Receptors Targeting to Complement Receptors and Fc Response By

Modulation of the Humoral Immune Response by Antibody-Mediated Antigen Targeting to Complement Receptors and Fc Receptors This information is current as of September 25, 2021. Dana C. Baiu, Jozsef Prechl, Andrey Tchorbanov, Hector D. Molina, Anna Erdei, Andrei Sulica, Peter J. A. Capel and Wouter L. W. Hazenbos J Immunol 1999; 162:3125-3130; ; http://www.jimmunol.org/content/162/6/3125 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 (Fc␥R). The role of CR1/2 and Fc␥R 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 Downloaded from mice with 7G6-OVA at a minimal dose of 1 ␮g 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 Fc␥RIIb 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 Fc␥RIIb.

In sum, 7G6-conjugated OVA, resembling a natural immune complex, induces an enhanced anti-OVA immune response that http://www.jimmunol.org/ involves at least CR1/2-mediated stimulation and that may be partially suppressed by Fc␥RIIb. The Journal of Immunology, 1999, 162: 3125–3130.

he generation of an immune response is controlled by immunization. Mice that are deficient in CR1 and CR2, which various humoral and cellular components of the innate were generated by targeted disruption of the Cr2 gene, mount im- T and acquired immune system. Immediately upon entering paired humoral responses to T cell-dependent Ags (12, 13). Evi- the body, foreign material can activate complement leading to the dence for the potent immunoregulatory activity of complement has deposition of C3 products, which in turn allows interaction with been provided using a recombinant fusion protein composed of by guest on September 25, 2021 complement receptors (CRs),4 initiating cellular responses. CRs C3d and a model Ag, which was highly immunogenic in mice, type 1 and 2 (CR1 and CR2), which are expressed on B cells and indirectly suggesting a role for CR1/2 in this process (14). Con- follicular dendritic cells in the mouse (1) as alternatively spliced sistent with that finding are results obtained using mice deficient in products of the single Cr2 locus (2, 3), recognize activated C3 and C3 and C4, which exhibited defective Ab responses against T cell- C4 products (4, 5). Various in vitro and in vivo studies support the dependent Ags (15). important role of these surface receptors in the regulation of the After the first contact with the innate immune system and the humoral immune response (reviewed in Ref. 6). Co-cross-linking initiation of the production of Ag-specific Igs, foreign material can CR1/2 and the Ag receptor reduces the threshold for activation of also be recognized by the acquired immune system and thus in- B cells in vitro (7, 8). The humoral response to T cell-dependent or teract with receptors for the Fc part of IgG (Fc␥R). B cells express -independent Ags can be strongly reduced by treating mice with Fc␥RIIb, which contains an immunoreceptor tyrosine-based inhib- the anti-CR1/2 mAb 7G6 (9, 10) or with soluble CR2 (11) before itor motif that is involved in the down-regulation of B cell func- tions (reviewed in Ref. 16). Fc␥RIIb transfected into an Fc␥R- *Department of Immunology, University Hospital Utrecht, Utrecht, The Netherlands; negative B cell line potently down-regulates B cell activation upon †Bucharest Center of Immunology, Bucharest, Rumania; ‡Department of Immunol- co-cross-linking with surface IgG (17). Mice deficient in Fc␥RIIb ogy, L. Eo¨tvo¨s University, Budapest, Hungary; and §Division of Rheumatology, Washington University School of Medicine, St. Louis, MO 63110 exhibit enhanced Ig production against T cell-dependent and Received for publication August 12, 1998. Accepted for publication December 4, -independent Ags (18). 1998. In the present study, we directly targeted Ag to CR1/2 using a The costs of publication of this article were defrayed in part by the payment of page complex consisting of OVA chemically conjugated to anti-CR1/2 charges. This article must therefore be hereby marked advertisement in accordance Abs. The conjugate was generated using intact IgG of the rat anti- with 18 U.S.C. Section 1734 solely to indicate this fact. CR1/2 mAb 7G6, allowing direct interaction not only with CR1/2 1 This work was supported by grants from the Copernicus Fixed Contribution Con- ␥ tract CIPA CT 94-0152, FKFP 0102 grant, and the Hungarian National Science Fund but also with Fc R, thus resembling a natural immune complex (OKTA) 017158. consisting of Ag coated with both complement and IgG. To ad- 2 Address correspondence and reprint requests to Dr. Peter J. A. Capel, Department dress the relative contributions of CR1/2 and Fc␥R in the immune of Immunology, University Hospital Utrecht, G04.614, Heidelberglaan 100, 3584 CX response, an isotype-matched IgG control, Fc␥R-transfected Utrecht, The Netherlands. IIA1.6 cells (an Fc␥R-negative murine B lymphoma cell line) and 3 Current address: Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. CR1/2-deficient mice were used for in vitro and in vivo studies. 4 Abbreviations used in this paper: CR, complement receptor; Fc␥R, IgG Fc receptor; We found that conjugation of 7G6 to OVA strongly enhanced the SATA, N-succinimidyl S-acetylthioacetate; mIg, membrane Ig. anti-OVA Ab response, and that this enhancement was dependent

upon interaction of the conjugate with CR1/2. We also found ev- Immobilon-P membranes (Millipore, Bedford, MA). The membranes were idence suggesting that interaction of the complex with Fc␥RIIb washed in PBS containing 0.1% Tween 20 (PBS-Tween), blocked with 1% ␮ could partially down-regulate this response. BSA in PBS-Tween, and probed with 0.8 g/ml of the anti- phosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY) for 1.5 h at room temperature. After washing three times with PBS-Tween, Materials and Methods membranes were incubated with 0.5 ␮g/ml peroxidase-labeled rabbit Cell cultures and Abs anti-mouse Ig (Dako A/S, Glostrup, Denmark); bound Abs were detected using the enhanced chemiluminescence system (Amersham, ␥ The murine B cell lymphoma line A20 (19) and its Fc R-negative variant Buckinghamshire, U.K.). 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 supple- Ag presentation in vitro mented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM glutamine, and 1 mM sodium pyruvate. Unless stated The effect of the interaction of Ab-Ag complexes with CR1/2 or Fc␥Ron otherwise, cells were suspended in the same medium during the in vitro the ability of IIA1.6 and A20 cells to present OVA Ag to OVA-specific Th assays. Fc␥R transfectants of the IIA1.6 cell line were maintained in the cells was studied using 7G6-conjugated OVA and OVA conjugated to the same medium supplemented with Geneticin (G418, 0.8 mg/ml; Life Tech- isotype control SHL45.6. The IIA1.6 or A20 cells, at a concentration of 5 nologies, Paisley, U.K.) or with Geneticin and methotrexate (10 ␮M; Phar- 2 ϫ 10 cells/ml, were incubated with OVA-specific T cell hybridoma (2 ϫ 4 machemie, Haarlem, The Netherlands). CTLL-L16 cells were cultured in 10 cells/ml) for 24 h at 37°C in the presence of various concentrations of RPMI 1640 medium supplemented with 100 U/ml rIL-2. Rat hybridoma Ag ranging from 1 to 300 ng/ml. As a positive control for T cell reactivity, 7G6 (21) (kindly provided by Dr. T. Kinoshita, Osaka University, Osaka, high concentrations of free OVA (Յ30 ␮g/ml) were used. As negative Japan) secreting IgG2b directed to murine CR1 and CR2 was cultured on controls, all experiments were also performed in the absence of T cells, a large scale in serum free Iscove’s modified Eagle’s medium (Life Tech- IIA1.6 cells, or Ag. The release of IL-2 by the Th cells in the culture

nologies). SHL45.6 (22) purified rat IgG2b mAb specific for human CD3 supernatants, as a measure of efficient Ag presentation, was determined Downloaded from was used as an isotype control (kindly provided by Dr. M. Clark, Cam- using a CTLL proliferation assay (17). CTLL-16 cells are T cells that are bridge University, Cambridge, U.K.). derived from C57BL/6 mice and that proliferate in an IL-2-dependent fash- ion. Briefly, supernatants were incubated with CTLL-16 for 24 h at 37°C; Preparation of IgG-OVA conjugates afterward, [3H]thymidine was added in a concentration of 1 ␮Ci/well. Af- ter4hofincubation, cells were lysed in water onto glass fiber filters Ab-OVA conjugates were prepared using N-succinimidyl S-acetylthioac- (Wallac, Turku, Finland), and incorporated radioactivity was measured in etate (SATA) (purchased from Pierce, Rockford, IL) as a chemical cross- a scintillation counter.

linker (reviewed in Ref. 23). Briefly, IgG at a concentration of Ն5 mg/ml http://www.jimmunol.org/ suspended in a 50 mM sodium phosphate buffer containing 1 mM EDTA Immunization protocol (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 For immunization experiments, female BALB/c of ϳ8 wk of age were same phosphate buffer to remove the excess of unbound SATA and sub- used. In some experiments, female CR1/2-deficient mice (12)A. A.B. B. sequently incubated with a 10-fold smaller volume of 0.5 M hydroxyl- were used; mice with a mixed genetic background of C57BL/6 ϫ 129Sv amine HCl (Pierce) in 50 mM sodium phosphate and 25 mM EDTA (pH were used as wild-type controls. Two i.p. injections with similar doses 7.5) for2hatroom temperature. Next, the solution was diluted four times were administered in five mice per group over 26-day intervals using var- in 0.1 M sodium phosphate, 0.15 M NaCl, and 0.1 M EDTA (pH 7.2) ious doses of Ag per mouse as indicated. In some cases, mice were injected containing maleimide-activated OVA (Pierce), giving a molar IgG:OVA with a mixture of OVA and an equal volume of CFA (Difco Laboratories, ratio of 1:1.5. The coupling reaction was allowed to proceed for 90 min at Detroit, MI), followed by a booster injection of the same concentration of room temperature and was stopped by the addition of 2-ME at a final OVA in IFA (Difco). The mice were bled at 3 wk after the first immuni- by guest on September 25, 2021 concentration of 10 mM. Conjugates were subsequently purified from un- zation and at 1 wk and 3 wk after the second immunization for analysis of conjugated proteins by fast protein liquid chromatography using a HiLoad the serum anti-OVA response. Superdex 200 HR column (Pharmacia LKB Biotech, Uppsala, Sweden) equilibrated with PBS. Fractions containing molecules with an estimated ELISA for serum anti-OVA Ab molecular mass ranging from 200 to 573 kDa (based on the retention time OVA was coated onto the surfaces of 96-well Maxisorp immunoplates of molecular mass markers) were pooled and used for the Ag presentation (Nunc, Roskilde, Denmark) by overnight incubation with 10 ␮g OVA per and immunization experiments. The concentration of the pooled fractions ml PBS at 4°C. The plates were washed and blocked with PBS containing was estimated by measuring the absorbance at 280 nm, and small aliquots 1%BSAfor1hatroom temperature, followed by two washes. Next, the were immediately frozen for long-term storage. Conjugates were analyzed plates were incubated with serial dilutions of sera of immunized mice or of by 6% SDS-PAGE under nonreducing conditions. preimmune serum as a control. After two washes, the plates were incubated Flow cytometry with alkaline phosphatase-conjugated goat anti-mouse Ig(HϩL) (Southern Biotechnology Associates, Birmingham, AL) to determine total Ig concen- The murine B lymphoma cell line IIA1.6 was used to analyze the capacity trations or with alkaline phosphatase-conjugated goat Ab specific for of 7G6-OVA conjugates to bind CR1/2 by flow cytometry. IIA1.6 cells mouse IgG1, IgG2a, or IgG2b (Southern Biotechnology Associates) to de- were washed twice and suspended at a concentration of 2 ϫ 107 cells/ml termine the isotype subclass of the responses. The plates were washed of PBS containing 2.5% FCS and 0.05% sodium azide. The cells were then twice and developed using p-nitrophenyl phosphate substrate (Kirkegaard incubated with nonconjugated 7G6 or with 7G6-OVA conjugates at dif- and Perry Laboratories, Gaithersburg, MD). Titers were determined by ferent concentrations for 30 min at 4°C, followed by two washes. Next, the calculating the dilution of each serum giving an absorbance at 405 nm that cells were incubated either with FITC-conjugated mouse anti-rat IgG con- was twice that of preimmune serum. jugate (Jackson ImmunoResearch Laboratories, West Grove, PA) or with polyclonal rabbit anti-OVA IgG (Cappel, Durham, NC) followed by incu- Results bation with FITC-conjugated goat anti-rabbit IgG; each incubation was performed for 30 min at 4°C. The cells were then washed twice and ana- Binding of 7G6-OVA to CR1/2 in vitro lyzed by flow cytometry. The ability of the 7G6-OVA conjugate to bind to CR1/2 was de- Tyrosine phosphorylation assay termined by flow cytometry using the CR1/2-expressing murine B lymphoma cell line IIA1.6. After incubation of these cells with 10 ϫ 7 IIA1.6 cells, at a concentration of 2 10 cells/ml, were incubated with 10 ␮g/ml 7G6-OVA and subsequently with an FITC-conjugated ␮g/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 mouse anti-rat Ab, a positive fluorescence was observed (Fig. 1b); 1640 medium and resuspended in aliquots of 20 ␮l containing 5 ϫ 105 this fluorescence was within the same range when unconjugated cells. The cells were then incubated in a waterbath at 37°C, and the reaction 7G6 instead of the whole conjugate was used (Fig. 1a). In addition, was stopped at various time periods by adding reducing sample buffer (v/v) at a lower concentration of 1 ␮g/ml 7G6-OVA, a significant flu- containing 8% sodium lauryl sulfate, 20% 2-ME, and 1 mM sodium or- thovanadate. After denaturing the samples by boiling, lysed cells were orescence was detected that was comparable with that seen when loaded onto a 10% SDS-polyacrylamide gel (2.5 ϫ 105 cells/lane), and the using nonconjugated 7G6 (data not shown). Cell-bound 7G6-OVA proteins were subsequently electrotransferred to polyvinylidene difluoride could be detected using a polyclonal rabbit anti-OVA Ab (Fig. 1d), The Journal of Immunology 3127

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 Downloaded from FIGURE 1. Binding of 7G6-conjugated OVA to IIA1.6 cells. Suspen- Fc␥RIa/␥-␥ (b), A20 cells (c), or IIA1.6 cells transfected with murine sions of IIA1.6 cells were incubated first with nonconjugated 7G6 Abs (a Fc␥RIIb1 (d). The release of IL-2 by the Th cells in the culture superna- and c) or with 7G6-OVA conjugate (b and d), followed by a second incu- tants, as determined using a CTLL proliferation assay, was used as a mea- bation with FITC-labeled mouse anti-rat Ab (a and b) or with rabbit IgG sure for Ag presentation efficiency. Results are expressed as relative units anti-OVA combined with FITC-labeled goat anti-rabbit Ab (c and d). The of IL-2 per ml. Mean values of triplicate samples of one representative fluorescence of the cells was analyzed by flow cytometry. Solid lines rep- experiment of four are shown. resent cells that were incubated with both the first and the secondary re- http://www.jimmunol.org/ agents; dotted lines represent control cells incubated with the secondary Abs only. This phosphorylation pattern is not visible after the incubation of cells with an isotype-matched control conjugated to OVA (Fig. 2), which did not recognize cell-bound unconjugated 7G6 (Fig. 1c). attesting that the binding of 7G6-conjugated OVA to CRs is re- These results indicate that the 7G6-OVA conjugate retained its sponsible for the phosphorylation of the proteins mentioned. Con- capacity to bind CRs and confirm the presence of OVA in the trol experiments with purified Ab 7G6 alone showed a similar conjugate. pattern of phosphorylation of 110 kDa and 74 kDa protein bands (data not shown), indicating that conjugation of 7G6 with OVA is by guest on September 25, 2021 Tyrosine phosphorylation basically not modifying the signaling pattern of the targeted Next, we questioned whether bound conjugates can initialize an receptors. intracellular response in murine B cells. The pattern of activation following the binding of 7G6-OVA to CRs on IIA1.6 cells was Ag presentation in vitro investigated by the detection of protein phosphorylation on ty- The ability of CRs to modulate the efficiency of Ag presentation in rosine residues. Strong phosphorylation of two protein bands of an vitro was studied using 7G6-conjugated OVA, IIA1.6 cells, and an estimated molecular mass of 110 kDa and 74 kDa was detectable OVA-specific T cell clone. When IIA1.6 cells were incubated with after 10 min of incubation at 37°C of cells treated with 7G6-OVA, 7G6-OVA, efficient Ag presentation to OVA-specific T cells was showing induction of an intracellular signaling response (Fig. 2). observed (Fig. 3a). Free OVA induced detectable Ag presentation by IIA1.6 cells only when using concentrations that were 50- to 100-fold higher (Յ15 ␮g/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 Fc␥RI were incubated with SHL45.6-OVA, which resulted in efficient Ag presentation (Fig. 3b), confirming the ability of this conjugate to interact with Fc␥R 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 effi- FIGURE 2. Induction of tyrosine phosphorylation in IIA1.6 cells by ciently by B cells when conjugated to 7G6, and that this process 7G6-OVA. IIA1.6 cells were incubated with 7G6-OVA (left panel) or with involves at least CR2 expressed on these cells. SHL45.6-OVA (right panel) for 20 min at 4°C, washed, and incubated at Next, murine Fc␥RIIb-transfected IIA1.6 cells were used to inves- 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 tigate whether an additional interaction of the 7G6-OVA conjugate ␥ phosphorylation. The cells were lysed, and proteins were separated by 10% with Fc RIIb on B cells would interfere with Ag presentation. Ag SDS-PAGE and transferred to a polyvinylidene difluoride membrane, presentation by IIA1.6 cells, when incubated with 7G6-OVA, was which was probed with anti-phosphotyrosine mAb. Results representative completely inhibited upon transfection with Fc␥RIIb (Fig. 3d). of three similar experiments are shown. This finding was supported by the inability of the natively 3128 REGULATION OF THE Ab RESPONSE BY COMPLEMENT RECEPTORS AND Fc RECEPTORS Downloaded from FIGURE 5. Dose-dependency of the secondary anti-OVA Ab response after two immunizations with Ab-conjugated OVA. Mice were injected i.p. FIGURE 4. Primary and secondary anti-OVA Ab response induced by twice (at 26-day intervals) with various doses of 7G6-OVA (E), noncon- immunization with 7G6-conjugated OVA. Mice were injected i.p. twice (at jugated OVA (‚), or SHL45.6-OVA (Ⅺ). Results are expressed as titers of 26-day intervals) with 5 ␮g of 7G6-OVA (E), nonconjugated OVA (‚), anti-OVA Ig in the serum at 3 wk after the second immunization; mean nonconjugated OVA in CFA (Ⅺ), or with saline (ϫ). Results are expressed values of five mice are shown.

as titers of anti-OVA Ig in the serum at 3 wk after the ﬁrst immunization http://www.jimmunol.org/ and at 1 and 3 wk after the second immunization. Mean values of ﬁve mice are shown. Arrows indicate days of injection.

OVA (Fig. 3d). Taken together, these results indicate that coupling OVA to the anti-CR Ab 7G6 strongly enhances the humoral im- ␥ Fc RIIb-expressing B cell line A20 to present Ag after incubation mune response to OVA, and that this enhancement is dependent with 7G6-OVA (Fig. 3c). These results imply that interaction of an upon CR1/2. Furthermore, the interaction of such an Ag-Ab com- ␥ Ag-Ab complex with Fc RIIb on B cells inhibits CR-mediated plex with Fc␥RIIb may suppress the Ab response. enhancement of Ag presentation by these cells. by guest on September 25, 2021 Immunization with 7G6-OVA conjugate Discussion Next, the effect of targeting OVA to CR1/2 and Fc␥R on the in The present results demonstrate that the Ab-mediated targeting of vivo response was investigated. When normal BALB/c mice were Ag to CR1/2 using OVA complexed to anti-CR1/2 mAb (7G6) immunized with 5 ␮g 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 Ն1 ␮g 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 FIGURE 6. Isotype specificity of the anti-OVA response induced by possibility that the response was down-regulated by interaction of immunization with 7G6-OVA. Mice were injected i.p. twice (at 26-day ␥ this conjugate with the inhibitory Fc RIIb on B cells. This obser- intervals) with 5 ␮g of nonconjugated OVA (ova), 7G6-OVA (7G6-ova), vation was supported by in vitro experiments showing that trans- or OVA in CFA (ova ϩ CFA). Results are expressed as titers of IgG1 fection of IIA1.6 cells with Fc␥RIIb inhibited the presentation of (hatched bars) or IgG2a (filled bars) anti-OVA at 3 wk after the second OVA epitopes to OVA-specific T cells after incubation with 7G6- immunization; mean values of five mice are shown. The Journal of Immunology 3129

Our results showed that the targeting of Ag to CR1/2 on IIA1.6 cells mediates efficient Ag presentation to specific Th clones with- out 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 2ϩ [Ca ]i, modifications in cell cycle (proliferation, apoptosis), se- cretion 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 2ϩ result in an increase in [Ca ]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 contrib- Downloaded from uting to enhanced Ag presentation (24). The significance of this FIGURE 7. Anti-OVA response in CR2-deficient mice induced by 7G6- restricted stimulatory effect could be related to lowering the thresh- OVA. CR2-deficient (CR2-KO) mice were injected i.p. twice (at 26-day old of required help from Ag-specific Th cells in early stages of intervals) with 5 ␮g of 7G6-OVA (E), nonconjugated OVA (‚), nonconjugated OVA in CFA (Ⅺ), or with saline (ϫ). Results are expressed as immune response. titers of anti-OVA Ig in the serum at 3 wk after the first immunization and The precise mechanism by which 7G6-mediated targeting of Ag at 1 and 3 wk after the second immunization. Mean values of five mice are to CR1/2 leads to an enhanced Ab response is currently unknown. http://www.jimmunol.org/ shown. Arrows indicate days of injection. 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 enhances the humoral immune response against this Ag. Interac- induce cross-linking between CR1/2 and the Ag receptor on OVA- tion of these Ag-Ab complexes with Fc␥RIIb may partially sup- specific B cells, resulting in enhanced intracellular signaling and/or press this response. Several lines of evidence support the above Ag internalization. Previously reported data indicated that human conclusions. ␥ CR2 is an amplifier of low intracellular signaling through the B First, incubation of the Fc R-negative B lymphoma cell line 2ϩ cell receptor, by showing synergistic increases in [Ca ] follow- by guest on September 25, 2021 IIA1.6 with 7G6-conjugated OVA resulted in the efficient presen- i ing cross-linking CR2 to mIgM at suboptimal activation doses for tation of OVA epitopes to an OVA-specific T cell line in vitro. the triggering of mIgM on B cells (7). Theoretically, it is possible This process was dependent upon interaction of the complex with that the binding of 7G6-OVA to CR1/2 provides an additional CR1/2, because SHL45.6-OVA as an isotype-matched control had no effect. Second, the anti-OVA Ab response was markedly en- stimulus by locally activating complement. Third, interaction be- hanced when mice were immunized with 7G6-OVA but not after tween the 7G6-OVA and CR1/2 expressed on Ag-nonspecific B immunization with SHL45.6-OVA. The enhanced anti-OVA re- cells may also lead to Ag presentation, thereby contributing to Ab sponse was dependent upon interaction of the 7G6-OVA complex production. This possibility is supported by our observation that with CR1/2, because such a response was not observed in CR1/2- 7G6-OVA induces tyrosine phosphorylation and Ag presentation deficient mice. These mice did respond to OVA in CFA, indicating by IIA1.6 cells, which do not recognize OVA via their surface they are able to mount an anti-OVA Ab response depending upon mIg. Finally, CR2 that is expressed on the surface of follicular the method used to stimulate the immune system. The 7G6-OVA- dendritic cells and is involved in the retention of complement- mediated enhancement was most pronounced during the secondary coated Ag and the maintenance of long-term B cell memory (25) response, suggesting the involvement of memory B cells or Th may contribute to the effect of 7G6-mediated targeting. cells. The observation that the enhanced response induced by 7G6- In separate experiments, we have injected mice with nonconju- OVA was primarily of the IgG1 subclass suggests that targeting to gated 7G6 Abs 24 h before immunization with free OVA. In these CR1/2 preferentially triggers Th2 responses, following the same mice, the primary anti-OVA Ab response was inhibited by 80% isotype specificity compared with OVA alone. The enhanced anti- and the secondary response by 30% compared with control mice OVA response in mice immunized with 7G6-OVA was accompa- injected with buffer before immunization with OVA (D.C.B. and nied by a strong IgG response against rat IgG (data not shown), W.L.W.H., unpublished observations). These results are in agree- suggesting efficient presentation of Ags in both parts of the com- ment with previous reports (9, 10) showing that the down-modu- plex. Mice immunized with the isotype control SHL45.6-OVA, lation of CR1/2 by pretreatment of mice with 7G6 before immu- which hardly raised an anti-OVA response, also mounted only a nization with keyhole limpet hemocyanin or FITC-haptenated weak specific anti-rat IgG response. Treating mice with 7G6-OVA bacteria strongly inhibits Ab production. These findings support did not increase the total Ig concentration in the serum (W.L.W.H., the crucial role of CR1/2 during a normal immune response against unpublished observations), indicating that the enhancement was a protein Ag. not associated with polyclonal B cell activation. The present re- Remarkably, when mice were immunized with OVA conjugated sults, based on the direct targeting of Ag to CR1/2, are in agree- to SHL45.6, an irrelevant isotype control, the anti-OVA response ment with the observation reported previously that coupling of a was impaired compared with immunization with nonconjugated model Ag to C3d, a ligand for CR1/2, enhances the immunoge- OVA, suggesting inhibition of the response. The possibility that nicity of this Ag (14). this inhibition was caused by interaction of the conjugate with the 3130 REGULATION OF THE Ab RESPONSE BY COMPLEMENT RECEPTORS AND Fc RECEPTORS down-regulatory Fc␥RIIb on B cells was supported by the obser- References vation that transfection with Fc␥RIIb rendered IIA1.6 cells, incu- 1. Kinoshita, T., G. Thyphronitis, G. C. Tsokos, F. D. Finkelman, K. Hong, bated with 7G6-OVA, unable to present OVA epitopes in vitro. H. Sakai, and K. Inoue. 1990. Characterization of murine complement receptor This result was confirmed by the inability of A20 cells, the original type 2 and its immunological cross-reactivity with type 1 receptor. Int. Immunol. 2:651. cell line from which IIA1.6 is derived and which naturally ex- 2. Molina, H., T. Kinoshita, K. Inoue, J.-C. Carel, and V. M. Holers. 1990. A presses Fc␥RIIb, to present Ag after treatment with 7G6-OVA. molecular and immunochemical characterization of mouse CR2: evidence for a Thus, it is conceivable that after immunization with 7G6-OVA, single gene model of mouse complement receptors 1 and 2. J. Immunol. 145: 2974. which allows interaction with CR1/2 and Fc␥R, the immune re- 3. Molina, H., W. Wong, T. Kinoshita, C. Brenner, S. Foley, and V. M. Holers. sponse is the result of stimulation via CR1/2 and suppression 1992. Distinct receptor and regulatory properties of recombinant mouse complement receptor 1 (CR1) and Crry, the two genetic homologues of human CR1. via Fc␥RIIb. J. Exp. Med. 175:121. In addition to the down-regulatory effects of Fc␥RIIb, interac- 4. Pramoonjago, P., J. Takeda, Y. U. Kim, K. Inoue, and T. Kinoshita. 1993. Ligand ␥ specificities of mouse complement receptors types 1 (CR1) and 2 (CR2) purified tion of an Ag with Fc R can also have stimulatory effects on the from spleen cells. Int. Immunol. 5:337. immune response. For example, in vitro experiments using Fc␥R- 5. Molina, H., T. Kinoshita, C. B. Webster, and V. M. Holers. 1994. Analysis of transfected cells have shown that the IgG-mediated interaction of C3b/C3d binding sites and factor I cofactor regions within mouse complement receptors 1 and 2. J. Immunol. 153:789. Ag with either murine Fc␥RII or Fc␥RIII can induce presentation 6. Carroll, M. C. 1998. The role of complement and complement receptors in in- to Ag-specific T lymphocytes (26). In addition, using mice ex- duction and regulation of immunity. Annu. Rev. Immunol. 16:545. ␥ 7. Carter, R. H., M. O. Spycher, Y. C. Ng, R. Hoffman, and D. T. Fearon. 1988. pressing transgenic human Fc RI, it has been demonstrated that Synergistic interaction between complement receptor type 2 and membrane IgM on B lymphocytes. J. Immunol. 141:457. Ag targeting to this receptor strongly enhances the Ab response Downloaded from 8. Mongini, P. K. A., M. A. Vilenski, P. Highet, and J. K. Inman. 1997. The affinity (27). This observation is consistent with our present finding that threshold for human B cell activation via the antigen receptor complex is reduced transfecting IIA1.6 cells with human Fc␥RI enhanced 7G6-OVA- upon coligation of the antigen receptor with CD21 (CR2). J. Immunol. 159:3782. induced Ag presentation. In contrast to nontransfected IIA1.6 cells, 9. Heyman, B., E. J. Wiersma, and T. Kinoshita. 1990. In vivo inhibition of the antibody response by a complement receptor-specific monoclonal antibody. these cells were able to present Ag after treatment with the isotype J. Exp. Med. 172:665. control SHL45.6-OVA. However, two lines of evidence indicated 10. Thyphronitis, G., T. Kinoshita, K. Inoue, J. E. Schweinle, G. C. Tsokos, ␥ E. S. Metcalf, F. D. Finkelman, and J. E. Balow. 1991. Modulation of mouse that interaction with Fc R was not responsible for the enhanced in complement receptors 1 and 2 suppresses antibody responses in vivo. J. Immunol. http://www.jimmunol.org/ vivo Ab production induced by 7G6-OVA. First, conjugation of 147:224. OVA to the isotype control Ab SHL45.6 resulted in suppression 11. Hebell, T., J. M. Ahearn, and D. T. Fearon. 1991. Suppression of the immune response by a soluble complement receptor of B lymphocytes. Science 254:102. rather than enhancement of the anti-OVA response. Second, the 12. Molina, H., V. M. Holers, B. Li, Y.-F. Fang, S. Mariathasan, J. Goellner, finding that immunizing CR1/2-deficient mice with 7G6-OVA did J. Strauss-Schoenberger, R. W. Karr, and D. D. Chaplin. 1996. Markedly im- ␥ paired humoral immune response in mice deficient in complement receptors 1 and not result in an enhanced response indicates that Fc R alone are 2. Proc. Natl. Acad. Sci. USA 93:3357. not sufficient to mediate the enhancement induced by 7G6-OVA. 13. Ahearn, J. A., M. B. Fischer, D. Croix, S. Goerg, M. Ma, J. Xia, X. Zhou, R. G. Howard, T. L. Rothstein, and M. C. Carroll. 1996. Disruption of the CR2 The results of the present study provide a better insight into the locus results in a reduction on B-1a cells and in an impaired B cell response to mechanism of the generation of the Ab response, which is under T-dependent antigen. Immunity 4:251. control of both the innate and the acquired immune system during 14. Dempsey, P. W., M. E. D. Allison, S. Akkaraju, C. C. Goodnow, D. T. Fearon. by guest on September 25, 2021 1996. C3d of complement as a molecular adjuvant: bridging innate and acquired a second contact of the organism with an Ag. The intensity and immunity. Science 271:348. duration of the Ab response induced by a complement- and IgG- 15. Fischer, M. B., M. Ma, S. Goerg, X. Zhou, J. Xia, O. Finco, H. Han, G. Kelsoe, R. G. Howard, T. L. Rothstein, et al. 1996. Regulation of the B cell response to containing immune complex may be controlled by a balance be- T-dependent antigens by classical pathway complement. J. Immunol. 157:549. tween stimulation via CR1/2 and down-regulation via Fc␥RIIb. 16. Vivier, E., and M. Dae¨ron. 1997. Immunoreceptor tyrosine-based inhibition mo- Most likely, stimulation of an immune reaction by CR1/2, which tifs. Immunol. Today 18:286. 17. Van den Herik-Oudijk, I. E., P. J. A. Capel, T. van der Bruggen, and depends upon the presence of activated complement factors in the J. G. J. van de Winkel. 1995. Identification of signalling motifs within human Ag complex, has an important function during the early phase of Fc␥RIIa and Fc␥RIIb isoforms. Blood 85:2202. 18. Takai, T., M. Ono, M. Hikida, H. Ohmori, and J. V. Ravetch. 1996. Augmented the response. At a later stage, dampening of the response would be humoral and anaphylactic responses in Fc␥RII-deficient mice. Nature 379:346. required to prevent uncontrolled cellular reactivity. This shift in 19. Jones, B., J. P. Tite, and C. A. Janeway. 1986. Different phenotypic variants of the mouse B cell tumor A20/2J are selected by antigen- and mitogen-triggered the balance toward down-regulation may be mediated by an in- cytotoxicity of L3T4-positive, IA-restricted T cell clones. J. Immunol. 136:348. creasing concentration of IgG which is induced after the onset of 20. Shimonkevitz, R., J. Kappler, P. Marrack, and H. Grey. 1983. Antigen recogni- the response; this IgG is able to interact with Fc␥RIIb. The present tion by H-2 restricted T cells. J. Exp. Med. 158:303. 21. Kinoshita, T., J. Takeda, K. Hong, H. Kozono, H. Sakai, and K. Inoue. 1988. data are relevant for the rational development of new vaccine strat- Monoclonal antibodies to mouse complement receptor type 1 (CR1): their use in egies. Targeting Ag to CR1/2 may provide the adjuvant effect nec- a distribution study showing that mouse erythrocytes and platelets are CR1-negative. J. Immunol. 140:3066. essary for the generation of an efficient immune response against 22. Clark, M., C. Bindon, M. Dyer, P. Friend, G. Hale, S. Cobbold. R. Calne, and various Ags. H. Waldmann. 1989. The improved lytic function and in vivo efficacy of mono- valent CD3 antibodies. Eur. J. Immunol. 19:381. 23. Graziano, R. F., C. Somasundaram, and J. Goldstein. 1995. The production of bispecific antibodies. In Bispecific Antibodies. M. W. Fanger, ed. Springer-Ver- lag, New York, p. 1–26. Acknowledgments 24. Kozono, Y., R. Abe, H. Kozono, R. G. Kelly, T. Azuma, and V. M. Holers. 1998. We thank Dr. M. Glennie (University of Southampton, Southampton, Cross-linking CD21/CD35 or CD19 increases both B7-1 and B7-2 expression on murine splenic B cells. J. Immunol. 160:1565. U.K.) for essential suggestions concerning the Ab-OVA coupling proce- 25. Liu, Y.-J., G. D. Johnson, J. Gordon, and I. C. M. MacLennan. 1992. Germinal dure; Dr. T. Kinoshita (Osaka University, Osaka, Japan) for providing the centers in T-cell-dependent antibody responses. Immunol. Today 13:17. 7G6 hybridoma; Dr. M. Clark (Cambridge University, Cambridge, U.K.) 26. Amigorena, S., D. Lankar, V. Briken, L. Gapin, M. Viguier, and C. Bonnerot. 1998. Type II and III receptors for immunoglobulin G (IgG) control the presen- for providing purified SHL45.6 Abs; and Dr. C. Bonnerot (Institute Curie, tation of different T cell epitopes from single IgG-complexed antigens. J. Exp. Paris, France) and Dr. I. van den Herik-Oudijk and M. van Vugt (Univer- Med. 4:505. sity Hospital Utrecht, Utrecht, The Netherlands) for providing the Fc␥R- 27. Heijnen, I. A. F. M., M. J. van Vugt, N. A. Fanger, R. F. Graziano, transfected cell lines. We also thank P. van Kooten for help with mAb T. P. M. de Wit, F. M. A. Hofhuis, P. M. Guyre, P. J. A. Capel, J. S. Verbeek, and J. G. J. van de Winkel. 1996. Antigen targeting to myeloid-specific human production, P. Aerts for consultation on fast protein liquid chromatography Fc␥RI/CD64 triggers enhanced antibody responses in transgenic mice. J. Clin. analysis, and A. van der Sar for animal care. Invest. 97:331.