Modulation of the Humoral Immune Response by Antibody-Mediated ...

Modulation of the Humoral Immune Response by Antibody-Mediated Antigen Targeting to Complement Receptors and Fc Receptors1 Dana C. Baiu,*† Jozsef Prechl,*‡ Andrey Tchorbanov,* Hector D. Molina,§ Anna Erdei,‡ Andrei Sulica,† Peter J. A. Capel,2* and Wouter L. W. Hazenbos3* During an ongoing immune response, immune complexes, composed of Ag, complement factors, and Igs, are formed that can interact with complement receptors (CRs) and IgG Fc receptors (FcgR). The role of CR1/2 and FcgR in the regulation of the immune response was investigated using OVA that was chemically conjugated to whole IgG of the rat anti-mouse CR1/2 mAb 7G6. FACS analysis using the murine B cell lymphoma IIA1.6 confirmed that the 7G6-OVA conjugate recognized CR1/2. Incubating IIA1.6 cells with 7G6-OVA triggered tyrosine phosphorylation and Ag presentation to OVA-specific T cells in vitro. Immunizing mice with 7G6-OVA at a minimal dose of 1 mg i.p. per mouse markedly enhanced the anti-OVA Ig response, which was primarily of the IgG1 isotype subclass. The 7G6-OVA did not enhance the anti-OVA response in CR1/2-deficient mice. OVA coupled to an isotype control Ab induced a considerably lower anti-OVA response compared with that induced by OVA alone, suggesting inhibition by interaction between the Fc part of the Ab and the inhibitory FcgRIIb on B cells. This finding was supported by the observation that IIA1.6 cells which were incubated with 7G6-OVA lost the ability to present Ag upon transfection with FcgRIIb. In sum, 7G6-conjugated OVA, resembling a natural immune complex, induces an enhanced anti-OVA immune response that involves at least CR1/2-mediated stimulation and that may be partially suppressed by FcgRIIb. The Journal of Immunology, 1999, 162: 3125–3130.

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he generation of an immune response is controlled by various humoral and cellular components of the innate and acquired immune system. Immediately upon entering the body, foreign material can activate complement leading to the deposition of C3 products, which in turn allows interaction with complement receptors (CRs),4 initiating cellular responses. CRs type 1 and 2 (CR1 and CR2), which are expressed on B cells and follicular dendritic cells in the mouse (1) as alternatively spliced products of the single Cr2 locus (2, 3), recognize activated C3 and C4 products (4, 5). Various in vitro and in vivo studies support the important role of these surface receptors in the regulation of the humoral immune response (reviewed in Ref. 6). Co-cross-linking CR1/2 and the Ag receptor reduces the threshold for activation of B cells in vitro (7, 8). The humoral response to T cell-dependent or -independent Ags can be strongly reduced by treating mice with the anti-CR1/2 mAb 7G6 (9, 10) or with soluble CR2 (11) before *Department of Immunology, University Hospital Utrecht, Utrecht, The Netherlands; † Bucharest Center of Immunology, Bucharest, Rumania; ‡Department of Immunology, L. Eo¨tvo¨s University, Budapest, Hungary; and §Division of Rheumatology, Washington University School of Medicine, St. Louis, MO 63110 Received for publication August 12, 1998. Accepted for publication December 4, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the Copernicus Fixed Contribution Contract CIPA CT 94-0152, FKFP 0102 grant, and the Hungarian National Science Fund (OKTA) 017158. 2 Address correspondence and reprint requests to Dr. Peter J. A. Capel, Department of Immunology, University Hospital Utrecht, G04.614, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. 3 Current address: Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.

Abbreviations used in this paper: CR, complement receptor; FcgR, IgG Fc receptor; SATA, N-succinimidyl S-acetylthioacetate; mIg, membrane Ig.

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Copyright © 1999 by The American Association of Immunologists

immunization. Mice that are deficient in CR1 and CR2, which were generated by targeted disruption of the Cr2 gene, mount impaired humoral responses to T cell-dependent Ags (12, 13). Evidence for the potent immunoregulatory activity of complement has been provided using a recombinant fusion protein composed of C3d and a model Ag, which was highly immunogenic in mice, indirectly suggesting a role for CR1/2 in this process (14). Consistent with that finding are results obtained using mice deficient in C3 and C4, which exhibited defective Ab responses against T celldependent Ags (15). After the first contact with the innate immune system and the initiation of the production of Ag-specific Igs, foreign material can also be recognized by the acquired immune system and thus interact with receptors for the Fc part of IgG (FcgR). B cells express FcgRIIb, which contains an immunoreceptor tyrosine-based inhibitor motif that is involved in the down-regulation of B cell functions (reviewed in Ref. 16). FcgRIIb transfected into an FcgRnegative B cell line potently down-regulates B cell activation upon co-cross-linking with surface IgG (17). Mice deficient in FcgRIIb exhibit enhanced Ig production against T cell-dependent and -independent Ags (18). In the present study, we directly targeted Ag to CR1/2 using a complex consisting of OVA chemically conjugated to anti-CR1/2 Abs. The conjugate was generated using intact IgG of the rat antiCR1/2 mAb 7G6, allowing direct interaction not only with CR1/2 but also with FcgR, thus resembling a natural immune complex consisting of Ag coated with both complement and IgG. To address the relative contributions of CR1/2 and FcgR in the immune response, an isotype-matched IgG control, FcgR-transfected IIA1.6 cells (an FcgR-negative murine B lymphoma cell line) and CR1/2-deficient mice were used for in vitro and in vivo studies. We found that conjugation of 7G6 to OVA strongly enhanced the anti-OVA Ab response, and that this enhancement was dependent 0022-1767/99/$02.00

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REGULATION OF THE Ab RESPONSE BY COMPLEMENT RECEPTORS AND Fc RECEPTORS

upon interaction of the conjugate with CR1/2. We also found evidence suggesting that interaction of the complex with FcgRIIb could partially down-regulate this response.

Materials and Methods Cell cultures and Abs The murine B cell lymphoma line A20 (19) and its FcgR-negative variant IIA1.6 (both expressing CR1 and CR2) as well as the Th OVA-specific hybridoma 3DO-54.8 (20) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM glutamine, and 1 mM sodium pyruvate. Unless stated otherwise, cells were suspended in the same medium during the in vitro assays. FcgR transfectants of the IIA1.6 cell line were maintained in the same medium supplemented with Geneticin (G418, 0.8 mg/ml; Life Technologies, Paisley, U.K.) or with Geneticin and methotrexate (10 mM; Pharmachemie, Haarlem, The Netherlands). CTLL-L16 cells were cultured in RPMI 1640 medium supplemented with 100 U/ml rIL-2. Rat hybridoma 7G6 (21) (kindly provided by Dr. T. Kinoshita, Osaka University, Osaka, Japan) secreting IgG2b directed to murine CR1 and CR2 was cultured on a large scale in serum free Iscove’s modified Eagle’s medium (Life Technologies). SHL45.6 (22) purified rat IgG2b mAb specific for human CD3 was used as an isotype control (kindly provided by Dr. M. Clark, Cambridge University, Cambridge, U.K.).

Preparation of IgG-OVA conjugates Ab-OVA conjugates were prepared using N-succinimidyl S-acetylthioacetate (SATA) (purchased from Pierce, Rockford, IL) as a chemical crosslinker (reviewed in Ref. 23). Briefly, IgG at a concentration of $5 mg/ml suspended in a 50 mM sodium phosphate buffer containing 1 mM EDTA (pH 7.5) was incubated with a 15-fold molar excess of SATA for 30 min at room temperature. The solution was dialyzed extensively against the same phosphate buffer to remove the excess of unbound SATA and subsequently incubated with a 10-fold smaller volume of 0.5 M hydroxylamine HCl (Pierce) in 50 mM sodium phosphate and 25 mM EDTA (pH 7.5) for 2 h at room temperature. Next, the solution was diluted four times in 0.1 M sodium phosphate, 0.15 M NaCl, and 0.1 M EDTA (pH 7.2) containing maleimide-activated OVA (Pierce), giving a molar IgG:OVA ratio of 1:1.5. The coupling reaction was allowed to proceed for 90 min at room temperature and was stopped by the addition of 2-ME at a final concentration of 10 mM. Conjugates were subsequently purified from unconjugated proteins by fast protein liquid chromatography using a HiLoad Superdex 200 HR column (Pharmacia LKB Biotech, Uppsala, Sweden) equilibrated with PBS. Fractions containing molecules with an estimated molecular mass ranging from 200 to 573 kDa (based on the retention time of molecular mass markers) were pooled and used for the Ag presentation and immunization experiments. The concentration of the pooled fractions was estimated by measuring the absorbance at 280 nm, and small aliquots were immediately frozen for long-term storage. Conjugates were analyzed by 6% SDS-PAGE under nonreducing conditions.

Flow cytometry The murine B lymphoma cell line IIA1.6 was used to analyze the capacity of 7G6-OVA conjugates to bind CR1/2 by flow cytometry. IIA1.6 cells were washed twice and suspended at a concentration of 2 3 107 cells/ml of PBS containing 2.5% FCS and 0.05% sodium azide. The cells were then incubated with nonconjugated 7G6 or with 7G6-OVA conjugates at different concentrations for 30 min at 4°C, followed by two washes. Next, the cells were incubated either with FITC-conjugated mouse anti-rat IgG conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA) or with polyclonal rabbit anti-OVA IgG (Cappel, Durham, NC) followed by incubation with FITC-conjugated goat anti-rabbit IgG; each incubation was performed for 30 min at 4°C. The cells were then washed twice and analyzed by flow cytometry.

Tyrosine phosphorylation assay IIA1.6 cells, at a concentration of 2 3 107 cells/ml, were incubated with 10 mg/ml 7G6-OVA or with the same concentration of SHL45.6-OVA for 20 min at 4°C. Next, the cells were washed twice with cold serum-free RPMI 1640 medium and resuspended in aliquots of 20 ml containing 5 3 105 cells. The cells were then incubated in a waterbath at 37°C, and the reaction was stopped at various time periods by adding reducing sample buffer (v/v) containing 8% sodium lauryl sulfate, 20% 2-ME, and 1 mM sodium orthovanadate. After denaturing the samples by boiling, lysed cells were loaded onto a 10% SDS-polyacrylamide gel (2.5 3 105 cells/lane), and the proteins were subsequently electrotransferred to polyvinylidene difluoride

Immobilon-P membranes (Millipore, Bedford, MA). The membranes were washed in PBS containing 0.1% Tween 20 (PBS-Tween), blocked with 1% BSA in PBS-Tween, and probed with 0.8 mg/ml of the antiphosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY) for 1.5 h at room temperature. After washing three times with PBS-Tween, membranes were incubated with 0.5 mg/ml peroxidase-labeled rabbit anti-mouse Ig (Dako A/S, Glostrup, Denmark); bound Abs were detected using the enhanced chemiluminescence system (Amersham, Buckinghamshire, U.K.).

Ag presentation in vitro The effect of the interaction of Ab-Ag complexes with CR1/2 or FcgR on the ability of IIA1.6 and A20 cells to present OVA Ag to OVA-specific Th cells was studied using 7G6-conjugated OVA and OVA conjugated to the isotype control SHL45.6. The IIA1.6 or A20 cells, at a concentration of 2 3 105 cells/ml, were incubated with OVA-specific T cell hybridoma (2 3 104 cells/ml) for 24 h at 37°C in the presence of various concentrations of Ag ranging from 1 to 300 ng/ml. As a positive control for T cell reactivity, high concentrations of free OVA (#30 mg/ml) were used. As negative controls, all experiments were also performed in the absence of T cells, IIA1.6 cells, or Ag. The release of IL-2 by the Th cells in the culture supernatants, as a measure of efficient Ag presentation, was determined using a CTLL proliferation assay (17). CTLL-16 cells are T cells that are derived from C57BL/6 mice and that proliferate in an IL-2-dependent fashion. Briefly, supernatants were incubated with CTLL-16 for 24 h at 37°C; afterward, [3H]thymidine was added in a concentration of 1 mCi/well. After 4 h of incubation, cells were lysed in water onto glass fiber filters (Wallac, Turku, Finland), and incorporated radioactivity was measured in a scintillation counter.

Immunization protocol For immunization experiments, female BALB/c of ;8 wk of age were used. In some experiments, female CR1/2-deficient mice (12)A. A.B. B. were used; mice with a mixed genetic background of C57BL/6 3 129Sv were used as wild-type controls. Two i.p. injections with similar doses were administered in five mice per group over 26-day intervals using various doses of Ag per mouse as indicated. In some cases, mice were injected with a mixture of OVA and an equal volume of CFA (Difco Laboratories, Detroit, MI), followed by a booster injection of the same concentration of OVA in IFA (Difco). The mice were bled at 3 wk after the first immunization and at 1 wk and 3 wk after the second immunization for analysis of the serum anti-OVA response.

ELISA for serum anti-OVA Ab OVA was coated onto the surfaces of 96-well Maxisorp immunoplates (Nunc, Roskilde, Denmark) by overnight incubation with 10 mg OVA per ml PBS at 4°C. The plates were washed and blocked with PBS containing 1% BSA for 1 h at room temperature, followed by two washes. Next, the plates were incubated with serial dilutions of sera of immunized mice or of preimmune serum as a control. After two washes, the plates were incubated with alkaline phosphatase-conjugated goat anti-mouse Ig(H1L) (Southern Biotechnology Associates, Birmingham, AL) to determine total Ig concentrations or with alkaline phosphatase-conjugated goat Ab specific for mouse IgG1, IgG2a, or IgG2b (Southern Biotechnology Associates) to determine the isotype subclass of the responses. The plates were washed twice and developed using p-nitrophenyl phosphate substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Titers were determined by calculating the dilution of each serum giving an absorbance at 405 nm that was twice that of preimmune serum.

Results Binding of 7G6-OVA to CR1/2 in vitro The ability of the 7G6-OVA conjugate to bind to CR1/2 was determined by flow cytometry using the CR1/2-expressing murine B lymphoma cell line IIA1.6. After incubation of these cells with 10 mg/ml 7G6-OVA and subsequently with an FITC-conjugated mouse anti-rat Ab, a positive fluorescence was observed (Fig. 1b); this fluorescence was within the same range when unconjugated 7G6 instead of the whole conjugate was used (Fig. 1a). In addition, at a lower concentration of 1 mg/ml 7G6-OVA, a significant fluorescence was detected that was comparable with that seen when using nonconjugated 7G6 (data not shown). Cell-bound 7G6-OVA could be detected using a polyclonal rabbit anti-OVA Ab (Fig. 1d),

The Journal of Immunology

FIGURE 1. Binding of 7G6-conjugated OVA to IIA1.6 cells. Suspensions of IIA1.6 cells were incubated first with nonconjugated 7G6 Abs (a and c) or with 7G6-OVA conjugate (b and d), followed by a second incubation with FITC-labeled mouse anti-rat Ab (a and b) or with rabbit IgG anti-OVA combined with FITC-labeled goat anti-rabbit Ab (c and d). The fluorescence of the cells was analyzed by flow cytometry. Solid lines represent cells that were incubated with both the first and the secondary reagents; dotted lines represent control cells incubated with the secondary Abs only.

which did not recognize cell-bound unconjugated 7G6 (Fig. 1c). These results indicate that the 7G6-OVA conjugate retained its capacity to bind CRs and confirm the presence of OVA in the conjugate. Tyrosine phosphorylation Next, we questioned whether bound conjugates can initialize an intracellular response in murine B cells. The pattern of activation following the binding of 7G6-OVA to CRs on IIA1.6 cells was investigated by the detection of protein phosphorylation on tyrosine residues. Strong phosphorylation of two protein bands of an estimated molecular mass of 110 kDa and 74 kDa was detectable after 10 min of incubation at 37°C of cells treated with 7G6-OVA, showing induction of an intracellular signaling response (Fig. 2).

FIGURE 2. Induction of tyrosine phosphorylation in IIA1.6 cells by 7G6-OVA. IIA1.6 cells were incubated with 7G6-OVA (left panel) or with SHL45.6-OVA (right panel) for 20 min at 4°C, washed, and incubated at 37°C for various time periods (0.5, 1, 5, and 10 min). Cells treated with medium alone (med) served as a negative control, revealing basal protein phosphorylation. The cells were lysed, and proteins were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane, which was probed with anti-phosphotyrosine mAb. Results representative of three similar experiments are shown.

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FIGURE 3. CR1/2-mediated presentation of OVA by B lymphocytes to OVA-specific Th cells. Nontransfected or transfected IIA1.6 cells were incubated with OVA-specific Th cells for 24 h at 37°C in the presence of various concentrations of 7G6-OVA (f), isotype-matched SHL45.6-OVA (E), or free OVA (Œ). The different panels depict OVA presentation by nontransfected IIA1.6 cells (a), IIA1.6 cells transfected with human FcgRIa/g-g (b), A20 cells (c), or IIA1.6 cells transfected with murine FcgRIIb1 (d). The release of IL-2 by the Th cells in the culture supernatants, as determined using a CTLL proliferation assay, was used as a measure for Ag presentation efficiency. Results are expressed as relative units of IL-2 per ml. Mean values of triplicate samples of one representative experiment of four are shown.

This phosphorylation pattern is not visible after the incubation of cells with an isotype-matched control conjugated to OVA (Fig. 2), attesting that the binding of 7G6-conjugated OVA to CRs is responsible for the phosphorylation of the proteins mentioned. Control experiments with purified Ab 7G6 alone showed a similar pattern of phosphorylation of 110 kDa and 74 kDa protein bands (data not shown), indicating that conjugation of 7G6 with OVA is basically not modifying the signaling pattern of the targeted receptors. Ag presentation in vitro The ability of CRs to modulate the efficiency of Ag presentation in vitro was studied using 7G6-conjugated OVA, IIA1.6 cells, and an OVA-specific T cell clone. When IIA1.6 cells were incubated with 7G6-OVA, efficient Ag presentation to OVA-specific T cells was observed (Fig. 3a). Free OVA induced detectable Ag presentation by IIA1.6 cells only when using concentrations that were 50- to 100-fold higher (#15 mg/ml) (Fig. 3a). OVA conjugated with the isotype control Ab SHL45.6 could not be presented by IIA1.6 cells (Fig. 3a), indicating that the observed effect of 7G6-OVA was specific for CR1/2. As a positive control, IIA1.6 cells transfected with human FcgRI were incubated with SHL45.6-OVA, which resulted in efficient Ag presentation (Fig. 3b), confirming the ability of this conjugate to interact with FcgR leading to the presentation of OVA epitopes. The Ag presentation by nontransfected IIA1.6 cells that was induced by 7G6-OVA could be blocked completely by the addition of free 7G6 but not by CR1-specific mAb 8C12 (data not shown), suggesting that this process involved at least CR2. These results indicate that OVA can be presented efficiently by B cells when conjugated to 7G6, and that this process involves at least CR2 expressed on these cells. Next, murine FcgRIIb-transfected IIA1.6 cells were used to investigate whether an additional interaction of the 7G6-OVA conjugate with FcgRIIb on B cells would interfere with Ag presentation. Ag presentation by IIA1.6 cells, when incubated with 7G6-OVA, was completely inhibited upon transfection with FcgRIIb (Fig. 3d). This finding was supported by the inability of the natively

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FIGURE 4. Primary and secondary anti-OVA Ab response induced by immunization with 7G6-conjugated OVA. Mice were injected i.p. twice (at 26-day intervals) with 5 mg of 7G6-OVA (E), nonconjugated OVA (‚), nonconjugated OVA in CFA (M), or with saline (3). Results are expressed as titers of anti-OVA Ig in the serum at 3 wk after the first immunization and at 1 and 3 wk after the second immunization. Mean values of five mice are shown. Arrows indicate days of injection.

FcgRIIb-expressing B cell line A20 to present Ag after incubation with 7G6-OVA (Fig. 3c). These results imply that interaction of an Ag-Ab complex with FcgRIIb on B cells inhibits CR-mediated enhancement of Ag presentation by these cells. Immunization with 7G6-OVA conjugate Next, the effect of targeting OVA to CR1/2 and FcgR on the in vivo response was investigated. When normal BALB/c mice were immunized with 5 mg 7G6-OVA conjugate, the anti-OVA Ab response was markedly enhanced compared with the response after immunization with free OVA (Fig. 4). The enhancing effect on the secondary response was most pronounced (Fig. 4). The effect of the 7G6-OVA conjugate on the Ab response was obvious when using doses of $1 mg per mouse (Fig. 5). The anti-OVA Ig response induced by 7G6-OVA was primarily of the IgG1 isotype subclass (Fig. 6). No significant enhancement was observed after immunization with maleimide-activated OVA (data not shown). When mice were immunized with OVA in CFA as a positive control, the primary and secondary anti-OVA responses were strongly enhanced (Fig. 4). Immunization with the 7G6-OVA conjugate had no effect in CR1/2-deficient mice (Fig. 7); however, this conjugate did enhance the anti-OVA response in wild-type controls of a similar genetic background (data not shown). As a positive control, CR1/2-deficient mice responded strongly to OVA in CFA (Fig. 7); the response was within the same range as that seen for wild-type control mice (data not shown). When mice were immunized with SHL45.6-OVA conjugate as an isotype control, the anti-OVA response was considerably lower compared with immunization with free OVA (Fig. 5), raising the possibility that the response was down-regulated by interaction of this conjugate with the inhibitory FcgRIIb on B cells. This observation was supported by in vitro experiments showing that transfection of IIA1.6 cells with FcgRIIb inhibited the presentation of OVA epitopes to OVA-specific T cells after incubation with 7G6-

FIGURE 5. Dose-dependency of the secondary anti-OVA Ab response after two immunizations with Ab-conjugated OVA. Mice were injected i.p. twice (at 26-day intervals) with various doses of 7G6-OVA (E), nonconjugated OVA (‚), or SHL45.6-OVA (M). Results are expressed as titers of anti-OVA Ig in the serum at 3 wk after the second immunization; mean values of five mice are shown.

OVA (Fig. 3d). Taken together, these results indicate that coupling OVA to the anti-CR Ab 7G6 strongly enhances the humoral immune response to OVA, and that this enhancement is dependent upon CR1/2. Furthermore, the interaction of such an Ag-Ab complex with FcgRIIb may suppress the Ab response.

Discussion The present results demonstrate that the Ab-mediated targeting of Ag to CR1/2 using OVA complexed to anti-CR1/2 mAb (7G6)

FIGURE 6. Isotype specificity of the anti-OVA response induced by immunization with 7G6-OVA. Mice were injected i.p. twice (at 26-day intervals) with 5 mg of nonconjugated OVA (ova), 7G6-OVA (7G6-ova), or OVA in CFA (ova 1 CFA). Results are expressed as titers of IgG1 (hatched bars) or IgG2a (filled bars) anti-OVA at 3 wk after the second immunization; mean values of five mice are shown.

The Journal of Immunology

FIGURE 7. Anti-OVA response in CR2-deficient mice induced by 7G6OVA. CR2-deficient (CR2-KO) mice were injected i.p. twice (at 26-day intervals) with 5 mg of 7G6-OVA (E), nonconjugated OVA (‚), nonconjugated OVA in CFA (M), or with saline (3). Results are expressed as titers of anti-OVA Ig in the serum at 3 wk after the first immunization and at 1 and 3 wk after the second immunization. Mean values of five mice are shown. Arrows indicate days of injection.

enhances the humoral immune response against this Ag. Interaction of these Ag-Ab complexes with FcgRIIb may partially suppress this response. Several lines of evidence support the above conclusions. First, incubation of the FcgR-negative B lymphoma cell line IIA1.6 with 7G6-conjugated OVA resulted in the efficient presentation of OVA epitopes to an OVA-specific T cell line in vitro. This process was dependent upon interaction of the complex with CR1/2, because SHL45.6-OVA as an isotype-matched control had no effect. Second, the anti-OVA Ab response was markedly enhanced when mice were immunized with 7G6-OVA but not after immunization with SHL45.6-OVA. The enhanced anti-OVA response was dependent upon interaction of the 7G6-OVA complex with CR1/2, because such a response was not observed in CR1/2deficient mice. These mice did respond to OVA in CFA, indicating they are able to mount an anti-OVA Ab response depending upon the method used to stimulate the immune system. The 7G6-OVAmediated enhancement was most pronounced during the secondary response, suggesting the involvement of memory B cells or Th cells. The observation that the enhanced response induced by 7G6OVA was primarily of the IgG1 subclass suggests that targeting to CR1/2 preferentially triggers Th2 responses, following the same isotype specificity compared with OVA alone. The enhanced antiOVA response in mice immunized with 7G6-OVA was accompanied by a strong IgG response against rat IgG (data not shown), suggesting efficient presentation of Ags in both parts of the complex. Mice immunized with the isotype control SHL45.6-OVA, which hardly raised an anti-OVA response, also mounted only a weak specific anti-rat IgG response. Treating mice with 7G6-OVA did not increase the total Ig concentration in the serum (W.L.W.H., unpublished observations), indicating that the enhancement was not associated with polyclonal B cell activation. The present results, based on the direct targeting of Ag to CR1/2, are in agreement with the observation reported previously that coupling of a model Ag to C3d, a ligand for CR1/2, enhances the immunogenicity of this Ag (14).

3129 Our results showed that the targeting of Ag to CR1/2 on IIA1.6 cells mediates efficient Ag presentation to specific Th clones without the requirement of OVA-specific membrane Ig (mIg). This correlated with the ability of IIA1.6 cells to generate and transduce intracellular responses consisting of the phosphorylation of cellular proteins when triggered with 7G6-conjugated OVA. However, treating IIA1.6 cells with 7G6-OVA did not induce an increase in [Ca21]i, modifications in cell cycle (proliferation, apoptosis), secretion of IL-4, or expression of MHC class II (D.C.B. and W.L.W.H., unpublished observations). In contrast, it has been reported that the co-cross-linking of CR1/2 with mIg on B cells does result in an increase in [Ca21]i (7). Thus, CR1/2-mediated Ag presentation can occur independently of mIg via an as yet unknown mechanism, which at least does not lead to a general pattern of B cell activation. One possibility may be provided by a recent report showing that the cross-linking of CR1/2 on murine splenic B cells enhances the expression of B7-1 and B7-2, which can provide costimulatory signals to T cells, thereby possibly contributing to enhanced Ag presentation (24). The significance of this restricted stimulatory effect could be related to lowering the threshold of required help from Ag-specific Th cells in early stages of immune response. The precise mechanism by which 7G6-mediated targeting of Ag to CR1/2 leads to an enhanced Ab response is currently unknown. The following possibilities can be envisaged. First, binding of the 7G6-OVA complex to CR1/2 may increase the local Ag concentration at the surface of Ag-specific B cells, facilitating the interaction between Ag and its receptor. Second, 7G6-OVA may induce cross-linking between CR1/2 and the Ag receptor on OVAspecific B cells, resulting in enhanced intracellular signaling and/or Ag internalization. Previously reported data indicated that human CR2 is an amplifier of low intracellular signaling through the B cell receptor, by showing synergistic increases in [Ca21]i following cross-linking CR2 to mIgM at suboptimal activation doses for the triggering of mIgM on B cells (7). Theoretically, it is possible that the binding of 7G6-OVA to CR1/2 provides an additional stimulus by locally activating complement. Third, interaction between the 7G6-OVA and CR1/2 expressed on Ag-nonspecific B cells may also lead to Ag presentation, thereby contributing to Ab production. This possibility is supported by our observation that 7G6-OVA induces tyrosine phosphorylation and Ag presentation by IIA1.6 cells, which do not recognize OVA via their surface mIg. Finally, CR2 that is expressed on the surface of follicular dendritic cells and is involved in the retention of complementcoated Ag and the maintenance of long-term B cell memory (25) may contribute to the effect of 7G6-mediated targeting. In separate experiments, we have injected mice with nonconjugated 7G6 Abs 24 h before immunization with free OVA. In these mice, the primary anti-OVA Ab response was inhibited by 80% and the secondary response by 30% compared with control mice injected with buffer before immunization with OVA (D.C.B. and W.L.W.H., unpublished observations). These results are in agreement with previous reports (9, 10) showing that the down-modulation of CR1/2 by pretreatment of mice with 7G6 before immunization with keyhole limpet hemocyanin or FITC-haptenated bacteria strongly inhibits Ab production. These findings support the crucial role of CR1/2 during a normal immune response against a protein Ag. Remarkably, when mice were immunized with OVA conjugated to SHL45.6, an irrelevant isotype control, the anti-OVA response was impaired compared with immunization with nonconjugated OVA, suggesting inhibition of the response. The possibility that this inhibition was caused by interaction of the conjugate with the

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down-regulatory FcgRIIb on B cells was supported by the observation that transfection with FcgRIIb rendered IIA1.6 cells, incubated with 7G6-OVA, unable to present OVA epitopes in vitro. This result was confirmed by the inability of A20 cells, the original cell line from which IIA1.6 is derived and which naturally expresses FcgRIIb, to present Ag after treatment with 7G6-OVA. Thus, it is conceivable that after immunization with 7G6-OVA, which allows interaction with CR1/2 and FcgR, the immune response is the result of stimulation via CR1/2 and suppression via FcgRIIb. In addition to the down-regulatory effects of FcgRIIb, interaction of an Ag with FcgR can also have stimulatory effects on the immune response. For example, in vitro experiments using FcgRtransfected cells have shown that the IgG-mediated interaction of Ag with either murine FcgRII or FcgRIII can induce presentation to Ag-specific T lymphocytes (26). In addition, using mice expressing transgenic human FcgRI, it has been demonstrated that Ag targeting to this receptor strongly enhances the Ab response (27). This observation is consistent with our present finding that transfecting IIA1.6 cells with human FcgRI enhanced 7G6-OVAinduced Ag presentation. In contrast to nontransfected IIA1.6 cells, these cells were able to present Ag after treatment with the isotype control SHL45.6-OVA. However, two lines of evidence indicated that interaction with FcgR was not responsible for the enhanced in vivo Ab production induced by 7G6-OVA. First, conjugation of OVA to the isotype control Ab SHL45.6 resulted in suppression rather than enhancement of the anti-OVA response. Second, the finding that immunizing CR1/2-deficient mice with 7G6-OVA did not result in an enhanced response indicates that FcgR alone are not sufficient to mediate the enhancement induced by 7G6-OVA. The results of the present study provide a better insight into the mechanism of the generation of the Ab response, which is under control of both the innate and the acquired immune system during a second contact of the organism with an Ag. The intensity and duration of the Ab response induced by a complement- and IgGcontaining immune complex may be controlled by a balance between stimulation via CR1/2 and down-regulation via FcgRIIb. Most likely, stimulation of an immune reaction by CR1/2, which depends upon the presence of activated complement factors in the Ag complex, has an important function during the early phase of the response. At a later stage, dampening of the response would be required to prevent uncontrolled cellular reactivity. This shift in the balance toward down-regulation may be mediated by an increasing concentration of IgG which is induced after the onset of the response; this IgG is able to interact with FcgRIIb. The present data are relevant for the rational development of new vaccine strategies. Targeting Ag to CR1/2 may provide the adjuvant effect necessary for the generation of an efficient immune response against various Ags.

Acknowledgments We thank Dr. M. Glennie (University of Southampton, Southampton, U.K.) for essential suggestions concerning the Ab-OVA coupling procedure; Dr. T. Kinoshita (Osaka University, Osaka, Japan) for providing the 7G6 hybridoma; Dr. M. Clark (Cambridge University, Cambridge, U.K.) for providing purified SHL45.6 Abs; and Dr. C. Bonnerot (Institute Curie, Paris, France) and Dr. I. van den Herik-Oudijk and M. van Vugt (University Hospital Utrecht, Utrecht, The Netherlands) for providing the FcgRtransfected cell lines. We also thank P. van Kooten for help with mAb production, P. Aerts for consultation on fast protein liquid chromatography analysis, and A. van der Sar for animal care.

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