mAb against hen egg-white lysozyme regulate its presentation to CD4+ T cells
Pierre Guermonprez,
Richard Lo-Man,
Christine Sedlik,
Marie-Jesus Rojas,
Roberto J. Poljak1 and
Claude Leclerc
Unit of Biology of Immune Regulation, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France
1 Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, MD 2085-3479, USA
Correspondence to:
C. Leclerc
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Abstract
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Specific antibodies increase antigen uptake and presentation by antigen-presenting cells via the B cell receptor in B cells or Fc
R in dendritic cells. To determine whether the interaction between antibody and antigen could influence the set of peptides presented by MHC II molecules, we analyzed the presentation of different CD4+ T cell epitopes of hen egg-white lysozyme (HEL) after the capture of immune complexes formed between HEL and seven different specific mAb. The 103117 T cell epitope (I-Ed) was specifically and selectively up-regulated by the D1.3 and F9.13.7 mAb that binds to proximal loops in the native structure of HEL. Furthermore, Ii-independent T cell epitopes exposed on the HEL surface (116129 and 3445, I-Ak restricted) which require a mild processing involving the recycling of MHC II molecules were selectively up-regulated by mAb that overlap those T cell epitopes (D1.3 and D44.1). However, F10.6.6, somatically derived from the same germ line genes as D44.1 and exhibiting an higher affinity for HEL, was without effect on the presentation of the 3445 epitope. An Ii-dependent T cell epitope buried into the tertiary structure of HEL (4561, I-Ak restricted) and requiring the neosynthesis of MHC II was up-regulated by high-affinity mAb recognizing epitopes located at the N- or C-terminus of the T cell epitope. These results strongly suggest that (i) the spatial relationship linking the T cell epitope and the B cell epitope recognized by the mAb, (ii) the intrinsic processing requirements of the T cell epitope, and (iii) the antibody affinity influences the presentation of a given T cell epitope.
Keywords: antigen presentation, antibodies, MHC class II
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Introduction
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Antibodies participate efficiently in antigen uptake by antigen-presenting cells (APC). The capture of antigen via the B cell receptor complex (1,2) or via immune complex (IC) uptake by Fc receptors (FcR) expressed by macrophages or dendritic cells (36) results in a MHC class II-restricted antigen presentation enhancement of 102- to 105-fold in comparison with non-specific uptake via fluid phase pinocytosis.
It has been suggested (7) that the fine specificity of antibodies implicated in the capture of antigen could regulate the nature of the peptides presented by MHC class II molecules. The antibody paratope could exert a steric hindrance during antigen proteolysis and thus interfere with the formation of complexes between peptide and MHC class II molecules. Several in vitro studies have shown that specific antibodies can differentially enhance antigen presentation to specific T cells in relation with their fine specificity; indeed, the binding of an antigen by an antibody could favor or inhibit the presentation of T cell epitopes which are located near to or within the antigenic determinant recognized by this antibody (811). It has also been shown (12,13) that specific EpsteinBarr virus-transformed B cell lines that recognize different epitopes of tetanus toxin generate different proteolytic degradation products of this antigen. Biochemical data concerning antigen proteolysis in the presence of specific mAb also support the hypothesis that the region of the antigen close to the epitope recognized by the mAb can be protected from proteolysis (14).
The goal of the present study was to determine if, following antibody-mediated antigen uptake by APC, the antigen presentation to T cells is influenced by the specificity of the antibody. We chose to address this question using the hen egg-white lysozyme (HEL) antigen since its three-dimensional structure and its antigenic recognition by several specific mAb have been structurally defined (1520). In addition, the affinity constant and the free energy of binding of these mAb have been determined by different techniques (21).
To determine if antibodies regulate antigen presentation from a qualitative point of view, we have analyzed the presentation to T cells of seven different monovalent HELanti-HEL IC by an Fc
R-bearing APC. The formation of different MHC class II moleculespeptide complexes corresponding to four different epitopes was followed using HEL-specific T cell hybridomas. The availability of well-characterized anti-HEL mAb and of T cells of different specificities allowed us to investigate whether the topologic relationship between the B cell epitope, recognized by the mAb, and a T cell epitope is a key parameter influencing the presentation of this epitope to T cells.
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Methods
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Antigens and mAb
HEL was purchased from Sigma (St Louis, MO). The HEL peptide p108116 (WVAWRNRCK) was purchased from Neosystem (Strasbourg, France). The MalE303C3:BT protein was prepared by genetic insertion of the 93116 residues of the poliovirus VP1 protein into the Escherichia coli MalE protein at position 303 (22) and was kindly provided by P. Martineau (Institut Pasteur, Paris, France). Anti-HEL B cell hybridomas are described in Table 1
. They all originated from BALB/c mice and are of the IgG1,
isotype. Ascitic fluids prepared from BALB/c mice were precipitated with ammonium sulfate, then resuspended in distilled water and extensively dialysed against PBS. Protein concentration was determined by a colorimetric assay (BioRad, Munich, Germany). mAb activity was checked in ELISA using HEL-coated microplates. Anti-MalE mAb 94.1 (IgG1,
) and anti-Borrelia burgdorferi mAb J8.3 (IgG1,
) were kindly provided by J.C. Mazié (Institut Pasteur, Paris, France). The latter mAb was used as an isotype-matched control for presentation experiments. Anti-Fc
RII/III 2.4G2 (23) was purchased from PharMingen (San Diego, CA).
Cell lines
Two B cell lymphomas were used as APC in this study. IIA1.6 (Ad/Ed) is a Fc
R variant originated from the A20 B cell lymphoma cell line and A6B9 is a stable transfectant of IIA1.6 for the Fc
RIIb2 (24). A6B9 cells were co-transfected with A
k and Aßk using NT plasmids (24) containing the hygromycine and zeocine resistance genes respectively and the Sr
promotor. A6B9 cells and NT plasmids were kindly given by C. Bonnerot (Institut Curie). The transfectant clone A6B9.k was tested for A
k and Aßk expression by FACS analysis using the anti-A
k 39J mAb and the anti-Aßk 10.3.62 mAb, and by functional antigen presentation assays (see Results).
The T cell hybridomas L3E10 and 45G10 specific respectively for HEL103117 (I-Ed) and the poliovirus VP1 protein epitope C3T:103116 (I-Ed), were previously derived from BALB/c mice (22,25). 3B11.1 (26) and 1D6.1 (27) T cell hybridomas specific for HEL 3445 (I-Ak) and HEL 116129 (I-Ak) respectively were a kind gift from Luciano Adorini (Milan). 3A9 T cell hybridoma specific for HEL 4861 (I-Ak) (28) was a kind gift of Paul Allen (Washington University).
Antigen-presentation assay
T cell hybridomas (105/well) were cultured in the presence of APC (105/well) in a final volume of 0.2 ml for 24 h in complete medium (RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine and 5x105 M 2-mercaptoethanol). For Fc
R blocking experiments, APC were incubated for 30 min at 4°C with the 2.4G2 mAb at 10 µg/ml in complete medium, then washed before the antigen presentation assay. mAb were mixed with the antigen for 1 h at 37°C, in a final volume of 0.1 ml before incubation with the cells. A final concentration of 0.17 µM (25 µg/ml) of the mAb was used in most experiments as indicated in figure legends. 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 a 100 µl aliquot of supernatant in 0.2 ml final volume. Two days later, [3H]thymidine (NEN Life Science, Boston, MA) (0.4 µCi, 20 µl) was added and the cells were harvested 18 h later with an automated cell harvester (Skatron, Lier, Norway). Incorporated thymidine was quantitated by scintillation counting. Each experiment was performed in duplicate.
Cytofluorimetric assay for the detection of immune complexes at the surface of APC
HEL was conjugated to biotin (D-biotinoyl-
-amidocaproic acid N-hydroxysuccinimide ester; Boehringer Mannheim, Mannheim, Germany) according to the supplier's protocol. Then 106 cells were incubated for 1 h at 4°C, with anti-HEL mAb (or an isotype-matched control) at 2 µg/105 cells and with HELbiotin at 10 µg/ml in 0.2 ml of PBS, BSA 5% (w/v) and 3 mM sodium azide. After washing with the same buffer, cells were stained with streptavidinFITC (Sigma). After two washes, cells were resuspended in the same buffer supplemented with propidium iodide (1 µg/ml) and analyzed on a FACScan (Becton Dickinson, Mountain View, CA). Living cells were gated using propidium iodide (Sigma) exclusion.
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Results
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Some anti-HEL mAb enhance HEL presentation to T cell hybridomas specific for the 103117, I-Ed-restricted T cell hybridoma
We first examined the ability of seven different anti-HEL mAb (Table 1
) to modulate in vitro the presentation of HEL to specific T cells. Various anti-HEL mAb were mixed with different doses of HEL and then incubated with the Fc
R-bearing APC, A6B9 (H-2d), a B cell lymphoma transfected for Fc
RIIb2 (24), and L3E10, a T cell hybridoma specific for the HEL immunodominant epitope 103117 restricted to I-Ed. The stimulation of L3E10 was assessed by the release of IL-2. As shown in Fig. 1
(A), the HEL presentation to L3E10 was significantly enhanced after incubation with two mAb among the seven tested: D1.3 and F9.13.7. About 10-fold less antigen was required to stimulate an optimum response of L3E10 in the presence of one of these two mAb, at the dose studied (0.17 µM). Similar results were obtained with two other T cell hybridomas of the same specificity (data not shown), suggesting that the effect observed was dependent on a regulation of the processing of the 103117 HEL peptide and not related to a peculiar property of this cell line. As expected, HELanti-HEL complexes did not induce IL-2 release by the A6B9 APC or by the T cell hybridoma cultured alone (data not shown).

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Fig. 1. Enhancement of HEL presentation to a specific T cell hybridoma by some anti-HEL mAb. The L3E10 T cell hybridoma (anti-HEL103117, I-Ed) and the A6B9 B cell lymphoma (Fc RIIb2+) were incubated with various concentrations of HEL, in the presence or absence of a constant dose (0.17 µM) of different anti-HEL mAb or isotype-matched control mAb (A) or with a constant, sub-optimal dose of HEL (0.1 µM) in the presence of various concentrations of anti-HEL mAb or isotype-matched control mAb (B). IL-2 released by the hybridoma was assessed by the proliferation of the CTLL cell line. Results are expressed in c.p.m.
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These results were confirmed in another antigen-presentation assay: a fixed dose of HEL (0.1 µM) was incubated with a broad range of mAb concentrations (Fig. 1B
). The HEL dose of 0.1 µM used in this experiment was too low to stimulate L3E10, but the addition of D1.3 or F9.13.7 mAb restored optimal L3E10 stimulation. Taken together, these results indicate that antigen recognition by mAb is necessary but not always sufficient to potentiate HEL presentation since only some anti-HEL mAb increase HEL presentation to specific T cells.
Specificity of the enhancement of HEL presentation by anti-HEL mAb
To verify the specificity of the enhancing effect of the mAb on HEL presentation, we tested the effect of anti-HEL mAb on the presentation of an unrelated antigen, the chimeric protein MalE303C3:BT to the C3:T-specific T cell hybridoma, 45G10. As shown in Fig. 2
(B), D1.3 did not modify the 45G10 T cell response to MalE303C3:BT. Similar results were obtained with other anti-HEL mAb (data not shown). Reciprocally, 94.1, an anti-MalE mAb which strongly stimulated the presentation of the MalE303C3:BT protein to 45G10 T cells did not modify HEL presentation to L3E10 T cells (Fig. 2A and B
). An isotype matched control mAb showed no effect on the presentation of these antigens to their specific T cell hybridomas.

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Fig. 2. Anti-HEL mAb enhance specifically the presentation of HEL without modifying the capacity of the A6B9 APC to present an heterologous antigen. (A) The L3E10 T cell hybridoma (anti-HEL103117, I-Ed) and the A6B9 B cell lymphoma (Fc RIIb2+) were incubated with various concentrations of HEL, in the presence or absence of a fixed dose (0.17 µM) of anti-HEL (D1.3), anti-MalE (94.1) or isotype-matched control mAb. (B) The 45G10 T cell hybridoma (anti-C3:T, I-Ed) and the A6B9 B cell lymphoma (Fc RIIb2+) were incubated with various concentrations of MalE303C3:BT, in the presence or absence of a fixed dose (0.17 µM) of anti-HEL (D1.3), anti-MalE (94.1) or isotype-matched control mAb. (C) The L3E10 T cell hybridoma (anti-HEL103117, I-Ed) and the A6B9 B cell lymphoma (Fc RIIb2+) were incubated with various concentrations of the HEL p108116 synthetic peptide, in the presence or absence of various anti-HEL mAb at a fixed concentration (0.17 µM). (D) The 45G10 T cell hybridoma (anti-C3:T, I-Ed) and the A6B9 B cell lymphoma (Fc RIIb2+) were incubated with various concentrations of the chimeric protein MalE303C3:BT, in the presence or absence of a fixed dose (0.17 µM) of various anti-HEL mAb plus a fixed dose of HEL (0.1 µM). IL-2 released by the hybridomas was assessed by the proliferation of the CTLL cell line. Results are expressed in c.p.m.
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Anti-HEL mAb, free or complexed to HEL, do not modify the intrinsic antigen presenting ability of A6B9 APC (Fc
RIIb2+)
The increased response of L3E10 T cell hybridoma to HEL in the presence of some anti-HEL mAb could have been due to a non-specific modification of the intrinsic antigen-presenting ability of A6B9 APC by IC. To test this hypothesis, we analyzed the effect of these antibodies on the response of the L3E10 hybridoma to the antigenic peptide p108116. IL-2 production by the L3E10 hybridoma in response to the peptide presented by the A6B9 APC was not modified by the anti-HEL mAb (Fig. 2C
). Furthermore, the addition of HEL and anti-HEL mAb did not modify the response of the I-Ed-restricted 45G10 T cell hybridoma to the MalE303C3:BT protein presented by the A6B9 APC (Fig. 2D
).
HEL presentation by anti-HEL mAb is enhanced by uptake via Fc
RIIb2
The mAb D1.3, which increases the efficiency of HEL presentation by A6B9, did not modify the efficiency of HEL presentation by IIA1.6 (Fc
R), the B lymphoma cell line transfected with Fc
RIIb2 to obtain A6B9 (Fig. 3A
). Similar results were obtained with other anti-HEL mAb (data not shown). Furthermore, the enhancing effect of mAb D1.3 on the L3E10 response to HEL presented by A6B9 can be blocked by the anti-Fc
RII/III mAb 2.4G2 (Fig. 3B
). Indeed, the response of L3E10 at 0.1 µM of HEL using A6B9 as APC was fully dependent on the mAb D1.3 as shown in Fig. 3
(B). However, preincubation of A6B9 APC with the mAb 2.4G2 at 4°C for 30 min totally abolished the L3E10 response. This treatment did not interfere with the L3E10 response to 10 µM of HEL in the absence of anti-HEL mAb. Taken together, these results indicate that the suppression of the D1.3 enhancing effect by mAb 2.4G2 is due to the hindrance of the D1.3Fc
R interaction and to the inhibition of the subsequent uptake of HELmAb complexes by the APC.
Failure of certain mAb to increase HEL presentation does not stem from deficient binding to Fc
R
The inability of certain mAb to enhance HEL presentation to the 103117 T cell hybridoma could be due to an inability of the complexes formed by these mAb with HEL to bind to the Fc
RIIb2 receptor expressed by the A6B9 APC. To test this hypothesis, we analyzed by cytofluorimetry the binding of D1.3HEL or D44.1HEL complexes at the surface of the APC. HEL was biotinylated and the bound complexes were detected by streptavidinFITC staining (Fig. 4
). Neither HELbiotin alone nor mAb D1.3 or D44.1 alone induced a signal in this assay. The addition of HELbiotin and mAb D1.3 or D44.1 were required to detect a positive staining by streptavidinFITC. This staining was inhibited by preincubating A6B9 with the anti-Fc
RII/III mAb 2.4G2 and was not observed with IIA1.6 (Fc
R). These results confirmed the specificity of our binding test. A similar signal was obtained after the addition of mAb D1.3 or D44.1 even though the former activates HEL presentation and the latter does not. Similar results were obtained with F9.13.7 and F10.6.6 mAb (data not shown). These data indicate that the lack of increase of HEL presentation in the presence of five of the seven mAb tested is not due to a less efficient binding and uptake of HELanti-HEL complexes by the A6B9 APC.
Synergistic enhancement of the presentation of HEL to an I-Ed-restricted, 103117-specific hybridoma by combination of non-activating and activating mAb
To examine whether the effect on HEL presentation was abrogated or increased using a mixture of mAb, we analyzed the presentation of HEL by A6B9 (Fc
RIIb2+) to the L3E10 hybridoma (anti-HEL103117, I-Ed) in the presence of two anti-HEL mAb. Different pairs of activating and non-activating mAb were tested. By pairing F9.13.7 and D11.15, F9.13.7 and D136.26, D1.3 and D44.1, and F9.13.7 and D44.1, a strong synergistic effect was observed: 100 to 1000 fold less HEL was required to obtain a half-optimal stimulation of the L3E10 hybridoma (Fig. 5
). This synergistic effect was observed for pairs of mAb which recognize non-overlapping epitopes and therefore bind together to the same molecule of HEL to form polyvalent IC (20,29,30). The polyvalent IC bind to the weak-affinity Fc
RIIb2 with an increased avidity compared to the monovalent IC (31). For other pairs of mAb (D1.3 and D136.26, D1.3 and D11.15) the enhancing effect of the mixture was equal to the one observed with the activating mAb alone. Thus, a lack of synergistic effect was observed for mAb which recognize overlapping epitopes and therefore cannot form polyvalent IC (29,30). We conclude that the lack of enhancing effect of D11.15 and D136.26 mAb when used in monovalent IC can be overcome when these mAb are included in polyvalent IC.

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Fig. 5. Synergistic enhancement of the presentation of HEL to a specific T cell hybridoma by combination of anti-HEL mAb. The L3E10 T cell hybridoma (anti-HEL103117, I-Ed) and the A6B9 B cell lymphoma (Fc RIIb2+) were incubated with various HEL concentrations and one or two anti-HEL mAb at 0.085 µM (1x) or 0.17 µM (2x). IL-2 released by the hybridomas was assessed by the proliferation of the CTLL cell line. For each mAb or pair of mAb, the HEL doseresponse of L3E10 was determined and the enhancing effect was quantified by the ratio: (HEL dose required for 50% maximal L3E10 IL-2 release without mAb)/(HEL dose required for 50% maximal L3E10 IL-2 release with mAb).
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Protective effect of mAb on the processing of Ii-independent I-Ak-restricted epitopes
To examine whether the effect of mAb on HEL presentation was dependent on the type of processing involved in the presentation of the HEL peptide, we took advantage of well-described I-Ak-restricted HEL epitopes. The 116129 and 3445 HEL epitopes are generated in an Ii-independent presentation pathway, involving the recycling of MHC class II molecules, whereas the 4861 epitope requires Ii and neosynthesized MHC class II (26,3234). We stably transfected the A6B9 APC for the
and ß chain of the I-Ak molecule to obtain an I-Ak-expressing line competent in Fc
R-mediated uptake of antigen. Expression of both
and ß chains was checked by flow cytometry (Fig. 6A
) and by a functional test of presentation using the I-Ak-restricted 3A9 T cell hybridoma (Fig. 6B
).
The presentation of HEL to the 116129-specific hybridoma was selectively up-regulated by the D1.3 mAb, whereas its presentation to the 3445-specific hybridoma was selectively up-regulated by the D44.1 mAb. The F9.13.7 had a significant enhancing effect on both epitopes, but this effect was very inferior to the one of D1.3 on the 116129 epitope or the one of D44.1 on the 3445 epitope. By contrast, the HEL presentation to the Ii-dependent anti-4861 hybridoma was up-regulated by the D1.3, F9.13.7, D74.3, D44.1 and F10.6.6 mAb.
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Discussion
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In the present study, we analyzed the regulation of antigen presentation by antibodies. IC formed between HEL and seven different mAb were presented to specific T cell hybridomas by the A6B9 APC, an Fc
RIIb2-transfected lymphoma B cell. Two out of the seven mAb tested, D1.3 and F9.13.7, increased significantly HEL presentation to a T cell hybridoma specific for the immunodominant 103117 epitope restricted to I-Ed. The 116129 epitope (I-Ak restricted) was also up-regulated by the mAb D1.3 with a striking efficiency (>50-fold) and by the mAb F9.13.7 with a reduced efficiency. The 3445 epitope (I-Ak-restricted) was efficiently up-regulated by the mAb D44.1, and less efficiently by the mAb D1.3 and F9.13.7. The enhancement of HEL presentation mediated by these mAb was found to be HEL-specific and was independent of an eventual bystander effect on APC. Moreover, the lack of enhancing effect observed with the IIA1.6 cell line devoid of Fc
R, as well as blocking experiments, demonstrate unambiguously that enhancement of HEL presentation depends on Fc
R-mediated uptake of IC.
Enhancement of antigen presentation to CD4+ T cells by antibodies has already been described in several studies concerning membrane Ig-mediated or Fc
R-mediated uptake of antigen (35). In addition to these previous findings, the present study shows that the modulation of HEL presentation by mAb is specific since up-regulation of HEL presentation was only observed with a limited number of mAb/T cell epitope combinations. Moreover, our results suggest that the enhancing effect of anti-HEL mAb is mediated by two independent phenomena: (i) an increase of antigen uptake and (ii) a modulation of epitope presentation. It is likely that all mAb tested participate in an efficient HEL uptake via Fc
RIIb2 since we demonstrate that D1.3, D44.1, F9.13.7 and F626.1 are capable of forming IC with HEL, and to bind to Fc
RIIb2 at the membrane of A6B9 cells. Furthermore, some of the mAb which did not affect HEL presentation to a 103117-specific hybridoma (D11.15, D136.26 and D44.1) were shown to synergize with activating mAb due to their ability to bind HEL simultaneously with the activating mAb and, thus, to form polyvalent IC. In these cases, HEL presentation can be enhanced up to 1000-fold. This phenomenon is likely due to an increased avidity of polyvalent IC for the weakly binding Fc
RIIb2 (36).
Antibodies regulate the processing of HEL according to their fine specificity
Despite the crucial role of antibody affinity for antibody-mediated uptake (37,38), affinity of the various mAb does not correlate positively or negatively with their ability to enhance HEL presentation (Table 1
). For example, both F10.6.6 which has the highest affinity (Ka > 109 M1) and D44.1 which has the lowest affinity (Ka = 1.3x107 M1) have no effect on HEL presentation. Thus, even if the affinity of the mAb could influence the efficiency of HEL uptake by APC and the HEL release in the endocytic pathway, differences in affinity are not sufficient to explain the different behaviors observed with the anti-HEL mAb. In this context, the antibody fine specificity appears as a key factor involved in the regulation of HEL processing.
Crystallographic studies of Fab and Fv fragments from the mAb D1.3 and D44.1 complexed to HEL have shown that the antigen is stabilized by interactions with the antibodies, as judged by the decreased overall mobility of the antigen atoms. NMR studies (39) have supported this conclusion and extended it to soluble complexes. Thus, it is reasonable to think that the bound mAb will confer some protection against proteolytic attack. However, the overall protective effect will be influenced by the location of the epitope and by the molecular mobility or tendency to unfold of the unprotected areas. These areas will be the first ones affected by proteolysis, leading to partial unfolding of the antigen, further proteolysis and eventual release of the bound epitopes. Release of bound epitopes, under the conditions of the intracellular processing compartments, will differ for the mAb depending on the nature of the interatomic contacts and of their intrinsic stability in the lower pH media. Evidently, some complexes will expose segments of HEL that are more suceptible to proteolysis and mAb could thus exert a steric hindrance on the proteolysis of HEL.
Each antibody binds different parts of the epitopes with different free energies, leading to differential dissociation of the hydrolysed segments. The antibody combining site will be also affected by these processes, further compounding the difficulty of a molecular modeling of the protection and peptide release events. However, biochemical studies have shown that a part of the antigen (larger than the strict B cell epitope) could be protected (`the footprint') by the mAb from proteolytic degradation (11,14). In these studies, purified proteolytic enzyme or cellular fractions were used. The relevance of these results for antigen processing has been pointed out by Watts and colleagues who have demonstrated that different B cell clones with different fine specificities gave different patterns of antigen degradation fragments (12). Furthermore, a large fraction of antigen fragments remained bound to the membrane Ig even when this latter had begun to be degradated: in this case, IC are a substrate for proteolytic enzyme of the endocytic pathway (13). These results are compatible with a regulatory role of the fine specificity of the antibodies in antigen processing, as initially speculated by Berzofsky (7). The availability of MHC class II molecules for loading of peptides located within the footprint is controversial. These peptides could be retained by the mAb which will prevent their binding to MHC class II molecules. However, this protection could increase their life time and their probability for MHC II loading. Watts et al. (10,11) have demonstrated that both suppressed and favored T cell epitopes fell within the region of antigen protected by the regulatory mAb. They speculated that a strict overlap between B and T cell epitopes results in suppression of the presentation of the T cell epitope, whereas the proximity of T and B cell epitopes results in increased presentation of the T cell epitope. For the 103117 T cell epitope, activating D1.3 and F9.13.7 mAb share a common feature: they both recognize an epitope near the 103117 T cell epitope in the native structure of HEL as defined by the crystallographic structure of HELanti-HEL complexes (16,20).
Processing requirements of the T cell epitopes may influence the sensitivity to the regulation exerted by antibodies
The presentation of the 4861, I-Ak-restricted T cell epitope was shown to be up-regulated at a moderate level by several high-affinity mAb (D1.3, F9.13.7 and F10.6.6). This peptide is not exposed to the surface of HEL and there is no evident topological relationship with the B cell epitopes recognized by these different mAb. The presentation of this peptide requires Ii and the neosynthesis of MHC class II molecules (26,33,34). Moreover, its processing involves an acidic and reductor compartment related to late endosome or MIIC.
Our main finding is that the 116129 and 3445 Ak-restricted T cell epitopes are strikingly up-regulated by the overlapping mAb D1.3 and D44.1 respectively.
D1.3 recognizes a discontinuous epitope encompassing two HEL loops. The 116129 T cell epitope belongs to one of these loops (Lys116, Gly117, Thr118, Asp119, Val120, Gln121, Ile124 and Arg125). Thus, the interaction of D1.3 with HEL could lead to the protection of a large region of HEL encompassing the 116129 T cell epitope. D44.1 failed to increase the presentation of the HEL 116129 T cell epitope. In these case, the mAb binds HEL in the ß-pleated sheet moiety of HEL which is separated by the active cleft from the 116129 T cell epitope. Thus, it can be envisaged it they will confer less protection to the
-helical moiety where the 116129 T cell epitope is located (19), whereas it would protect the 3445 region due to its recognition of residues Gln41 and Arg46.
However, it is worth noting that the F10.6.6 mAb (recognizing the same epitope as D44.1) is without effect on 3445 epitope presentation. This may be due to its higher affinity. In this case, the protected paratope would not be released by the mAb, resulting in the inhibition of MHC II loading.
The 116129 and 3445 T cell epitopes share common features: (i) they are exposed in the surface of the molecule, (ii) they do not require Ii for their presentation (26,32) and (iii) they do not require neosynthesized MHC class II molecules as determined by resistance of their presentation to Brefeldin A (26,33) inhibition. Furthermore, it is well established that the presentation of the 116129 epitope involves recycling of MHC class II molecules (33). All these features evoke a processing involving early compartments of the endocytic pathway. In this context it was speculated that the generation of these epitopes required a moderate proteolytic attack. Indeed, it was shown that inhibition of endosomal proteolysis (by chloroquine or leupeptin inhibition) results in an increase of their presentation (33).
Taken together, our results are in favour of a model in which overlapping antibodies regulate the presentation of solvent-exposed T cell epitopes by protecting them from an excessive proteolytic degradation. In this context, the fine specificity of the capturing antibody could play a major regulatory role: depending on the antibody affinity, the outcome of the antigenantibody interaction might be positive or negative. In contrast, solvent-buried peptide requiring a more aggressive processing may be less sensitive to antibody-mediated regulation because of their efficient targeting to the late endosomal compartment. In this case, the affinity of antibody may play a major role since the affinity should be sufficiently high to avoid dissociation before reaching late endosomes.

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Fig. 7. Differential regulation of presentation of Ii-independent and Ii-dependent HEL epitopes presentation by anti-HEL mAb. I-Ak-restricted, anti-HEL T cell hybridomas (1D6.1, anti-HEL116129, in A; 3B11.1, anti-HEL 3445, in B; 3A9, anti-HEL 4861, in C) and the A6B9.k cells (Fc RIIb2+, I-Ak) were incubated with various concentrations of HEL, in the presence or absence of a constant dose (0.17 µM) of different anti-HEL mAb or isotype-matched control mAb. IL-2 released by the hybridoma was assessed by the proliferation of the CTLL cell line. For each mAb, the HEL doseresponse of the T cell hybrid was determined and the enhancing effect on HEL presentation was quantified by the ratio: (HEL dose required for 50% maximal T cell hybridoma IL-2 release without mAb)/(HEL dose required for 50% maximal maximal T cell hybridoma IL-2 release with mAb). For each T cell hybridoma, the enhancing effect ratio (fold) is plotted against the mAb tested. Enhancing effect ratio represents the mean of at least two experiments, each including all experimental points in duplicate.
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Acknowledgments
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We would like to acknowledge Luciano Adorini (Roche Milano Ricerche, Milan) and Paul Allen (Washington University, Washington) for kind the gift of T cell hybridomas. We would like to express our gratitude to Christian Bonnerot and Armelle Regnault for gifts of material and for their valuable help in the preparation of A6B9.k.
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Abbreviations
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APC antigen-presenting cell |
Fc R receptor for the Fc portion of IgG |
Fc RIIb2 b2 isoform of murine type II Fc R |
HEL hen egg-white lysozyme |
IC immune complex |
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Notes
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Transmitting editor: H. Bazin
Received 19 May 1999,
accepted 5 August 1999.
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References
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