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*
Unité de Biologie des Régulations Immunitaires and
Groupe d’ingénierie des protéines (Centre National de la Recherche Scientifique URA1129), Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R) by other APC has been shown to increase greatly
Ag presentation to T cells (3, 5). In the course of the T cell-dependent humoral response to many haptens, the detailed analysis of Ab affinity (at the serum level, or at the clonal level) revealed a decrease in the dissociation constant (Kd) from 10-5/10-6 M in the primary response to 10-7/10-8 M in the late response after booster injections (6). This progressive increase of affinity has not been observed for other T cell-dependent Ag such as monomeric hen egg white lysozyme (HEL). In these cases, the Ab affinity reaches a high level (Kd = 10-7/10-10 M) a few days after a primary injection and does not increase after booster injections (7).
In the present study, we analyzed the role of the affinity of Ab for Ag
on the efficiency of Ab-mediated Ag presentation to CD4+ T
cells. We chose to address this question with HEL, which is a monomeric
protein Ag. We have previously demonstrated in vitro the enhancing
effect of the anti-HEL mAb D1.3 on the presentation of HEL to a
specific T cell hybridoma (anti-HEL 108–116, I-Ed) by
an FcR-expressing APC (P.Guermonprez et al., manuscript in
preparation). In the present study, HEL was captured by a bifunctional
hybrid protein (Fv-MalE) (8) formed between the variable fragment (Fv)
of the anti-HEL mAb D1.3 and the Escherichia coli
maltose-binding protein (MalE) in the presence of two anti-MalE
mAbs. Different Fv-MalE hybrids were prepared with mutations in the
D1.3 complementarity determining regions (CDR), leading to reduced
affinities for HEL (9). Thus, in this experimental model, the
internalization of Fv-MalE hybrids is therefore fixed to a stable level
that is independent of the HEL concentration. Affinity
variations between Fv-MalE and HEL were determined with precision,
opening the possibility of a quantitative analysis of the role of
affinity on Ab-mediated Ag presentation. The effects of these
hybrids on HEL presentation to a specific T cell hybridoma were then
compared with those obtained with the wt D1.3 Fv-MalE. The results of
this study show that an increase of HEL presentation to T cells
occurred through the interaction of the Fc regions of the anti-MalE
mAbs with the FcR of the APC. This experimental system allowed us to
show that the affinity of anti-HEL Abs determines the efficiency of
Ab-mediated Ag presentation to T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HEL was purchased from Sigma (St. Louis, MO). Peptide 108–116
from the HEL sequence (p108–116, WVAWRNRCK) was synthetized by
Neosystem (Strasbourg, France). The D1.3 anti-HEL B cell hybridoma
(IgG1, 
) was a kind gift from Dr. Poljak (Center for Advanced
Research in Biotechnology, Rockville, MD) (10). The mAb D1.3 was
obtained from ascitic fluid from BALB/c mice and precipitated with
ammonium sulfate, then resuspended in purified water and extensively
dialyzed against PBS. Protein concentration was determined with a
colorimetric assay (Bio-Rad, Münich, Germany). The anti-MalE
mAbs 94.1 and 56.5 (IgG1, ) were a kind gift of Dr. J. C.
Mazié (Institut Pasteur, Paris, France).
Fv-MalE hybrids and their mutants
Fully functional variable fragments (Fv) of D1.3, a mouse mAb
directed against HEL (10), were produced as hybrids (Fv-MalE) with the
maltose-binding protein of E. coli (MalE). The production
and purification of the Fv-MalE hybrids and their mutants has been
previously described (8, 9). In the case of
VL::VH-MalE hybrids, the variable
region of the H chain (VH) is genetically coupled to the N
terminus of MalE and noncovalently associated with the variable domain
of the L chain (VL). In the opposite case
(VH::VL-MalE), the variable region of
the L chain (VL) is genetically coupled to the N terminus
of MalE and noncovalently associated with the variable domain of the H
chain (VH). For both constructs, site-directed mutations
have been constructed in the complementarity determining regions (CDR)
of the H or L chain of D1.3. The kinetics of interaction between HEL
and the wt or mutant hybrids have already been described (9). The
characteristics of the different Fv-MalE hybrids used in this study are
summarized in Table I
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HEL was covalently immobilized on the carboxymethylated dextran surface of a CM5 sensorchip to a level of 2700 resonance units (RU), using the Amine Coupling Kit (Pharmacia Biosensor, Uppsala, Sweden). The resulting derivatized surface, CM5-HEL, was equilibrated with buffer M at a temperature of 20°C and a flow rate of 5 µl/min, conditions that were used in all subsequent steps. Buffer M was 10 mM phosphate buffer (pH 7.4), 2.7 mM KCl, 137 mM NaCl, 0.005% detergent P20 (Pharmacia), and 1 mM maltose. The addition of maltose prevents any dimerization of MalE (9). For all binding experiments, samples were diluted in buffer M.
Cell lines
A20 (Ad/Ed), a B cell lymphoma line
originated from BALB/c mice (11), and IIA1.6, an FcR-deficient
variant of A20 (12), have been described. A6B9, a stable transfectant
of IIA1.6 for the b2 isoform of the murine type II weak affinity
receptor for IgG (FcRIIb2) (13), was used as APC and was kindly
provided by C. Bonnerot and S. Amigorena (Institut Curie, Paris,
France). The L3E10 T cell hybridoma, which is specific for the HEL
108–116 T cell epitope (WVAWRNRCK), I-Ed restricted, was
produced using BALB/c mice immunized with HEL (14). The CTLL cell line
was purchased from the American Type Culture Collection (ATCC,
Manassas, VA).
Ag presentation assay
HEL and/or anti-HEL Fv-MalE and/or a mixture of the two
anti-MalE mAbs 94.1 and 56.5 were mixed together for 1 h at
37°C, in a final volume of 0.1 ml of complete medium (RPMI 1640
medium supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml
streptomycin, 2 mM L-glutamine, and 5 x
10-5 M 2-ME) in 96-well flat-bottom culture microplates.
The concentrations used in each experiment are indicated in the figure
legends. Then, L3E10 T cell hybridoma cells (105
cells/well) were cocultured with A6B9 (105 cells/well) and
the preformed complexes (HEL, anti-HEL Fv-MalE, and anti-MalE
mAbs) for 24 h in 0.2 ml (final volume) of complete medium. After
24 h, the supernatants were frozen for at least 2 h at
-70°C. Then, 104 cells/well of the CTLL cell line, which
proliferates specifically in response to IL-2 but not IL-4, were
cultured with 100 µl of supernatant in 0.2 ml final volume. Two days
later, [3H]thymidine (NEN Life Science, Boston, MA) was
added, and the cells were harvested 18 h later with an automated
cell harvester (Skatron, Lier, Norway). Incorporated thymidine was
detected by scintillation counting. To block FcR, the A6B9 APC were
incubated 30 min at 4°C with the anti-FcRII/III mAb 2.4G2 (15)
(PharMingen, San Diego, CA) at 10 µg/ml, then washed before use for
Ag presentation assay. In all experiments, each point was done at least
in duplicate and more often in triplicate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a previous study, we analyzed the effect of the anti-HEL
mAb D1.3 and other anti-HEL mAbs on the presentation of HEL to the
specific T cell hybridoma L3E10 (anti-HEL 108–116,
I-Ed restricted) by the A6B9 APC
(Ad/Ed), which is a B cell lymphoma transfected
with the b2 isoform of the type II murine FcR (FcRIIb2).
FcRIIb2 is implicated in Ag presentation, unlike FcRIIb1, and is
physiologically expressed by professional APC (13). We have shown that
the anti-HEL mAb D1.3 increases significantly the efficiency of HEL
presentation. This effect is HEL specific, T cell epitope specific,
independent of a nonspecific activation of the APC, and dependent on
FcRIIb2-mediated uptake (P.Guermonprez et al., manuscript in
preparation). In the present report, we analyzed the effect of the
anti-HEL D1.3 Fv-MalE on the presentation of HEL to the same T
cells by the same APC, in the presence of two anti-MalE mAbs (Fig. 1). Fv-MalE hybrids are formed by
genetically fusing the Fv of the anti-HEL mAb D1.3 with the MalE
protein.
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A revealed that the
concentration of HEL required to obtain a half optimal T cell
stimulation (assessed by IL-2 release) was about 50-fold less in the
presence of the anti-HEL mAb D1.3 than in its absence. A similar
enhancement of HEL presentation was obtained with a mixture of an
anti-HEL Fv-MalE, derived from mAb D1.3, and of the two
anti-MalE mAbs 94.1 and 56.5. Anti-HEL Fv-MalE alone or the mixture
of the anti-MalE mAbs 94.1 and 56.5 alone was without effect on the
presentation of HEL to the L3E10 T cell hybridoma. The mixture of
anti-HEL Fv-MalE plus the two anti-MalE mAbs 94.1 and 56.5, or
the anti-HEL mAb D1.3 alone, were without effect on the
presentation of the synthetic HEL peptide 108–116 to the specific T
cell hybridoma L3E10 (Fig. 2B). These results demonstrated
that the enhancement effect observed with Fv-MalE in the presence of
anti-MalE mAbs was specific of HEL presentation and involved the
antigenic recognition of HEL by the Fv-MalE molecule.
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The lack of effect of anti-HEL Fv-MalE on HEL presentation in
the absence of anti-MalE mAbs suggested that the complexes formed
between anti-HEL Fv-MalE and HEL were recognized by the
anti-MalE mAbs. The reactivity of the anti-MalE mAb 56.5 toward
the MalE component of anti-HEL Fv-MalE has already been described
(9). We further analyzed the ability of the two different anti-MalE
mAbs to bind the complex formed between anti-HEL Fv-MalE and HEL by
using the BIAcore technology (Fig. 3).
The simultaneous recording of signals obtained in each experiment, both
on a control surface (mock-derivatized) and on a surface derivatized
with HEL, allowed us to subtract the signal, due only to changes in the
refraction index of the buffers and/or to the nonspecific binding of
compounds on the dextran matrix of the sensorchip, from the specific
signal obtained on the HEL surface. The comparison of curves 1 and 2
demonstrated that Fv-MalE specifically associated with HEL. The
comparison of curves 2 and 3 showed the specific association of the
anti-MalE mAb 94.1 to the HEL/Fv-MalE complex. The comparison of
curves 3 and 4 showed the specific binding of the anti-MalE mAb
56.5 to the HEL/Fv-MalE/94.1 complex. Similar results were obtained
when inverting the order of binding of the two anti-MalE mAbs (data
not shown). These results therefore showed that both anti-MalE mAbs
could bind simultaneously to the HEL/anti-HEL Fv-MalE complex.
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RIIb2 mediated
We reported above that the enhancement of HEL presentation by
Fv-MalE depended on the anti-MalE mAbs. In particular, the
stimulation of L3E10 by A6B9 at a suboptimal concentration of HEL (0.1
µM) was fully dependent on the presence of the Fv-MalE/anti-MalE
mAb complex (see Fig. 2
A). This dependence suggested that
the complex formed between Fv-MalE and HEL was efficiently captured by
the APC via the FcRIIb2 receptor. To test this hypothesis, we
blocked FcRIIb2 by preincubating the A6B9 APC with the
anti-FcRII/III mAb 2.4G2. This mAb has been shown to inhibit the
Fc/FcR interaction (15). The L3E10 response to the mixture of 0.1
µM HEL, Fv-MalE, and anti-MalE mAbs was completely abolished by
the preincubation of the A6B9 APC with mAb 2.4G2 (Fig. 4). In a previous study, we showed that
this mAb also totally and specifically blocked the D1.3-mediated
increase of HEL presentation by A6B9 (P. Guermonprez et al., manuscript
in preparation). In contrast, the preincubation treatment of A6B9 with
mAb 2.4G2 did not interfere with the stimulation of L3E10 at an optimal
concentration of free HEL (20 µM). These results showed that
formation of complexes between HEL, anti-HEL Fv-MalE, and the two
anti-MalE mAbs 94.1 and 56.5 resulted in the efficient uptake of
HEL via the FcR of APC, which, in turn, led to a more efficient
presentation of HEL to the L3E10 T cell hybridoma.
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To quantitatively measure the contribution of the affinity between
Fv-MalE and HEL to the enhancement of HEL presentation, we used mutants
of the anti-HEL Fv-MalE. The mutations affected residues of the
light or heavy chain CDRs (Table I). All
the mutant Fv-MalE are derived from the anti-HEL mAb D1.3, they had
the same specificity, and their reduced affinity for HEL was mainly due
to an increase of their dissociation rate constant
(koff) (9). We measured the stimulation of the
L3E10 T cell hybridoma in the presence of HEL at various
concentrations, of each mutant anti-HEL Fv-MalE at a fixed
concentration (0.2 µM), and of the anti-MalE mAbs 94.1 and 56.5
also at fixed concentrations (0.13 µM each). A representative
experiment is shown in Figure 5A. The two wt hybrids, which
have virtually the same affinity for HEL, gave an identical enhancement
of HEL presentation. These hybrids have the Fv of the anti-HEL mAb
D1.3 in different configurations,
VH::VL-MalE (wt) or
VL::VH-MalE (wt). Thus, the fusion of
either the VH or the VL region to the N
terminus of MalE does not interfere with the enhancement of HEL
presentation by the anti-HEL Fv-MalE hybrids. We then examined the
effects of six different mutations in VL (Y50F or
Y32F or W92A) or in VH (Y101F or D100N or D54A).
The enhancement of HEL presentation observed in the presence of
VH::VL-MalE(Y50F) or
VH::VL-MalE(Y32F) or
VL::VH-MalE(D54A) was similar to that
induced by VH::VL-MalE (wt) or
VL::VH-MalE (wt) (Fig. 5A
and data not shown for
VL::VH-MalE(D54A)). The enhancement
was significantly lower for
VL::VH-MalE(Y101F) than for the wt
hybrids. VL::VH-MalE(D100N) and
VH::VL-MalE(W92A) virtually did not
enhance the HEL presentation (Fig. 5A and data not shown for
VH::VL-MalE(W92A)). In these
experiments, the internalization of the different Fv-MalE mutants by
the APC did not vary since it depended on the complexation of the
Fv-MalE with the anti-MalE mAbs as shown above. The
Kd values of the interaction between mAbs 94.1
and 56.5 and MalE are, respectively, equal to 7 x
10-10 M and 8 x 10-10 M. Thus, assuming
two independent equilibriums in solution between Fv-MalE and each
anti-MalE mAb, we calculated that, under the experimental
conditions used, 100% of the Fv-MalE molecules were complexed by at
least one anti-MalE mAb and that 42% were simultaneously complexed
by the two anti-MalE mAbs. Thus, our experimental system was
appropriate for evaluating the role of the affinity between Fv-MalE and
HEL on the presentation of HEL since the capture of the different
Fv-MalE hybrids by the FcR-bearing APC was identical for all the
hybrids tested. We then examined whether the efficiency of presentation
to the T cells depended only on the concentration of bound HEL or also
on the specific mutation of Fv-MalE. To address this question, the
results of Figure 5A were plotted vs the amount of HEL
theoretically complexed to the different Fv-MalE hybrids, assuming an
equilibrium in solution. This representation showed clearly that, for
the same concentration of complexed HEL, the Fv-MalE mutants did not
increase HEL presentation to the T cell hybridoma with the same
efficiency (Fig. 5B). In particular,
VL::VH-MalE(Y101F) and
VL::VH-MalE(D100N) were significantly
less efficient in stimulating HEL presentation than the other Fv-MalE
hybrids tested.
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were tested in at
least two independent experiments. Figure 6 represents a synthesis of the results
obtained with the different Fv-MalE hybrids. Each point represents the
enhancing effect of one Fv-MalE hybrid on HEL presentation obtained in
an independent experiment. The EFs observed for the different
anti-HEL Fv-MalE hybrids were plotted against their respective
dissociation constant K'd (nM) or
koff (10-4 s-1) or
t1/2 (s) measured with the BIAcore apparatus
(Fig. 6, A, B, and C, respectively).
The EFs corresponding to the lowest values of
K'd (between 8.6 and 357 nM) were comparable and
induced a 150- to 200-fold increase of HEL presentation. The EF rapidly
decreased with affinity for intermediate values of
K'd. For the highest values of
K'd (around 5000 nM), the presentation of HEL
was almost not affected by Fv-MalE. A similar relation between the EF
and koff was observed. This similarity is due to
the fact that the increase in K'd is mainly due
to an increase of koff (see Table I). The
half-life (t1/2) of the different
complexes is inversely proportional to koff and
was consequently inversely related to the EF.
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). For
the three plots, the transition between the virtual absence of
enhancement effect and the plateau value was highly cooperative. The
half transition values were K'd = 643 nM,
koff = 622 10-4 s-1
and t1/2 = 11 s. These values corresponded
precisely to the binding parameters of
VL::VH-MalE(Y101F) (Table I).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we analyzed the role of Ab affinity on
Ab-mediated Ag uptake and presentation by APC using a new experimental
system. In this system, the Ag (i.e., HEL) was captured by an
anti-HEL Fv-MalE hybrid, which, in turn, was captured by
anti-MalE mAbs via the FcRIIb2 cellular receptor (Fig. 1). The
subsequent FcRIIb2-mediated endocytosis of these complexes resulted
in the presentation of HEL by MHC II molecules. Indeed, the complexes
formed between the T cell epitope (HEL 108–116) and the
I-Ed molecule are recognized by the specific T cell
hybridoma L3E10.
The stimulation of the T cells was measured as a function of the
concentration of HEL present in the culture, using fixed concentrations
of anti-HEL Fv-MalE hybrids and anti-MalE mAbs. For these
reasons, we could consider that the internalization of the Fv-MalE by
the anti-MalE mAbs was constant and independent of HEL capture. We
calculated an EF to compare the efficiencies of HEL presentation by the
FcRIIb2-mediated endocytosis of the anti-MalE/Fv-MalE/HEL
complex (arrow b in Fig. 1) and by the pinocytosis of HEL in fluid
phase (arrow a in Fig. 1). We used Fv-MalE mutants with different
affinities for HEL to analyze the relationship between their binding
parameters and the EF. The results showed that only Fv-MalE hybrids
having a sufficient affinity for HEL corresponding to a
K'd below a transition value equal to 643 nM
enhanced the presentation of HEL with a maximal efficiency. In this
range of K'd, the maximum EF was approximately
148-fold. We found similar thresholds for koff
and t1/2 due to the weak changes in
kon between the different hybrids tested. The
saturation of the EF at the lowest K'd values
likely stemmed from a limitation of the Fv-MalE uptake by the A6B9 APC.
Indeed, we observed, in the same antigenic system (HEL and L3E10
hybridoma) and with the same APC, EFs rising up to 1000-fold when HEL
was captured by a mixture of two anti-HEL mAbs of high affinity (P.
Guermonprez et al., manuscript in preparation). This last result rules
out the possibility that the saturation of the EF observed here comes
from a limitation of the sensitivity of the T cell assay that we used.
The K'd and koff of the Fv-MalE hybrids could affect the presentation at several levels. K'd determines the efficiency of HEL complexation with Fv-MalE in the culture medium and, thus, the amount of HEL that is internalized. However, we found that, for a given amount of bound HEL, some hybrids triggered HEL presentation with lower efficiencies. This finding suggested strongly that the mutations influenced not only the level of HEL complexation but also later steps of its uptake and intracellular processing by the APC. Therefore, the binding characteristics of Fv-MalE and HEL could interfere either with the endocytosis or with the intracellular targeting and delivery of HEL.
Binding characteristics of Fv-MalE and HEL endocytosis
The half-life of the interaction between the Fv-MalE/anti-MalE
complex and FcR at the surface of the APC could constrain the rate
of dissociation between Fv-MalE and HEL. The internalization of the
Fv-MalE/anti-MalE complex by the APC occurred at a constant rate
since Fv-MalE and the anti-MalE mAbs were at fixed concentrations
in the presentation experiments. Therefore, HEL was internalized
efficiently only if it did not dissociate before the internalization of
the Fv-MalE/anti-MalE immune complexes via anti-MalE
mAbs/FcR interactions. Thus, the rate of HEL/Fv-MalE dissociation
should be compared with the rate of the clearance of immune complexes
(Fv-MalE/anti-MalE mAbs) from the membrane by the FcR-mediated
endocytosis. If the t1/2 of the HEL/Fv-MalE
complex is inferior to the t1/2 of the
Fv-MalE/anti-MalE mAbs complex at the surface of the cell (before
endocytosis by FcRIIb2), some of the Fv-MalE/anti-MalE mAbs
complexes would not participate in HEL uptake, and, therefore, some HEL
molecules that have been complexed with Fv-MalE/anti-MalE mAbs and
fixed to FcRIIb2 would not be internalized. Mellman et al. (16) have
found that the t1/2 of polyvalent immune
complexes bound to the FcR of macrophages is approximately 105
s. The half transition values of t1/2 that we
found here for the HEL/Fv-MalE complex is 11 s. The comparison of
these two values strongly suggests that the half-life of the complexes
between the Fv-MalE hybrids and HEL could be a factor limiting the
internalization of HEL by the constitutive endocytosis of the Fv-MalE
hybrids. This parameter could also influence the BCR-mediated
internalization of Ag by specific B cells. Indeed, Watts and Davidson
(17) have determined that the half-life of membrane Ig in human EBV-B
cell lines is 8 min (480 s). This half-life would correspond to a
limiting koff of 14 x 10-4
s-1. If the hypothesis developed above for the
internalization of Fv-MalE through anti-MalE mAbs and FcRIIb2
can be extrapolated to the internalization through BCR, then it would
impose a rather high affinity for the BCR-mediated internalization of
monovalent and soluble Ag by specific B cells for presentation to T
cells at the beginning of a T cell-dependent humoral response.
Binding characteristics of Fv-MalE and intracellular targeting or delivery of HEL
The fate of internalized HEL could differ between the different Fv-MalE mutants, and this could influence the efficiency of HEL presentation. There still exists a controversy on the exact compartment where Ag processing takes place, and the physico-chemical environment where HEL is processed is not precisely known (3). Nevertheless, the mutations in the Fv regions of the hybrids could differentially affect the dynamics of intracellular HEL release. Indeed, Aluvihare et al. (18) showed that the quality of the Ag/BCR interaction could impose different degrees of dependence on the rapid intracellular delivery to the MHC II-processing compartment. In our case, this kind of limitation could explain why, at the same level of bound HEL, all Fv-MalE mutants did not trigger HEL presentation with the same efficiency. For Fv-MalE with the higher koff (the shorter t1/2), HEL could dissociate from Fv-MalE before reaching the right compartment for Ag processing. The mutations could also affect differentially the pH stability of the HEL/Fv-MalE interaction in the acid and reducing environment of the endocytic pathway and, thus, affect the efficiency of HEL processing (19).
Concluding remarks
The K'd value that was required for the half optimal EF on HEL presentation (6.43 x 10-7 M) determined in this study corresponds approximately to the intermediate range of affinities that can be observed during the humoral immune response (6). This value cannot be extended to the internalization of Ag by the BCR due to possible differences between the behaviors of this receptor and of the immune complexes formed between Fv-MalE and anti-MalE mAbs. Thus, it should be relevant to test the impact of the mutations we studied here on the BCR (formed with the D1.3 Ig)-mediated presentation of HEL to T cells. Nevertheless, our results support the idea that affinity maturation may influence the Ag presentation to T cells by specific B cells. The hypothetical constraint on Ab koff (or t1/2) for Ag internalization due to the rate of receptor internalization (or half-life on cell surface of BCR) could be decreased for a polyvalent Ag such as hapten-carrier conjugates, as compared with a monovalent one such as HEL for two reasons. First, the avidity of an Ag for the BCR is increased by its polyvalence and, second, the receptor aggregation could accelerate the internalization of the complexes (20). These constraints on koff, if physiologically relevant, could influence the affinity of B cell clones that are recruited in the preimmune repertoire by a T cell-dependent Ag. The fact that HEL Abs reach a high affinity in primary response (7) is especially relevant for the choice of this antigenic model. One can ask whether this fact relies on a bias on the naive B cell repertoire toward this Ag or whether it depends on a selective process that favors the expansion of high affinity B cell clones. Our results support the second hypothesis: weak affinity B cell clones might not be selected due to their inability to present the monomeric HEL to specific anti-HEL T helper cells to receive help for Ab production. Thus, priming of high affinity B cells in primary response could be a more general feature of strictly monovalent Ags (which is not the case of the well-described hapten-carrier systems as discussed above). The identification of this kind of constraint, even in an unphysiologic experimental system like the one we used, thus appears particularly relevant for the understanding of the dynamic of the Ab response.
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Footnotes |
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2 Abbreviations used in this paper: BCR, B cell receptor complex; FcRIIb2, b2 isoform of the type II receptor for the constant region of IgG; HEL, hen egg white lysozyme; MalE, maltose-binding protein of Escherichia coli; Fv-MalE, hybrid formed between the MalE protein and the variable regions of the anti-HEL D1.3 mAb; Kd, dissociation constant; K'd, dissociation constant calculated with the BIAcore apparatus; koff, rate of dissociation; kon, rate of association; VH, V region heavy chain; VL, V region light chain; CDR, complementarity determining region; wt, wild-type; EF, enhancement factor.
Received for publication March 17, 1998. Accepted for publication June 24, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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