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

1 Departments of Medicine and
2 Pathology, and
3 Biomedical Instrumentation Center, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA
4 Rheumatology Service, Department of Medicine, Walter Reed Army Medical Center, Washington, DC 20307, USA
5 Medical Research Service, Durham Veterans Administration Hospital, Division of Immunology, Division of Rheumatology and Immunology, Duke University Medical Center, Durham, NC 27710, USA
6 Retroviral Immunology Section, Center for Biologics Evaluation, Food and Drug Administration, Bethesda, MD 20892, USA

Correspondence to: B. E. Goeckeritz


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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 5' purines and two 3' pyrimidines induce high levels of B cell activation, while ODN whose CpG are methylated or flanked by sequences other than the optimal two 5' purines and two 3' 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 multi-epitope-expressing microorganism in the context of mammalian (methylated) or microorganism (non-methylated) DNA can lead to dysregulated B cell proliferation and Ig secretion.

Keywords: antibodies, B lymphocytes, cellular activation, CpG, oligodeoxynucleotides, rodent, spleen


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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-{alpha} production (28). 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 5' end by two purines and on the 3' 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-{kappa}B-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 (1315). 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, anti-IgD–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
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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 (CBAxC57B16) 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 (5'-AAAAAAAAAAAAAACGTTAAAAAAAAAAA-3'), 1082 (5' GCCGAGGTCCATGTCGTACGC-3') and 2105 (5'-TTGCTTCCATCTTCCTCGTC-3'). 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 (5'-GCXGAGGTCCATGTXGTAXGC-3', 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 in the oligonucleotide was accomplished using 5-Me-dC-ß cyanoethyl-diisopropyl phosphoramidite (Cruachem, Dulles, VA). This phosphoramidite delivers a methylated dC at any site (other that the terminal 3' 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 30-H12) (18) and anti-CD4 (clone GK 1.5) (19), were grown as ascitic fluid in nude mice. The mouse mAb against rat IgG {kappa} chain, MAR 18.5 (20), was produced by cells grown in tissue culture. The mAb H{delta}a/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 H{delta}a/1 mAb to dextran was done as described (23) and the H{delta}a/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 {kappa} 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 goat-anti-mouse {kappa} 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-chloro-indolyl-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
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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 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. 1Go). 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. 2Go). 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 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.



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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 ± 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).

 


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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 anti-IgD–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 ± 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).

 
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 cross-linking 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. 3Go). 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 anti-IgM at concentrations 1000-fold greater than was required with anti-IgD–dex (data not shown). These results demonstrate 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 cross-linking 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.



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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 ± SE of triplicate cultures and are representative of three similar experiments.

 
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. 4aGo). With lower concentrations of anti-IgD–dex significant synergy was seen only with the stimulatory but not with the non-stimulatory ODN (Fig. 4bGo). To determine whether bivalent mIg cross-linking agonists would synergize with the poorly stimulatory ODN 1082 and 2105, anti-IgD and anti-IgM 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. 5Go). 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 CpG motifs, many motifs that have been described as non-stimulatory can in fact synergize to induce B cell proliferation.



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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 ± SE of triplicate cultures and are representative of three similar experiments.

 


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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 ± SE of triplicate cultures and are representative of three similar experiments.

 
Anti-IgD–dex enhances the Ig-secretory ability of both unmethylated and methylated ODN
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 7GoGo). 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. 7Go).



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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 (•) or absence ({circ}) 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.

 


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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.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The data in this manuscript show that ODN that are poorly stimulatory can, in the context of a multivalent mIg cross-linking 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 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 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 anti-IgD–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- 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.


    Abbreviations
 
anti-IgD–dexanti-IgD conjugated to high mol. wt dextran
CD40LCD40 ligand
CpGcytosine–guanine dinucleotide
LPSlipopolysaccharide
ODNoligodeoxynucleotide
SNsupernatant

    Notes
 
Transmitting editor: Z. Ovary

Received 25 May 1999, accepted 8 July 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Krieg, A. M., Yi, A., Matson, S., Waldschmidt, T. J., Bishop, G. A., Teasdale, R., Koretzky, G. A. and Klinman, D. M. 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374:546.[ISI][Medline]
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