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Modulation of the Humoral Immune
Response by Antibody-Mediated Antigen
Targeting to Complement Receptors and Fc
Receptors
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
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Copyright © 1999 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
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*
T
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. Eötvös 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.
4
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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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.
3126
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
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
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),
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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.).
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.).
The Journal of Immunology
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.
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 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.
3127
3128
REGULATION OF THE Ab RESPONSE BY COMPLEMENT RECEPTORS AND Fc RECEPTORS
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-
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.
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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.
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.
The Journal of Immunology
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).
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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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.
3129
3130
REGULATION OF THE Ab RESPONSE BY COMPLEMENT RECEPTORS AND Fc RECEPTORS
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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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.