulatory ODN (Fig. 4b). To determine whether
bivalent mIg cross-linking agonists would synergize with the
poorly stimulatory ODN 1082 and 2105, anti-IgD and antiIgM were co-cultured with 1082 and 2105 across a range of
doses. Inconsistent synergistic responses were observed with
both agents (data not shown). When the CpG ODN 1082
was methylated synergistic proliferative responses were still
observed after stimulation with anti-IgD–dex but only at the
higher concentrations of ODN (Fig. 5). The occasional variation that was observed in the concentration of ODN that
induced optimal synergy with anti-Ig-dex may reflect the
variation in the level of proliferative response that was stimulated by the anti-Ig-dex only. These data demonstrate that in
the presence of multivalent cross-linking stimuli, while there
is a clear distinction between stimulatory and non-stimulatory
The only previous report showing enhanced in vitro Ig secretion in response to stimulation with a CpG ODN, in combination
with mIg stimulation, used a B cell line (1). In view of our
current data demonstrating the ability of multivalent mIg
to synergize with methylated ODN for induction of B cell
proliferation we wished to determine whether a multivalent
mIg cross-linking agonist, at concentrations which were a
1000-fold lower than was required by unconjugated anti-Ig
antibodies, would enhance IgM-secretory responses as well
as IgG in normal splenic B cells. B cells were cultured with
the CpG-containing ODN 74 and its methylated counterpart
74m in the presence of anti-IgD–dex and the number of
IgM-secreting cells or total IgM secretion was measured after
6 days (Figs 6 and 7). In concert with anti-IgD–dex, synergistic
responses were seen with the stimulatory ODN at concentrators as low as 1 µg/ml and levels of enhancement in the
numbers of IgM-secreting cells were as great as 400-fold
increase at low concentrations of the stimulatory ODN.
Increases in secreted IgM were not as great as the increase
seen in IgM-secreting cells and were on average 2.5- to
5.0-fold increased. There was also an average 5-fold increase
in secreted IgG. Methylation of the stimulatory CpG ODN 74
did not significantly alter its ability to synergize with
anti-IgD-dex to induce IgM secretion but had an effect in
reducing its stimulation of IgG secretion (Fig. 7).
Discussion
The data in this manuscript show that ODN that are poorly
stimulatory can, in the context of a multivalent mIg crosslinking signal, induce high levels of B cell proliferation. Thus,
ODN that by themselves induce low levels of B cell proliferation
either because they do not possess a stimulatory motif or
because the stimulatory motif has been methylated, as seen
in mammalian DNA, can in the presence of anti-IgD-dex
induce high levels of B cell proliferation. Thus, ODN 1082
and 2105 or 74m that either do not contain an optimally
stimulatory motif or contain a stimulatory motif that is methylated respectively, and by themselves induce low levels of
B cell proliferation, induce significant B cell proliferation and
IgM and IgG secretion in the presence of anti-IgD-dex. This
pronounced synergy was not observed when unconjugated
anti-IgD or anti-IgM mAb were used to cross-link the antigen
receptor. Nor was it observed when B cells were cultured in
the presence of trivalent membrane CD40L. Although in
previous studies CD40–CD40L cross-linking has been shown
to enhance antigen induced B cell activation, it is not clear
that it should be synergistic with ODN. The absence in vitro
of a synergistic response to trivalent membrane CD40L does
not preclude the possibility that in vivo this antigen, were it
presented in a different array, as on an activated T cell, may
be synergistic for B cell proliferation. Our data suggest that
antigen cross-linking mediated by stimuli that are monovalent,
bivalent or paucivalent may be incapable of synergizing with
poorly stimulatory alternative DNA motifs that are present in
1698 B cell stimulating activities of ODN of various motifs
Fig. 4. Anti-IgD–dex synergizes with stimulatory and poorly stimulatory non-methylated CpG-containing ODN for B cell proliferation. B cells
from DBA/2 mouse spleen were stimulated with 1.0 µg/ml of ODN 74, 2105 or 1082 in the (a) presence or (b) absence of a fixed (1.0 ng/ml)
or varying dose of anti-IgD–dex. [3H]Thymidine was added 24 h after initiation of culture for an 18 h period and cultures were then harvested
for scintillation spectroscopy. Data are expressed as the mean 6 SE of triplicate cultures and are representative of three similar experiments.
Fig. 5. Anti-IgD–dex synergizes with stimulatory and poorly stimulatory ODN when in either an unmethylated or methylated state for B cell
proliferation. B cells from DBA/2 mouse spleen were stimulated with varying concentrations of ODN 74, 74m, 1082 or 1082m in the presence
or absence of anti-IgD–dex (1.0 ng/ml). [3H]Thymidine was added 24 h after initiation of culture for an 18 h period and cultures were then
harvested for scintillation spectroscopy. Data are expressed as the mean 6 SE of triplicate cultures and are representative of three similar
experiments.
microorganisms and mammals. In contrast, microorganisms
that display multiple and repeating epitopes, as are found on
the viral capsids or on the polysaccharides of encapsulated
organisms, could easily synergize with these poorly stimulatory motifs to induce B cell proliferation and Ig secretion. This
finding could be exploited in using ODN to adjuvant responses
to poorly immunogenic epitopes. Thus, converting a poorly
stimulatory antigen to a multivalent format would increase the
likelihood that it could induce high titer humoral antibody in
the presence of ODN as adjuvants. The fact that there was a
400-fold increase in the number of Ig-secreting cells that was
stimulated indicates that the synergistic stimulation that was
induced in the presence of a low concentration of multivalent
mIg cross-linking and ODN is both at the level of B cell
proliferation and Ig secretion. The discrepancy in the amount
of secreted IgM as compared to the numbers of Ig-secreting
cells may reflect the absence of cytokines or factors in
our in vitro cultures which could enhance the Ig-secretory
response.
We demonstrate here for the first time that even methylated
motifs as are found in mammalian cells can, in the presence
of a multivalent mIg cross-linking stimulus, induce B cell
B cell stimulating activities of ODN of various motifs 1699
proliferation. The observable differences between the methylated and the non-methylated ODN are the ability of the latter
to synergize at concentrations that are ~100-fold lower than
that of the methylated ODN and with concentrations of antiIgD–dex that are lower as well. This suggest that even
mammalian DNA can in an appropriate context of mIg signaling mediate dysregulated B cell activation. In addition to
inducing cellular proliferation, we show that IgM and IgG
secretion are induced by the combination of anti-IgD–dex
and ODN.
We have previously shown that conjugation of anti-IgD to
a high mol. wt dextran produces a conjugate that mimics the
activity of a multivalent mIg cross-linking stimulus and reflects
signals that are induced by T cell-independent antigens (28).
As compared to unconjugated anti-IgD, the anti-IgD–dextran
conjugate stimulates B cells at concentrations that are 1000-
Fig. 6. Anti-IgD–dex synergizes with the CpG-containing ODN 74 for
enhancement of IgM-secreting cells. B cells from CBA.C57BL/6F1
mouse spleen were stimulated with varying concentrations of ODN
74 in the presence (d) or absence (s) of anti-IgD–dex (1.0 ng/ml).
After a 6 day incubation the number of IgM-secreting cells per
106 cultured cells was determined using spot-ELISA as previously
discussed (Methods). Data are expressed as the mean of triplicate
cultures and are representative of two similar experiments.
to 10,000-fold lower. While other investigators have shown
that cross-linking of mIg in the context of stimulatory ODN
induces higher levels of B cell proliferation, this occurred only
at concentrations of ODN which were in and of themselves
stimulatory (1). We show that in the context of multivalent mIg
cross-linking stimuli concentrations of ODN that are 100-fold
less than optimally stimulatory concentrations when used
alone are still able to induce significant synergy. Furthermore,
the level of enhancement that was observed was in the range
of 5- to 10-fold increase above that anticipated from their
additive responses.
Another important difference between the published reports
showing synergistic proliferative responses and the data in
this manuscript is our finding that the concentration of the
mIg cross-linking stimuli required to induce synergy was
10,000-fold lower than that which was reported by others (1).
This suggests that B cell proliferation which is initiated at the
time of an infection by an organism expressing multivalent
epitopes could be sustained for some time after infection
even when the concentration of residual antigen and residual
DNA may be extremely low. It also suggests that B cell
proliferation can be induced at a time distant from the actual
invasion of the microorganism, when antigenic concentrations
are extremely low.
Taken together, these data demonstrate that the range of
ODN motifs that can enhance B cell stimulation is broader
than previously reported. It also supports our earlier findings
that demonstrated that the mode of B cell activation is an
important factor in determining subsequent responsiveness
of the B cell to other stimuli.
Acknowledgments
This work was supported by National Institutes of Health Grant
AI36588 and Walter Reed Army Medical Center, Department of
Clinical Investigation, work unit 3723. Opinions and assertions contained herein are the private ones of the authors and are not to be
construed as official or reflecting the views of the Department of
Defense, Walter Reed Army Medical Center or the Uniformed Services
University of the Health Sciences.
Fig. 7. Anti-IgD–dex enhances the Ig-secretory ability of both unmethylated and methylated ODN. B cells from DBA/2 mouse spleen were
stimulated with either ODN 74 or 74m (1.0 µg/ml) in the presence or absence of anti-IgD–dex (1.0 ng/ml). Culture SN were harvested 6 days
after initiation of culture for measurement of secreted concentrations of IgM and IgG by ELISA. Data are expressed as the mean of triplicate
cultures and are representative of two similar experiments.
1700 B cell stimulating activities of ODN of various motifs
Abbreviations
anti-IgD–dex
CD40L
CpG
LPS
ODN
SN
anti-IgD conjugated to high mol. wt dextran
CD40 ligand
cytosine–guanine dinucleotide
lipopolysaccharide
oligodeoxynucleotide
supernatant
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