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*
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R). The role of
CR1/2 and FcR 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 µg 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 FcRIIb 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
FcRIIb. 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 FcRIIb. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 (FcR). B cells express FcRIIb,
which contains an immunoreceptor tyrosine-based inhibitor motif that is
involved in the down-regulation of B cell functions (reviewed in 16 . FcRIIb transfected into an FcR-negative B cell line potently
down-regulates B cell activation upon co-cross-linking with surface IgG
17 . Mice deficient in FcRIIb 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 anti-CR1/2 mAb
7G6, allowing direct interaction not only with CR1/2 but also with
FcR, thus resembling a natural immune complex consisting of Ag
coated with both complement and IgG. To address the relative
contributions of CR1/2 and FcR in the immune response, an
isotype-matched IgG control, FcR-transfected IIA1.6 cells (an
FcR-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 upon interaction of the conjugate with
CR1/2. We also found evidence suggesting that interaction of the
complex with FcRIIb could partially down-regulate this response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine B cell lymphoma line A20 19 and its FcR-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. FcR 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 µM; 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 cross-linker (reviewed in 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 x 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 x 107 cells/ml, were incubated with 10 µg/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 µl containing 5 x 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 x 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 µg/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, membranes were incubated with 0.5 µg/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
FcR 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 x 105 cells/ml, were incubated
with OVA-specific T cell hybridoma (2 x 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 µg/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
µCi/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 x 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 µg 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(H+L) (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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
µg/ml 7G6-OVA and subsequently with an FITC-conjugated mouse
anti-rat Ab, a positive fluorescence was observed (Fig. 1
b); 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 µg/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), 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.
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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). 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.
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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 µg/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 FcRI were
incubated with SHL45.6-OVA, which resulted in efficient Ag presentation
(Fig. 3b), confirming the ability of this conjugate to
interact with FcR 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.
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RIIb-transfected IIA1.6 cells were used to
investigate whether an additional interaction of the 7G6-OVA conjugate
with FcRIIb 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 FcRIIb (Fig. 3
d). This finding was supported by the inability of the
natively FcRIIb-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 FcRIIb 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 FcR on the in
vivo response was investigated. When normal BALB/c mice were immunized
with 5 µg 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 µg 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).
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); 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).
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), raising the possibility that the
response was down-regulated by interaction of this conjugate
with the inhibitory FcRIIb on B cells. This observation was
supported by in vitro experiments showing that transfection of IIA1.6
cells with FcRIIb 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 FcRIIb
may suppress the Ab response. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIb may partially suppress this response. Several
lines of evidence support the above conclusions.
First, incubation of the FcR-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
CR1/2-deficient 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-OVA-mediated
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 7G6-OVA 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 anti-OVA 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 [Ca2+]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 [Ca2+]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 OVA-specific 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 [Ca2+]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 complement-coated 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 down-regulatory
FcRIIb on B cells was supported by the observation that transfection
with FcRIIb 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 FcRIIb, 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 FcR, the
immune response is the result of stimulation via CR1/2 and suppression
via FcRIIb.
In addition to the down-regulatory effects of FcRIIb, interaction of
an Ag with FcR can also have stimulatory effects on the immune
response. For example, in vitro experiments using FcR-transfected
cells have shown that the IgG-mediated interaction of Ag with either
murine FcRII or FcRIII can induce presentation to Ag-specific T
lymphocytes 26 . In addition, using mice expressing transgenic human
FcRI, 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
FcRI enhanced 7G6-OVA-induced 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 FcR 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 FcR 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 IgG-containing immune complex
may be controlled by a balance between stimulation via CR1/2 and
down-regulation via FcRIIb. 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 FcRIIb. 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.
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Acknowledgments |
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R-transfected 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. |
Footnotes |
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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.
4 Abbreviations used in this paper: CR, complement receptor; FcR, IgG Fc receptor; SATA, N-succinimidyl S-acetylthioacetate; mIg, membrane Ig.
Received for publication August 12, 1998. Accepted for publication December 4, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIa and FcRIIb isoforms. Blood 85:2202.RII-deficient mice. Nature 379:346.[Medline]
RI/CD64 triggers enhanced antibody responses in transgenic mice. J. Clin. Invest. 97:331.[Medline]
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E. Lunde, K. H. Western, I. B. Rasmussen, I. Sandlie, and B. Bogen Efficient Delivery of T Cell Epitopes to APC by Use of MHC Class II-Specific Troybodies J. Immunol., March 1, 2002; 168(5): 2154 - 2162. [Abstract] [Full Text] [PDF]
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A. Cherukuri, P. C. Cheng, and S. K. Pierce The Role of the CD19/CD21 Complex in B Cell Processing and Presentation of Complement-Tagged Antigens J. Immunol., July 1, 2001; 167(1): 163 - 172. [Abstract] [Full Text] [PDF]
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J. L. Gommerman, D. Y. Oh, X. Zhou, T. F. Tedder, M. Maurer, S. J. Galli, and M. C. Carroll A Role for CD21/CD35 and CD19 in Responses to Acute Septic Peritonitis: A Potential Mechanism for Mast Cell Activation J. Immunol., December 15, 2000; 165(12): 6915 - 6921. [Abstract] [Full Text] [PDF]
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S. E. Applequist, J. Dahlstrom, N. Jiang, H. Molina, and B. Heyman Antibody Production in Mice Deficient for Complement Receptors 1 and 2 Can Be Induced by IgG/Ag and IgE/Ag, But Not IgM/Ag Complexes J. Immunol., September 1, 2000; 165(5): 2398 - 2403. [Abstract] [Full Text] [PDF]
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R. Merica, A. Khoruts, K. A. Pape, R. L. Reinhardt, and M. K. Jenkins Antigen-Experienced CD4 T Cells Display a Reduced Capacity for Clonal Expansion In Vivo That Is Imposed by Factors Present in the Immune Host J. Immunol., May 1, 2000; 164(9): 4551 - 4557. [Abstract] [Full Text] [PDF]
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), isotype-matched
SHL45.6-OVA (
), or free OVA (
). The different panels depict OVA
presentation by nontransfected IIA1.6 cells (a), IIA1.6
cells transfected with human Fc
),
nonconjugated OVA in CFA (
), or with saline (x). 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.
