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The Journal of Immunology, 1998, 160: 1759-1766.
Copyright © 1998 by The American Association of Immunologists

Immunodominance Does Not Result from Peptide Competition for MHC Class II Presentation1

Richard Lo-Man2,*, Jan P. M. Langeveld{ddagger}, Pierre Martineau{dagger}, Maurice Hofnung{dagger}, Robert H. Meloen{ddagger} and Claude Leclerc*

* Unité de Biologie des Régulations Immunitaires, et {dagger} Unité de Programmation Moléculaire et Toxicologie Génétique (CNRS-URA1444), Institut Pasteur, Paris, France; and {ddagger} Institute for Animal Science and Health (ID-DLO), Lelystad, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Competition for binding to MHC class II molecules between processed peptides derived from a single protein Ag is considered an important parameter leading to the presentation of a limited set of peptides by APCs. We tested the relevance of this competition process in a model Ag, the MalE protein, by deleting T cell epitopes or by introducing a competitor T cell peptide. We identified in DBA/1 (I-Aq) mice six immunodominant T cell determinants in the MalE sequence, 89–95, 116–123, 198–205, 211–219, 274–281, and 335–341. Synthetic peptides carrying these determinants were classified in three groups as weak, intermediate, or strong I-Aq binders in competition experiments with the PreS:T peptide of hepatitis B surface Ag. In vivo, synthetic MalE peptides with weak and intermediate MHC binding capacity were inhibited in their capacity to stimulate proliferative response in the presence of the PreS:T competitor peptide, whereas the strongest MHC binder was not. Strikingly, the insertion of the potent competitor PreS:T peptide into the MalE sequence, as a single copy or as four copies, did not inhibit the proliferative response to the six immunodominant peptides of the recipient protein. Moreover, deletion in the protein sequence disrupting either the weak (198–205) or strong (335–341) MHC binding determinant of MalE did not modify the proliferative response to the remaining T cell determinants as compared with wild-type MalE protein. Altogether, these results show that peptide competition for MHC binding may not represent the most important event in processes leading to immunodominance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Following Ag uptake by APCs, exogenous protein Ags undergo a mild proteolytic degradation through the endocytic route resulting in the presentation of a small number of selected Ag-derived peptides by class II molecules available for CD4+ T cell activation (1). The class II-associated peptides are heterogeneous in length, ranging from 13 to 25 amino acid residues, and the core binding sequence is extended at either end (2, 3). The rules that govern the determinant selection at the level of MHC presentation mainly involve the generation of peptides by proteases and the ability of peptides to bind MHC molecules, but both may be intimately linked to each other through a MHC-guided processing (4). So far, however, the sequence of the different processing steps remains barely understood.

Recent studies have increased our knowledge of the mechanisms of the intracellular transport of MHC class II molecules with escorting invariant chain and HLA-DM/H-2 M molecules (5). However, the complexity of Ag processing resides in the intersection of the retrograde transport of the Ag under proteolyis in the endocytic route with the anterograde transport of MHC class II molecules to the plasma membrane. Therefore, the unanswered question concerning Ag processing is how protein Ag deals with both proteases and class II molecules. The identification of several compartments rich in stable class II/peptide complexes, such as lysosome-like MIIC (6, 7, 8) or endosome-like CIIV (9), which are thought to be the peptide-loading vesicles, suggests that the egress of peptide/MHC complexes to the cell membrane can take place at many points in the endocytic route (10).

Originally, it was proposed that the hierarchy of peptide presentation was due to the affinity of peptides for class II molecules, but also resulted from a competition process among these different peptides for binding to class II molecules (11). The intrinsic properties of the processed peptides are of most importance for the formation of stable peptide/MHC complexes with long half-lives (12, 13). However, does the competition for class II molecules between the peptides contained within a single protein Ag act as a driving force for their presentation to T cells? If such a mechanism plays a major role in T cell determinant selection, then the emergence of a T cell response to several determinants must be an equilibrium of presentation by MHC molecules among the different peptides, and it is expected that the addition or deletion of a given peptide within the protein sequence would result in a noticeable perturbation of the previous equilibrium.

The hypothesis of competition relies on experiments performed with free synthetic peptides that bypass the intracellular processing events for binding to class II molecules (14). We have reinvestigated the question of intramolecular competition between peptides belonging to the same antigenic molecule using chimeric proteins for which the intracellular processing is a prerequisite for MHC presentation. We have previously shown that immunogenic peptides genetically inserted in internal positions of a recipient protein can be efficiently presented by class II molecules and can activate specific T cells (15, 16, 17, 18, 19, 20, 21). The success of this strategy in many different systems (22, 23, 24, 25) suggests that the competition between the foreign inserted peptide and the recipient protein does not influence the processing and presentation of the inserted epitope, unless the MHC binding of the foreign peptide affects the presentation of the peptides of the recipient protein.

Here, we used recombinant Escherichia coli MalE proteins carrying the PreS:T T cell determinant from hepatitis B surface Ag (HBsAg)3 (18). We first demonstrated that the PreS:T peptide and the immunogenic peptides of the MalE recipient protein can compete in vitro for binding to I-Aq class II molecule. In vivo, after immunization of DBA/1 (I-Aq) mice with an equimolar mixture of the PreS:T and the MalE peptides, the PreS:T peptide was shown to compete with several MalE peptides for the induction of proliferative responses. In contrast, when the potent competitor PreS:T peptide was introduced in different permissive insertion sites into MalE, as a single or as multiple copies, it was unable to interfere in vivo with the presentation of the T cell determinants of the recipient protein. Our results clearly show that within chimeric MalE-PreS:T proteins, the immunologic behavior of the inserted viral determinant and of the T cell determinants of the MalE protein are independent of each other, indicating that at the protein level these T cell determinants do not compete for MHC presentation and T cell activation in vivo. This view is also supported by results obtained with mutant MalE proteins carrying deletions of MalE I-Aq binding sequences, since such modifications of the protein sequence did not modulate the immunogenicity of the remaining MalE T cell determinants. Altogether, our results indicate that under real in vivo conditions, peptide competition may not play a major role in the immunodominance phenomenon.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Eight- to twelve-wk-old DBA/1 inbred mice were used in all experiments and were purchased from the animal colony of the Pasteur Institute (Paris, France).

Peptides

Based on the sequence of MalE (26), a complete set of 385 overlapping 15-mer peptides was synthesized on polyethylene pins according to standard PEPSCAN procedures (27, 28). The final peptides were released from their support under basic conditions and contained a free amino terminus and an amidated C terminus. In addition to this mg scale synthesis, selected regions of the MalE sequence were synthesized at 20 mg scale according to standard synthesis procedures for peptides using Wang resin (p-alkoxybenzylalcohol resin, Bachem, Bubendorf, Switzerland) resulting in peptides with free amino and carboxyl termini. The PreS:T peptide corresponding to the 120- to 132-amino acid sequence of the PreS2 region of HBsAg (29) and the C3 peptide corresponding to the sequence of a neutralizing B cell epitope of poliovirus type 1 (30) were both synthesized by Neosystem (Strasbourg, France).

MalE mutant proteins

All of the E. coli-derived MalE chimeric proteins used in this study were previously described (18, 31). Mutant proteins were developed using a two-step genetic procedure. A BamHI linker was inserted into the malE gene, which was used to further insert a virus-derived peptide sequence. For all of the proteins used here, the first insertion step led to a short deletion in the malE gene sequence. Proteins were named according to the residue number of the mature MalE preceding the insert. MalE206C3, MalE211C3, and MalE339C3 correspond, respectively, to the deletion of amino acids 207 to 216, 212 to 220, and 340 to 357, followed by the second-step insertion of the C3 sequence coding for amino acids DNPASTTNKDK of VP1 protein from poliovirus type 1 (31). MalE133 and MalE303 mutant proteins carry deletions, respectively, of amino acids 134 to 142 and 304 to 309. Derived chimeric proteins MalE133-PreS:T and MalE303-PreS:T correspond to insertion of the PreS:T amino acid sequence (15). MalE133-PreS:T4B and MalE303-PreS:T4B proteins correspond to the insertion of four copies of PreS:T followed by 132–145 sequence of HBsAg (18). MalE133-PreS:B corresponds to the insertion at site 133 of the PreS:B epitope from HBsAg and was used as a control. Production and affinity purification of all proteins were done as previously described (15).

Cell lines

M12C10 (H-2d/q) B-lymphoma cell was obtained by fusion of DBA/1 mice spleen cells with M12 cells (21). The I-Aq-restricted 52A12 T cell hybridoma was derived from DBA/1 mice and was specific for the PreS:T peptide (18).

In vitro competition assay

M12C10 cells (5 x 104) were fixed using glutaraldehyde and plated in RPMI 1640 supplemented with 10% FCS, antibiotics, 2 mM L-glutamine, and 5 x 10-5 M 2-ME, and were next incubated at 37°C for 4 h with 1 µM of peptide PreS:T in the absence or the presence of 0.5 to 125 µM of MalE competitor peptides. Cells were then washed three times and used as APCs to stimulate 105 PreS:T-specific T cell hybridoma 52A12. Eighteen-hour supernatants were tested using 104 cells/well of the IL-2-dependent CTLL cell line. Two days later, [3H]thymidine (0.4 µCi/well) was added, and the cells were harvested 18 h later with an automated cell harvester. Incorporated thymidine was detected by scintillation counting. Results are expressed as the percentage of response of the 52A12 T cell hybridoma to 1 µM of the peptide PreS:T in the absence of competitor peptide.

Proliferation assay

Mice were immunized s.c. with peptides or chimeric MalE proteins emulsified in CFA (Sigma Chemical Co., St. Louis, MO). Seven to ten days later, draining inguinal lymph nodes were removed, and single-cell suspensions were prepared and cultured in HL-1 medium (Hycor Biomedical Inc., Irvine, CA) with 2 mM L-glutamine. Lymph node cells (8 x 105) (LNC)/well were plated onto 96-well microtiter plates (TPP, Trasadingen, Switzerland) in duplicate or triplicate with the indicated peptides. After 3 days at 37°C, cells were pulsed for 18 h with [3H]TdR (NEN, Boston, MA). Incorporated radioactivity was measured by scintillation counting. Results were expressed as mean cpm from duplicate or triplicate culture wells. Results are representative of two to three experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mapping of dominant MalE T cell determinants in DBA/1 mice

To map the dominant T cell determinants of the MalE protein, we used 15 amino acid-long synthetic peptides that walk over the MalE protein sequence with a single amino acid step. DBA/1 mice were immunized with 0.25 nmol of MalE protein, and proliferation of LNC from these mice was determined after in vitro stimulation with MalE synthetic peptides. MalE synthetic peptides were first tested as pools of 12 consecutive peptides to determine the dominant proliferative regions of the molecule (Fig. 1GoA). Eight pools of peptides positively restimulated MalE-primed LNC, corresponding to MalE sequences 71–96, 83–108, 107–132, 191–216, 203–228, 263–288, 323–348, and 335–360. As a second step, peptides from these positive series were tested individually for MalE-primed LNC proliferation to accurately map these T cell determinants. As seen in Figure 1GoB, six nonoverlapping dominant determinants were detected in DBA/1 mice, which can be defined by their core sequences as 89–95, 116–123, 198–205, 211–219, 274–281, and 335–341. We choose peptides p85–99, p112–126, p195–209, p207–221, and p330–344 among the different MalE peptides to further study these T cell determinants. Each of these peptides contains a single core sequence and gives a strong proliferative response (Fig. 1GoB). All of these peptides are probably presented to T cells by the I-Aq molecule, since only DBA/1 mice express this unique MHC class II restriction element (32).



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  		FIGURE 1. Mapping of the dominant T cell determinants of the MalE protein in DBA/1 mice. DBA/1 mice were primed s.c. with 0.25 nmol of MalE protein emulsified in CFA. Then, LNC were stimulated in vitro with overlapping 15-mer peptides covering the entire MalE sequence in a single amino acid step. A, Series of 12 peptides were pooled (2 µM of each peptide) and tested for reactivity. The peptide pools are referred to as the sequence encompassed by a peptide series. B, Overlapping peptides were tested individually at 6.5 µM and are referred as the NH2-terminal amino acid of each peptide. Proliferation was determined by [3H] incorporation at day 4. Proliferation of LNC with medium alone was 1281 cpm (A) and 6284 cpm (B).

 
In vitro competition between MalE peptides and PreS:T peptide for binding to I-Aq molecule

To verify that peptides carrying these MalE determinants were able to bind to I-Aq molecule, we performed an in vitro competition experiment in which MalE peptides were assayed for their capacity to inhibit the response of the 52A12 T cell hybridoma specific for the PreS:T peptide of HBsAg in association with the I-Aq molecule (18). As shown in Figure 2Go, the stimulation of the 52A12 T cell hybridoma by 1 µM of the PreS:T peptide was determined in the absence or the presence of various amounts of the different MalE peptides (0.5 to 125 µM). The p330–344 peptide competed strongly with the PreS:T peptide for binding to I-Aq molecule, since 50% inhibition of the maximal stimulation induced by 1 µM of the PreS:T peptide was obtained with 4 µM of this peptide (IC50 = 4 µM). Peptides p85–99 and p112–126 competed moderately for I-Aq binding (IC50 = 39 µM), whereas p195–209, p207–221, and p271–285 competed weakly with the PreS:T peptide (IC50 values of 67, 64, and 81 µM, respectively). All of the IC50 values were calculated from the experiment shown in Figure 2Go. These values vary slightly among the three experiments performed, but the classification of these peptides in three distinct categories was always identical. The control MalE peptide p40–64 did not modify the stimulation of 52A12 cells by the PreS:T peptide. These results show that all of the six MalE peptides containing DBA/1-reactive T cell determinants inhibited the binding of the PreS:T peptide to I-Aq MHC molecule, but with various efficacy, underlining a hierarchy in their capacity to bind I-Aq.



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  		FIGURE 2. Inhibition by MalE peptides of the 52A12 T cell hybridoma response to the PreS:T peptide restricted by I-Aq. 52A12 T cell hybridoma was cultured in the presence of glutaraldehyde-fixed M12C10 lymphoma B cells preincubated with 1 µM of the PreS:T peptide mixed with serial dilution (0.5 to 125 µM) of the indicated MalE peptide. After 24 h, IL-2 production was assessed with the CTLL cell line. Results are presented as the percentage of the response to 1 µM of PreS:T peptide alone.

 
In vivo competition between MalE and PreS:T synthetic peptides for T cell activation

To confirm these results obtained in vitro, we next assessed the capacity of the MalE peptides to compete in vivo with the PreS:T peptide for I-Aq binding by analyzing the T cell response of DBA/1 mice immunized with the PreS:T peptide mixed with the MalE peptides. To do so, we choose the peptides p207–221, p85–99, and p330–344, which, respectively, displayed weak, intermediate, and strong relative inhibitory capacity in vitro. DBA/1 mice were immunized either with the PreS:T peptide or the MalE peptides alone, or with an equimolar mixture of the peptide PreS:T together with one of the three MalE peptides. LNC from these mice were then tested for proliferative response to the immunizing peptides. After immunization with peptide PreS:T mixed with peptide p85–99 (Fig. 3GoA) or with peptide p207–221 (Fig. 3GoB), the proliferative response to p85–99 and p207–221 was reduced 50% as compared with the response induced by these peptides injected alone, whereas the response to PreS:T was unchanged in all cases. In contrast, as shown in Figure 3GoC, coimmunization with the peptides PreS:T and p330–344 induced a proliferative response to both peptides similar to or even slightly higher than the one obtained with the peptides injected separately. These results show that at a 1:1 molar ratio, the PreS:T peptide impairs the in vivo presentation of MalE peptides of weak (p207–221) and intermediate (p85–99) in vitro inhibitory capacity, indicating that these two MalE peptides bind more weakly to I-Aq than the PreS:T peptide. In contrast, the fact that coimmunization with the p330–344 and the PreS:T peptides triggered a similar proliferative response than after separate immunization suggests that both peptides bind I-Aq with a similar efficiency.



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  		FIGURE 3. In vivo competition between the PreS:T peptide and the MalE peptides in DBA/1 mice. DBA/1 mice were immunized separately with 0.25 nmol of the PreS:T peptide ({blacktriangleup} in A, B, and C) or 0.25 nmol of one of the following MalE peptides alone ({blacksquare}): p85–99 (A), p207–221 (B), and p330–344 (C); and LNC were restimulated with the homologous peptide. Other groups of mice were immunized with 0.25 nmol of the peptide PreS:T mixed with 0.25 nmol of the following MalE peptide (open symbols): p85–99 (A), p207–221 (B), and p330–344 (C); and LNC were restimulated with the PreS:T peptide ({triangleup}) or the immunizing MalE peptide ({square}). In D, mice were primed with 0.25 nmol of MalE peptide p207–221 ({blacksquare}) or p330–344 ({blacktriangleup}), and LNC were restimulated with the homologous peptide. Alternatively, mice were primed with a combination of peptides (open symbols) containing 0.25 nmol of p207–221 peptide mixed with 0.25 nmol of either p330–344 ({square}, {triangleup}) or C3 ({circ}), and LNC were restimulated with p207–221 ({square}, {circ}) or with p330–344 peptide ({triangleup}). Proliferation was determined by [3H] incorporation at day 4.

 
To verify this hierarchy of MHC binding between MalE peptides, mice were immunized with the peptide p207–221 alone or mixed with either the peptide p330–344 or the control peptide C3 (Fig. 3GoD). The response to p207–221 was reduced in the presence of the p330–344 peptide compared with the response induced with the p207–221 peptide alone or mixed with the control C3 peptide. By contrast, identical anti-p330–344 proliferative responses were observed in mice immunized with the p330–344 peptide alone or mixed with the p207–221 peptide. Altogether, the results obtained in vivo with synthetic peptides are in agreement with their relative inhibitory capacity for I-Aq binding observed in vitro.

Deletion of MalE amino acid sequences containing a given T cell determinant does not alter the immunogenicity of the remaining T cell determinants

To investigate the phenomenon of competition for MHC presentation between the different peptides emerging from the processing of the same protein Ag, we used insertion/deletion mutants of the MalE protein in which T cell determinant sequences were partially deleted. The MalEC3 chimeric proteins are mutants of the MalE protein in which amino acid sequences were deleted and replaced by the 11-amino acid sequence of the peptide C3 of poliovirus (31). If peptide competition in relation to MHC binding plays an important role in the selection of T cell determinants during Ag processing, it can be expected that immunization with such mutant proteins would lead to quantitative modifications of the peptides displayed by MHC molecules and of the subsequent T cell responses.

Mice were immunized with either wild-type MalE protein or MalE206C3, MalE211C3, or MalE339C3 chimeric proteins. Then, the proliferation of LNC was examined in response to the six MalE peptides containing the different T cell determinants previously characterized (Fig. 4Go). As expected, after priming with MalE206C3 and MalE211C3, which carry, respectively, the deletion of sequence 207–216 and 212–220, disrupting the same T cell determinant core sequence 211–219 in two different ways, no proliferative response to the peptide p207–221 was observed (Fig. 4GoD). In contrast, the responses to the remaining T cell determinants using peptides p85–99, p112–126, p195–209, p271–285, and p330–344 were unaffected as compared with the responses obtained after priming with wild-type MalE protein. Likewise, the deletion of amino acids 340 to 357 in MalE339C3 abolished the induction of the p330–344-specific T cell response (Fig. 4GoF), whereas the responses to peptides p85–99, p112–126, p195–209, p207–221, and p271–285 were similar to that induced by the wild-type MalE protein.



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  		FIGURE 4. Deletion of MalE amino acid sequences containing T cell determinants does not modify the immunogenicity of the remaining determinants. Mice were immunized s.c. with 0.25 nmol of mutant proteins MalE206C3 ({circ}), MalE211C3 (•), or MalE339C3 ({triangleup}), or with 0.25 nmol of wild-type MalE protein ({square}) emulsified in CFA. Then, LNC were stimulated in vitro with different concentrations of the indicated MalE peptide. Proliferation was determined by [3H] incorporation at day 4.

 
Therefore, despite the fact that peptide competition was observed in vivo using synthetic MalE peptides, the 211–219 and 335–341 T cell determinants do not influence each other within the MalE molecule. It is noteworthy that the presentation of the 198–205 T cell determinant did not benefit by the deletion of the adjacent 211–219 T cell determinant in MalE206C3 and MalE211C3 proteins. Altogether, these results show that modifications in the amino acid sequence of two different MalE T cell determinants did not introduce any change in the immunogenicity of the remaining T cell determinants, indicating that they behave as autonomous elements at the peptide-MHC-binding level. It should be noted that we also scanned the T cell responses with series of overlapping MalE peptides (as in Fig. 1GoA) after priming with mutant MalEC3 proteins to determine whether determinant disruption led to the emergence of new T cell reactivities in these proteins (but this was not the case; data not shown).

Lack of competition between MalE peptides and the PreS:T peptide when associated within the same molecule

Since in vitro and in vivo competition for I-Aq binding between the PreS:T and the MalE peptides was shown using synthetic peptides, we next tested whether this process occurs when these peptides are part of the same molecule. The insertion of the peptide PreS:T into MalE was realized in two internal insertion sites, positions 133 and 303 (18). DBA/1 mice were immunized with 0.25 nmol of MalE, MalE133-PreS:T, or MalE303-PreS:T protein and tested for proliferative responses to the peptide PreS:T and the MalE peptides (Fig. 5Go). In these conditions, both MalE133-PreS:T and MalE303-PreS:T induced an efficient PreS:T-specific proliferative response, showing that the PreS:T-inserted peptide was available on I-Aq molecules and that it was efficiently presented to T cells. Furthermore, the eight-peptide series that induced proliferation to MalE-primed LNC also stimulated LNC from mice primed with either MalE133-PreS:T or MalE303-PreS:T, demonstrating that the peptide specificity of the MalE-primed T cells was not affected by the presence of the heterologous PreS:T peptide. Proliferative responses obtained after restimulation with single MalE peptides p85–99, p112–126, p195–209, p271–285, and p330–344 displayed similar dose curve responses after priming DBA/1 mice with MalE-PreS:T and with wild-type MalE protein (data not shown).



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  		FIGURE 5. The insertion of the PreS:T peptide within MalE protein does not alter the proliferative response to MalE T cell determinants. Mice were immunized s.c. with 0.25 nmol of either MalE133-PreS:T (hatched bars), MalE303-PreS:T (filled bars), or wild-type MalE (open bars). Then, LNC were stimulated in vitro with MalE protein (0.1 µM), PreS:T peptide (10 µM), or MalE peptides pooled in series of 12 overlapping 15-mer peptides with a one-amino acid step (2 µM of each peptide). Proliferation was determined by [3H] incorporation at day 4.

 
An important feature of competition between antigenic peptides for MHC-binding relies on the stoichiometry of processed peptides available after Ag degradation (33). To determine whether the stoichiometry of the heterologous peptide generated after the processing of MalE-PreS:T proteins could modify the presentation of MalE T cell determinants, four consecutive copies of the PreS:T peptide were introduced in MalE at site 133 or 303. These proteins are referred to as MalE133-PreS:T4B and MalE303-PreS:T4B (18). In our previous work, we performed a detailed comparison of in vivo and in vitro immunogenicity of the MalE-PreS:T and MalE-PreS:T4B proteins showing that the efficiency of the PreS:T presentation was highly increased when inserted as four copies (18). As shown in Figure 6Go, the PreS:T determinant inserted at site 133 into MalE as a single copy stimulated a PreS:T-specific T cell hybridoma as efficiently as the synthetic free peptide, but the insertion of four copies markedly increased the presentation of the PreS:T insert. The presentation of the PreS:T insert was increased at least ~30-fold, but regarding the PreS:T epitope stoichiometry in MalE133-PreS:T4B, this corresponds to a 10-fold increase. This result shows that the loading of the PreS:T epitope on I-Aq molecules is strongly increased when MalE-PreS:T4B proteins are used as Ag.



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  		FIGURE 6. Comparison of the in vitro presentation of the PreS:T peptide inserted into MalE as a single copy or as four copies. Irradiated DBA/1 spleen cells were incubated with graded concentration of the PreS:T peptide, MalE133-PreS:T, MalE133-PreS:T4B, or MalE133-PreS:B proteins. These APC were then used to stimulate a PreS:T-specific T cell hybridoma. IL-2 production was assessed with the CTLL cell line.

 
Mice were immunized with MalE133-PreS:T4B or MalE303-PreS:T4B proteins, and proliferative responses to MalE peptides were compared with the response of MalE-primed mice. As shown in Figure 7Go, the MalE-PreS:T4B induced a very strong proliferative response specific for PreS:T. However, the responses to peptides p85–99, p112–126, p195–209, p207–221, and p330–344 were very similar after priming with either wild-type MalE or MalE-PreS:T4B proteins. It should be noted that the PreS:T peptide proliferative response induced by the MalE-PreS:T4B proteins was higher than the response to the different MalE peptides (Fig. 7Go), whereas the PreS:T response induced by the MalE-PreS:T proteins was weaker than the response to most of the MalE T cell determinants (Fig. 5Go), confirming that the level of presentation of the PreS:T peptide is strongly increased with the multicopy insert as compared with the single-copy insert.



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  		FIGURE 7. Lack of peptide competition between MalE peptides and the PreS:T peptide after insertion of four copies of the PreS:T peptide into MalE. Mice were immunized s.c. with 0.25 nmol of either MalE133-PreS:T4B ({triangleup}), MalE303-PreS:T4B ({circ}), or MalE ({blacksquare}). Then, the proliferative responses of LNC were determined after in vitro recall with different concentrations of the PreS:T peptide (A) or the indicated MalE peptide (B–G). Proliferation was determined by [3H] incorporation at day 4.

 
Altogether, these results show that the introduction of a competitor peptide as a single copy or as four copies into the MalE protein sequence does not affect the presentation of the T cell determinants of the recipient MalE protein, indicating that the competition for MHC presentation between peptides originating from the same molecule does not account for determinant selection.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Among the parameters controlling the immunodominance of T cell epitopes, it has been proposed that there exists a competition for binding to MHC class II molecules between the processed peptides contained within a single antigenic molecule (14). Experimentally, when mice were immunized with a mixture of two immunogenic peptides of hen egg lysozyme (HEL) having differences in their relative binding capacity to the same class II molecule, the T cell proliferative responses to the weakest binder was inhibited by the strongest binder as a result of MHC occupancy (11). Such in vivo competition between synthetic peptides for binding to MHC was extensively demonstrated (33, 34, 35) and support the hypothesis of peptide competition. However, this hypothesis is based on the artificial situation of immunizations with free peptides, which represent a processed state of the Ag.

In the present study, we tested this hypothesis with bound T cell peptide sequences in a model Ag, MalE. The central question is whether the competition process really drives the peptide presentation following intracellular processing events. After priming DBA/1 (I-Aq) mice with MalE protein, proliferative response to overlapping peptides revealed six distinct T cell determinants in this strain of mice. After priming with mutant MalE proteins carrying deletions within two of these determinants, the immunogenicity of the remaining T cell determinants was unchanged compared with the wild-type protein. Likewise, the insertion of a single copy or several copies of the PreS:T peptide from HBsAg into the MalE protein did not affect the immunogenicity of the MalE T cell epitopes, although we demonstrated that the PreS:T synthetic peptide was able to compete in vivo with some of these MalE peptides in conjunction with their relative I-Aq binding capacity. Altogether, our results indicate that in a single protein Ag, the presentation of each determinant occurs independently of the others.

These experiments were performed by immunizing mice for a given T cell determinant with the same molarity of either the protein or the synthetic peptides. One explanation for the discrepancies we observed would be that synthetic peptides enable the saturation of available class II binding sites, whereas chimeric protein-derived peptides do not. Conversely, the half-life of a protein Ag in the animal body is much higher than the half-life of a 15-mer synthetic peptide. In both cases, therefore, it is difficult to determine whether we reached saturating conditions for the available class II molecules, but following a local immunization, one might expect that the Ag dosage required to saturate APC in the vicinity of the injection site can be achieved without a very large amount of Ag.

There is growing evidence that synthetic peptides may not totally mimic physiologically processed peptides, which may therefore explain the discrepancies between the results we obtained using synthetic peptides and those obtained with chimeric proteins.

First, it is now well established that a given antigenic T cell determinant is displayed by MHC molecules as a set of peptides extended at either end of the core sequence (36, 37). Therefore, features belonging to a particular synthetic peptide may not represent the heterogeneity of the naturally processed peptides. Second, free peptides may not activate the same T cell repertoire than intracellularly derived peptides (38, 39). For instance, Viner et al. (39) have shown that the same peptide/MHC complex, HEL peptide 47–62 bound to I-Ak, stimulated a particular set of T cell hybridomas when this complex was formed using extracellularly added free peptide but not when it was formed following intracellular processing. To explain these results, it was proposed that the peptide accommodation in the MHC-binding groove may vary depending on parameters linked to the local environment of MHC molecules at the time of peptide acquisition, such as the pH level or the presence of Ii or HLA-DM/H-2 M as accessory molecules. Indeed, APC fed with synthetic free peptides do not complex nascent class II molecules, since these peptides are mainly available for MHC binding at the cell surface or in recycling vesicles, and they accumulate poorly in intracellular compartments (40). Therefore, our data reveal that the competition process observed in vivo between MalE and PreS:T synthetic peptides is ineffective when these peptides are carried by chimeric MalE-PreS:T proteins, probably because of intracellular Ag processing steps required for presentation of these different T cell determinants.

It has also been proposed that the processing steps may be under the control of MHC class II proteins leading to a MHC-guided processing (4). Following internalization, the Ag travels through a pH gradient and a reducing environment in the endocytic pathway. In these conditions, the protein is partially unfolded, displaying more or less its determinant sequences, which may be selected by class II molecules and protected from degradation allowing their further presentation at the cell surface. The molecular basis of this hypothesis relies on: 1) the ability of class II to protect immunogenic peptides from in vitro proteolytic degradation (41, 42); and 2) the ability of class II molecules to bind unfolded protein Ags (43, 44, 45). Through this mechanism, it is expected that the capture of antigenic determinants is driven by their affinity for MHC molecules and is thus subjected to competition, unless they are still buried within the antigenic molecule in a partially unfolded state.

Based on the structural features of the MalE protein, the MalE-PreS:T proteins should be favorable for such a determinant capture process. First, the permissive insertion sites into MalE correspond to regions of the protein that are not involved in its folding (46, 47) and are surface exposed so that the peptide insertions do not disturb the overall structure of MalE. The exposition of an inserted peptide at site 133 of MalE was confirmed recently by the crystal structure of such chimeric proteins (48). Second, replacing the determinant core sequences on the MalE 3D-structure (49), the six determinants are also surface exposed on the molecule. Therefore, despite the fact that either the PreS:T insert or the MalE T cell determinants are apparently easily available on the intact molecule for binding to I-Aq molecules, we did not detect any competition between these determinants that might result from determinant capture.

In vivo evidence for a class II-mediated determinant selection was provided by Deng et al. (50) in the HEL system. This study shows that in F1(NOD x BALB/c) mice, the response to the 95–102 determinant of HEL (restricted by I-ANOD) was subjugated by the closely located 108–116 determinant (restricted by I-Ed). The disruption of the peptidic bond into the HEL protein between these two determinants using cyanogen bromide was able to restore the response to the 95–102 determinant. Interestingly, this competition process arises between two closely located binding sequences, since these determinant cores are separated by only six residues. The 95–102 and 108–116 HEL determinants are contained in a short sequence of 22 residues, and therefore, upon strong affinity differences, this neighborhood may force a determinant choice by class II molecules, in agreement with the length of processed peptides bound to class II, which ranges from 13 to 28 residues (37). Thus, the competition for class II presentation is likely to occur at the protein level, but may also occur at the peptide level. Effectively, it was previously demonstrated that competition for binding to class II molecules is likely to occur within a single peptide containing two synthetically linked epitopes (51, 52, 53). Furthermore, the cyanogen bromide cleavage of HEL breaks the continuity of the HEL sequence, but the two determinants still belong to the same molecule, since the four disulfide bonds maintain the integrity of the HEL molecule. Therefore, this strongly suggests that the competition for MHC presentation between T cell determinants within a single molecule does not take place at long distance along the molecule. In MalE-PreS:T chimeric proteins, the potential PreS:T competitor peptide is separated from the nearest MalE core determinant by 12 and 24 residues at site 133 and 303, respectively, without inducing any competition. This feature strengthens the idea that if competition happens, it only occurs between very closely linked binding sequences.

Another aspect pointed out by recent studies concerns the possibility that some determinants are presented by nascent class II in an Ii- and HLA-DM-dependent manner, whereas others are presented by mature class II in recycling vesicles (44, 54, 55, 56, 57). In a single protein Ag, it is expected that the different T cell determinants that will be presented by class II molecules require more or less catabolic activity to be generated. Thus, during the intracellular trafficking of the Ag, there might be a compartment-associated processing in which some determinants will be available for class II in early endocytic vesicles (55), whereas others will be generated in late endocytic vesicles (54) depending upon the acidification level and the protease content of the local environment. Moreover, some determinants that are very sensitive to proteolysis may be available only in early endosomes, since they may be degraded in later endocytic compartments. Therefore, the model of Ag processing so-proposed will be that distinct T cell determinants carried by the same protein Ag will be produced in distinct compartments through the endosomal/lysosomal route (58).

We have some pieces of information concerning the processing requirements of the PreS:T insert, but not for the presentation of MalE determinants. Indeed, the stimulation of the PreS:T-specific T cell hybridomas by APC pulsed with MalE-PreS:T proteins is chloroquine sensitive and cycloheximide sensitive (data not shown), demonstrating that the presentation of the PreS:T insert requires intracellular processing and newly synthesized class II molecules, which suggests a late processing compartment. If the MalE determinants are produced earlier in the endocytic route, they may not compete with the PreS:T peptide. This possibility cannot be totally excluded, although it would be surprising to find that all six of the MalE determinants were processed in a compartment different from the one used for the PreS:T insert. If this were true, however, a competition could still occur between the MalE determinants, and the retrieval of determinant sequences in deleted MalE proteins would lead to a noticeable modification of the presentation of the remaining determinants. However, this was not the case. Therefore, the lack of competition we observed cannot be explained merely by the fact that the different determinants may be generated in distinct endocytic compartments.

It was recently shown in the HEL system that feeding APC with an excess of antigenic molecules led to the internalization of a small fraction of Ag (0.3%), and that finally only 1 of 750 internalized HEL molecules was converted into the dominant 48–62 peptide/I-Ak complex (59). This poor presentation efficiency suggests that if there is peptide competition, this occurs most likely with self protein-derived peptides rather than with other antigenic peptides located in the same molecule. Therefore, parameters other than intramolecular peptide competition for MHC binding—such as the yield of processed peptides or the ability of these peptides to replace endogenous self peptides (including class II-associated Ii chain peptide (CLIP)) to load and stabilize MHC molecules—may be much more important for peptide presentation.

The results presented in this study indicate that peptides that arise from the intracellular processing of chimeric MalE-PreS:T proteins do not compete for MHC binding and presentation by APC, whereas they are able to compete, either in vitro or in vivo, when given to APC as exogenous free synthetic peptides. Therefore, these results demonstrate that competition between peptides belonging to the same antigenic molecule for MHC binding may not represent an important parameter in the selection of peptides that can be presented to T lymphocytes.


   Acknowledgments
 
We thank W. C. Puijk and Dr. W. M. M. Schaaper for their excellent work in synthesizing peptides.


   Footnotes
 
1 This work was supported by a grant from the European Economic Community (EEC) Biotechnology Program, Contract PL920349. R.L.M. was supported by a fellowship from the CANAM. Back

2 Address correspondence and reprint requests to Dr. Richard Lo-Man, Institut Pasteur, 25 rue du Docteur Roux 75724 Paris cédex 15, France. E-mail address: Back

3 Abbreviations used in this paper: HBsAg, hepatitis B surface Ag; LNC, lymph node cells; IC50, 50% inhibition of maximal stimulation; HEL, hen egg lysozyme; [3H] incorporation, [3H]thymidine incorporation. Back

Received for publication August 4, 1997. Accepted for publication October 31, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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