Invariant chains with the class II binding site replaced by a sequence from influenza virus matrix protein constrain low-affinity sequences to MHC II presentation

Cornelia Carstens, Debra K. Newman1, Heribert Bohlen2, Angelika König and Norbert Koch

Division of Immunobiology, University of Bonn, Römerstrasse 164, 53117 Bonn, Germany
1 The Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, WI 53201-2178, USA
2 Clinic I for Internal Medicine, University of Cologne, 50924 Cologne, Germany

Correspondence to: N. Koch


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Presentation of antigenic peptides by MHC II molecules is required to initiate CD4 Th cell responses. Some peptides, however, because of low affinity for MHC II, are not efficiently presented. A segment of the MHC II chaperon molecule, invariant chain (Ii), is known to bind early in biosynthesis with low affinity to the peptide binding groove. Here we have exploited the properties of Ii to manipulate the MHC II-loading pathway and to present low-affinity sequences. We used a deletion mutant of Ii where the promiscuous binding site to MHC II, which is adjacent to the groove binding segment, was deleted. A recombinant Ii (rIi) chimera, derived from this construct, was made in which the class II binding segment was exchanged for wild-type or single amino acid substitution variants of an HLA-DR1-restricted sequence from influenza matrix protein (MAT), which leads to MHC II allotype-specific binding. This rIi was expressed in antigen-presenting cells (APC) and introduced the MAT sequence into the MHC II-processing pathway. As expected, rIiMAT elicited antigen-specific, DR1-restricted T cell cytokine production and proliferation. Significantly, rIiMAT, that binds the HLA-DR4 allele with low affinity, elicited DR4-restricted IL-2 production but not proliferation. In contrast, exogenously provided MAT peptide failed to elicit any responses from DR4-restricted T cells. Compatible results were obtained with a single amino acid substitution variant (MATT), which binds with high affinity to DR4 but low affinity to DR1. We conclude that loading of MHC II with antigenic peptides from endogenously synthesized rIi chimeras allows presentation of low-affinity sequences that cannot be presented if provided exogenously as peptides. Ii fusion proteins containing low-affinity antigenic sequences might be useful for vaccination with tumor antigens to overcome deficiencies in antigen presentation.

Keywords: antigen processing, antigen presentation, epitope, gene vaccination, MHC


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Introduction of foreign genes into antigen-presenting cells (APC) is frequently conducted to challenge the immune system. One aim of this gene transfer is to elicit an immune response when such a response is deficient under normal physiological conditions. An impaired immune response can be the result of imperfect MHC–TCR contact or inefficient antigen processing and presentation. In addition, some antigens are not presented because of the low affinity of their interaction with MHC-encoded restricting elements, although amino acid residues for interaction with the TCR are available.

Presentation of exogenous antigens is a natural task of MHC II molecules (1). Before antigens are recognized by the TCR, they are internalized and degraded by APC, after which fragments of processed antigen are bound to MHC II {alpha}ß heterodimers (2). The intracellular encounter of antigens with MHC II molecules is well characterized by previous studies showing the critical role of invariant chain (Ii), HLA-DM and HLA-DO in peptide loading (35). Ii attains direct contact with MHC II {alpha}ß heterodimers in the endoplasmic reticulum. During biosynthesis it assembles with MHC II molecules by binding to the peptide binding cleft (6). The most important role of Ii is to guide the MHC II complex on its biosynthetic route to endocytic vesicles (7). In endocytic compartments, such as the CIIV and MIIC vesicles, MHC II molecules undergo a conformational change (8,9). A regulated degradation of Ii produces MHC II heterodimers transiently receptive to binding of antigenic peptides (10). Interaction with HLA-DM, which edits the peptides, generates stable MHC II–peptide complexes (11).

The properties of Ii have been employed to introduce antigens into the MHC II-processing pathway (1216). Recombinant replacement of the class II binding site of Ii by an antigenic sequence yields an Ii–antigen fusion protein (rIi) that assembles in the endoplasmic reticulum with MHC II heterodimers (17). There, the inserted antigenic sequence is accommodated in the MHC II cleft. Further deletion of a promiscuous binding site of rIi leads to MHC II allotype-specific binding of the antigenic sequence (17). Intracellular degradation of rIi generates MHC II–peptide complexes that can be transported to the cell surface of APC and detected by antigen-specific CD4+ T cells (18). An early delivery of T cell epitopes to MHC II molecules has the advantage that, after binding of the sequence to MHC II dimers, the antigen is protected against proteolytic destruction. Moreover, the abundant loading of MHC II dimers by rIi could promote presentation of low-affinity sequences.

We show here that an Ii-inserted T cell epitope of influenza virus A matrix protein (MAT) with allotype-specific binding to DR1 is presented to antigen-specific T cells. Mutation of one DR1 anchor position of MAT almost abolishes binding to DR1, but strongly elevates its binding to DR4. While APC incubated with MAT peptides with low DR affinities elicit no T cell response, endogenously expressed rIiMAT with mismatched sequences for binding to DR1 or to DR4 stimulates antigen-specific T cells to secrete IL-2.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
DNA constructs
The DNA encoding rIiMATA and rIiMATT have been described before (17). In rIiMATT, amino acids 81–101 of murine Ii are replaced by SGPLKAEITQRLEDV (MATaa17–31). rIiMATA and rIiMATT differ in Ala or Thr in position 25 of MAT. DNA for recombinant Ii and DR chains were expressed under SV40 promoter control in the pcExV3 expression vector (7,19).

APC
COS7 (DSMZ ACC 60) cells were cultivated in high glucose DMEM supplemented with 10% inactivated FCS, 100 µg/ml penicillin, 100 U/ml streptomycin, 1 mM sodium pyruvate, 10 mM HEPES and 2 mM L-glutamine. The B cell lymphoma lines JESTHOM, BM92 (kindly provided by J. G. Bodmer, ICRF, London) and Epstein–Barr virus-transformed B cell lines (BLCL) from healthy donors were kept in RPMI 1640 supplemented as described above. Transient transfection of suspended cells was performed by liposome-mediated transfer using 1 µg uncleaved DNA and 10 µg DOSPER (Boehringer, Mannheim, Germany) per 5x105 cells (20). In brief, 5x105 cells were incubated in the liposome formulation of 1 µg uncleaved DNA and 10 µg cationic lipid (DOSPER) in serum-reduced essential medium (OptiMEM; Gibco/BRL, Eggenstein, Germany) with 10–5 M ß-mercaptoethanol. The fusion of the lipid complex with cell membranes was accomplished after 20 min by addition of 10% FCS. At 48 h after incubation, the uptake and expression of DNA was assessed by immunofluorescent staining with the mAb In1 that recognizes the N-terminal region of murine Ii.

Responding T cells
Peripheral blood lymphocytes (PBL) from healthy adult donors were obtained by sedimentation of heparinized blood in Ficoll-Isopaque solution (Pharmacia, Uppsala, Sweden). DR restriction and MAT responsiveness were investigated in a 5 day antigen-presentation assay using MATA and MATT peptide-loaded autologous BLCL, and human B cell lines homozygous for DR1 (JESTHOM) or DR4 (BM92) as APC. PBL from donors BC13 and BC17 were responsive to MATT and MATA respectively, without showing a detectable allotype response towards JESTHOM or BM92. The DR1-restricted CD4+ T cell clone with specificity to MATA (MP10.4) has been described (21). MP10.4 re-stimulation was performed every 10 days using equal amounts of stimulator cells. Stimulators were DR1+ PBL and JESTHOM pulsed with 1 µg/ml MATA peptide. Stimulators were fixed by 1 h incubations in 0.25% para-formaldehyde in PBS (v/v), and subsequently in PBS with 5% glycine and 1% human AB serum (Sigma-Aldrich, Deisenhofen, Germany). After stimulation, 0.5 U/ml IL-2 (Biotest, Dreieich, Germany) and 10% human AB serum were added. Only for the first T cell expansion, phytohemagglutinin (Difco, Hamburg, Germany) was added at a submitogenic concentration (100 ng/ml). Human T cells were grown in RPMI 1640 with 10% FCS. The murine IL-2-dependent indicator cell line CTLL-2 (DSMZ ACC 27) was kept in IMDM with 5% FCS and 2 U/ml human IL-2. Other supplements were as described for B cells.

Antigen-presentation assays
APC were JESTHOM (DR1+) or BM92 (DR4+), and COS7 cells transfected with DNA for DR1 or DR4. For antigen presentation, APC were transfected with DNA for wild-type Ii, rIiMATA or rIiMATT. Transfection rates were ~35% for B cells and 60% for COS7 cells. To investigate antigen presentation, 1x105 UVB-irradiated (20,200 J/m2) APC were co-cultivated with 4x103 MP10.4 cells or 1x105 PBL. After 48 h, proliferation and IL-2 secretion was determined. To assess proliferation, samples were pulsed with 0.5 µCi [3H]thymidine (Amersham, Braunschweig, Germany) during the last 18 h of culture. Cells were harvested and incorporated radioactivity was determined by liquid scintillation spectrometry. [3H]Thymidine incorporation of irradiated APC and medium control valued <200 c.p.m. To assess IL-2 secretion, supernatants were harvested and incubated with 1x104 CTLL-2 indicator cells. [3H]Thymidine incorporation in the last 18 h of 24 h co-cultivation was measured. To calculate the amount of IL-2, CTLL-2 proliferation obtained with sample supernatant was compared to the CTLL-2 proliferation obtained with defined concentrations of human IL-2 (Biotest). In this case, 1 IU IL-2/ml corresponds to the incorporation of 50,000 c.p.m. into 1x104 CTLL-2 cells in 100 µl. To obtain control values for the presentation capacity of transfected APC, half of the transfected APC were additionally pulsed with 100 nmol MATA peptide. Peptide was added 2–6 h before UVB irradiation. MATA (SGPLKAEIAQRLEDV) and MATT (SGPLKAEITQRLEDV) peptides were produced by the peptide synthesis facilities of the Imperial Cancer Research Fund (London, UK). The peptide extMATT (LRMKLSGPLKAEITQRLEDVSMDNM) was obtained from Genosys (Cambridge, UK).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
DR allotype-mismatched antigen elicits an IL-2 response
The MHC II binding segment of Ii was replaced by the wild-type antigenic sequence amino acids 17–31 of MAT (rIiMATA) (Fig. 1Go). rIiMATA lacks the promiscuous MHC II binding site of Ii. Thus binding to MHC II is exclusively mediated by the MAT sequence. We recently showed that the mutation Ala25 to Thr in MAT (rIiMATT) directs binding of rIiMATT to the DR4 allotype (17). B lymphoma cells homozygous for DR1 or DR4 and COS7 cells transiently expressing DR1 or DR4 were transfected with constructs encoding rIiMATA, rIiMATT or Ii. These transfected cells were used as APC to activate the DR1/MATA-specific T cell clone MP10.4 (21). IL-2 release by MP10.4 was assessed by stimulation of CTLL-2 indicator cells. Secretion of IL-2 was observed upon stimulation with APC expressing DR1/MAT but not with DR4/MAT or without antigen (Fig. 2AGo, solid bars). Significantly, we observed IL-2 secretion after challenge with rIiMATT. This indicates that the DR1-mismatched sequence MATT is presented by DR1.



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Fig. 1. rIiMAT construct and encoded fusion protein. The genetic information for MAT-derived antigenic sequence 17SGPLKAEIAQRLEDV31 was introduced into an expression vector containing an Ii deletion mutant. DNA and protein of rIiMATA are presented schematically. The black section shows the antigenic sequence MATaa17–31 inserted in position of the Ii class II-binding site (Iiaa81–101). The mutated construct rIiMATT contains the amino acid substitution Ala25 to Thr in position MATaa25. This mutation alters an anchor residue for binding to DR1. Exon boundaries are indicated by vertical lines. The transmembrane region is shown by diagonal strips.

 


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Fig. 2. DR1+ APC expressing rIiMAT are recognized by a MAT-specific T cell clone. Presentation of MAT was investigated with the DR1-restricted CD4+ T cell clone MP10.4. APC were human B cell lines homozygous for DR1 or DR4, and COS7 cells transfected with DR1 cDNA. For antigen presentation, APC were transfected with DNA for Ii, rIiMATA or rIiMATT, as indicated on the left. MP10.4 cells were stimulated with transfected APC (filled bars) or with APC additionally pulsed with 100 nM MATA (open bars). MP10.4 cells (4x103) were co-cultivated with 1x105 UVB-irradiated APC. After 24 h, IL-2 production (A) and proliferation (B) was determined. To assess IL-2 production, 1x104 CTLL-2 indicator cells were cultivated in APC/MP10.4 supernatant. Proliferation was measured by [3H]thymidine incorporation. The mean + SD of triplicates is shown.

 
Subsequently, we tested the proliferative response of MP10.4 challenged with rIiMATA and rIiMATT. Proliferation was only achieved with rIiMATA-expressing DR1+ APC (Fig. 2BGo, solid bars). rIiMATT/DR1+ APC or DR4+ APC did not induce a proliferative T cell response. This confirms the originally reported specificity of MP10.4 T cells for MATA (21). The APC that were employed to stimulate MP10.4 were able to elicit a maximal response. This is shown by loading of the APC with MATA peptide (Fig. 2A and BGo, open bars).

The incompetence of rIiMATT to induce proliferation is in contrast to its ability to induce IL-2 release. Possibly a partial T cell activation is achieved with the MATT sequence albeit the peptide shows deficient binding to DR1 (22). Accessory molecules are not likely to be important for the observed IL-2 release because COS cells are non-professional APC that do not express co-stimulatory molecules (23), yet DR1/MATA-expressing COS7 cells could induce both IL-2 release (Fig. 2AGo) and proliferation (Fig. 2BGo). At this stage we could not rule out that the MP10.4 T cell clone has an unusual property that gives rise to IL-2 secretion upon stimulation with rIiMATT.

Presentation of rIiMAT-derived peptides by mismatched DR allotypes elicits IL-2 production by T cells from DR1- and DR4-matched donors
Infections with influenza A virus are very common. Thus it is likely that many individuals possess MAT-specific T cells. We tested PBL from 16 donors for the presence of T cells responsive to MATA or MATT peptides presented by DR1+ or by DR4+ B lymphoma cells. Two donors were selected that either responded to MATA and DR1 or to MATT and DR4 (not shown). This response was antigen specific and no allotype reactivity was observed (not shown). A deficient allotype response against lymphoma cells has been described previously and might be explained by the lack of accessory molecules on the tumor cells (24). PBL from the two donors containing MAT-specific T cells were incubated with rIiMAT-expressing DR1+ or DR4+ APC. IL-2 secretion by the T cells was monitored. The polyclonal DR1-restricted T cells secreted IL-2 upon challenge with rIiMATA and with rIiMATT (Fig. 3AGo, left). These T cells did not respond to presentation by DR4+ APC. This result shows that polyclonal DR1-restricted T cells are activated to secrete IL-2 by the DR1-mismatched MATT. Nevertheless, MATT failed to induce a proliferative response by the polyclonal DR1-restricted T cells (Fig. 3AGo, right). Since the substitution of Ala25 by Thr alters an anchor position for binding of MAT to DR1, the residues presented to the TCR remain unchanged. However, it is still possible that the conformation of the T cell epitope is altered by the mutation of the anchor position.





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Fig. 3. Stimulation of DR1+ and DR4+ polyclonal T cells by rIiMAT. MAT-specific IL-2 production (left) and proliferation (right) were examined for DR1-restricted polyclonal T cells (A), DR4-restricted polyclonal T cells (B) and the DR1-restricted T cell clone MP10.4 (C). APC were B cell lines homozygous for DR1 or DR4 (DR1+BLCL or DR4+BLCL). APC were transfected with DNA encoding Ii, rIiMATA or rIiMATT, or were pulsed with 100 nM MATA or MATT peptides as indicated.

 
The availability of polyclonal DR4-restricted T cells enabled us to study the presentation of rIiMAT-derived antigens by DR4 molecules. Figure 3Go(B, left) shows IL-2 secretion of DR4-restricted T cells after challenge with rIiMATA or rIiMATT. Both DR4/MATT and DR4/MATA APC elicited an IL-2 response, whereas MATT but not MATA was able to induce proliferation of DR4-restricted polyclonal T cells (Fig. 3BGo, right). This is important, because it shows another DR allotype and different antigen-specific T cells responding to rIiMAT antigen. In addition, the use of donor PBL suggests that antigen presentation and T cell activation conducted in vitro resembles an in vivo immune response.

The observed dissimilarity in IL-2 secretion and proliferation could be explained by different affinities of the DR-matched and unmatched MAT sequences to DR molecules. We tested whether this result can be reproduced by presentation of synthetic peptides. APC incubated with MATA or MATT peptides were used to stimulate DR1- or DR4-restricted polyclonal T cells or the DR1-restricted MP10.4 T cell clone (Fig. 3AGo–C). DR1-restricted polyclonal T cells (Fig. 3AGo) or T cell clones (Fig. 3CGo) responded only to the MATA synthetic peptide, whereas DR4-restricted polyclonal T cells responded only to the MATT synthetic peptide (Fig. 3BGo). No cross-activation of T cells by the DR-mismatched peptides was obtained, as the synthetic MATT peptide failed to induce IL-2 secretion from either polyclonal DR1-restricted T cells (Fig. 3AGo) or the DR1-restricted T cell clone (Fig. 3CGo) and polyclonal DR4-restricted T cells failed to secrete IL-2 upon challenge with synthetic MATA peptide (Fig. 3BGo). The deficiency in proliferation upon activation with mismatched rIiMAT is explained by diminished IL-2R expression on the responding T cells (not shown). The cytokine pattern secreted by both MP10.4 cells and by polyclonal T cells shows that all rIiMAT constructs and the DR-matched peptide stimulate a Th1 rather than a Th2 response (not shown).

In contrast to rIiMAT, DR-mismatched MAT peptides elicit no T cell response
The ability of DR-mismatched rIiMAT, but not MAT synthetic peptide, to induce a T cell response may be attributable to higher occupancy of the MHC II cleft upon endogenous expression of rIiMAT than can be achieved by incubation of APC with 100 nM of MAT peptide. In support of this possibility, peptide binding studies revealed that affinities of MATA peptide to DR1 exceed that to DR4 by at least 2 orders of magnitude (25). Another report demonstrated high- and low-affinity binding of MATT peptide to DR4 and to DR1 respectively (22). Low-affinity binding of the MAT mutants to the mismatched DR allotypes might be compensated by the high concentration of rIiMAT available from biosynthesis. If the observed IL-2 secretion depends on the number of the presented MAT epitopes, an increased concentration of the mismatched MAT peptide should elicit elevated IL-2 secretion.

Similarly, high levels of occupancy of the MHC II cleft might be achieved by providing synthetic peptide at concentrations >100 nM. Thus, we tested the dose dependence of the T cell response to peptides that differ with respect to the presence of Ala or Thr at position 25 of MAT over a concentration range from 1 pM to 10 µM. T cell responses to peptide-loaded APC were compared to those elicited by rIiMAT-expressing APC. The levels of IL-2 secretion and proliferation induced by high concentrations of MATA peptide were similar to those induced by rIiMATA, suggesting that maximal stimulation of T cells was achieved (Fig. 4Go). In contrast, IL-2 secretion was observed in response to stimulation with rIiMATT but not synthetic MATT peptide, even at the highest peptide concentration (10 µM) tested.



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Fig. 4. Peptide dose–response curves of MP 10.4 T cells. DR1+ B cell lines were pulsed with increasing amounts of MATA, MATT or extMATT peptides. MP10.4 stimulation by peptide-loaded APC was compared to the MP10.4 response obtained with rIiMATA- and rIiMATT-expressing APC (bars).

 
Another possibility is that the flanking sequences of the endogenously processed antigen may contribute to stabilize binding to MHC II dimers. A peptide with an extended sequence was synthesized that contains five Ii-derived amino acids each at the N- and C-terminus of MATT (extMATT). Incubation of the peptide with APC will allow internalization of the 25mer. Intracellular degradation of extMATT may produce similar sized fragments as obtained after processing of rIiMATT. IL-2 secretion and proliferation of MP10.4 T cells was determined after challenge with APC (Fig. 4Go). The extMATT was not able to induce IL-2 production or proliferation. With amounts of the extMATT up to 10 µM no IL-2 secretion was observed. In a parallel experiment extMATT stimulated DR4-restricted T cells, verifying that the antigen is displayed by DR4 molecules (not shown).


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We demonstrate here that low-affinity antigenic sequences elicit an IL-2 response, when expressed as Ii chimeras. The observed allotype-specific binding of rIiMAT is achieved because Ii sequences adjacent to the MHC II groove binding segment are lacking in the Ii fusion proteins. These flanking sequences mediate binding to various MHC II allotypes despite the variable affinity of the groove binding segment of Ii to different MHC II alleles (17). As a consequence, the antigenic sequence of MAT determines the specificity of rIiMAT binding to DR allotypes. Allotype-specific binding of MAT leads to appropriately DR-restricted stimulation of T cells, which was demonstrated using a DR1-restricted T cell clone as well as DR1- and DR4-matched polyclonal T cells. The specificity of the T cells contained in the PBL is concordant with a random infection of the donors with influenza A virus. Thus the re-stimulation of these T cells by APC in vitro might resemble an in vivo challenge by re-infection with virus.

It was an unpredicted observation that rIiMATT elicits a DR1-restricted IL-2 response that was not obtained with the MATT peptide. Moreover, rIiMATA, but not MATA peptide, stimulates DR4-restricted T cells to secrete IL-2. Thus, two independent combinations of rIiMATA/DR4 and rIiMATT/DR1 elicit IL-2 secretion by antigen-specific T cells. This response appears not to be restricted to a single antigen/DR/TCR combination. It might be promising to conduct similar experiments with different antigens and with other DR allotypes for which it is desirable that a deficiency in antigen presentation be overcome. Thus, antigenic sequences with low affinity for class II binding might acquire the ability to stimulate T cells if they are presented to the MHC II antigen-processing/presentation pathway as a component of Ii. Th responses elicited with constructs such as these might be protective against infection or against tumor growth.

The data suggests binding of rIiMAT to a mismatched DR peptide binding cleft. We observed presentation of rIiMATT-derived peptide but not synthetic MATT peptide, in association with DR1, and of rIiMATA-derived peptide, but not synthetic MATA peptide, in association with DR4. Why are MAT sequences presented by mismatched DR molecules when they are provided as rIiMAT but not peptides? Early in biosynthesis DR {alpha} and ß chains assemble in the ER with Ii (26). The Ii chains promote folding of the DR dimers and prevent aggregation of MHC II with unfolded polypeptides that are available from biosynthesis in the endoplasmic reticulum (27,28). Antigen processing and peptide binding to MHC II molecules are highly controlled processes (29). When the MHC II-associated Ii is degraded in endocytic vesicles, a fragment of Ii, class II-associated Ii peptide (CLIP), remains bound to the MHC II cleft (30). CLIP has a moderate affinity for most MHC II allotypes (31). Dissociation of CLIP is enhanced by HLA-DM molecules (32). At this transient stage empty MHC II dimers are susceptible for binding of peptides (33). In vitro studies showed that binding of peptides at this receptive stage leads to stable and long-lasting MHC II–peptide complexes (10). Binding of mismatched rIiMAT to DR dimers might promote the generation of some stable DR complexes despite the low affinity of the MAT sequence. Possibly, stable DR complexes are not formed when low-affinity peptides are exogenously added. It was also possible that N- and C-terminal extensions of the processed rIiMAT exhibit different binding properties than synthetic MAT peptides. However, testing a synthetic MAT peptide with flanking sequences of Ii (cf. Fig. 4Go) could not substantiate this assumption.

One may speculate that the premature loading of the MHC II dimers by processed rIiMAT impedes further transport to acidic compartments but allows expression on the cell surface. This would explain why on its intracellular route DR-bound MAT fragments with low affinity are not released at low pH or exchanged by the catalytic action of DM. DM appears not to be critical for presentation of rIiMAT-derived sequences because we obtained no difference in presentation by DM+ B lymphoma cells and DM COS cells.

We have shown that rIiMAT associates in the endoplasmic reticulum with MHC II (17). MHC II binding of rIiMAT early in biosynthesis yields a high level of occupancy of the MHC II peptide cleft. It is likely that, after degradation of rIiMAT in endosomes, the antigenic sequence remains bound to MHC II molecules. The continuous production of rIiMAT may lead to enhanced loading of MHC II molecules. A high level of occupancy of DR dimers by processed rIiMAT could explain why presentation of low-affinity MAT peptides was observed under these conditions. This high number of presented T cell epitopes increases the avidity to the TCR. In comparison, it is possible that the number of bound ligands after incubation of APC with low-affinity MAT peptides is marginal and not sufficient to induce clustering of the TCR. It has been shown that even at high concentration of peptide only a few percent of surface MHC II are charged with the ligand (34). In addition, the level of the T cell epitope presented declines after pulsing the APC with synthetic peptide. Targeting antigens to the MHC II pathway has been shown to be more efficient in loading MHC II molecules than using exogenous peptides (35).

Other interpretations also may be considered. A preference for recognition by T cells for a peptide epitope was achieved after immunization with synthetic peptide in contrast to immunization with hen egg lysozyme (HEL) protein (36). A conclusion was that the interaction of free peptides with MHC II molecules could generate complexes that are antigenically dissimilar to those resulting from intracellular processing of intact HEL (37). Different to these experiments where the peptides stimulate the T cells, in our experiments the processed antigen elicits a T cell response. It is possible that the endogenously produced epitope shows a conformation that resembles the native antigen and is different to the MHC II–peptide epitope that is formed with synthetic peptide.

We show here that fusion proteins composed of Ii and a T cell epitope (MAT) mediate binding of low-affinity sequences to the peptide binding groove of DR molecules. Presentation of these MAT sequences elicited an IL-2 response but not proliferation, indicating a partial activation of T cells. This could suggest that activation of T cells with rIiMAT induces anergy such as has been shown with altered TCR ligands (38). However, the T cells partially activated by rIiMAT could be stimulated to proliferate by MAT peptide (Fig. 2Go). This result suggests that no anergy was induced.

A stepwise activation of T cells by rIiMAT may allow investigation of the TCR signal transduction pathway that is important for dissection of a T cell response. Since we used non-professional APC in our in vitro studies, the employment of dendritic cells that express additional co-stimulatory molecules could lead to an abundant activation of T cells.

The Ii–antigen fusion protein employed in this study describes a novel way to induce an immune response. Since Ii constructs can elicit a primary T cell response in mice (35), similar constructs might be used as vaccines against cancer or to overcome other immunodeficiencies.


    Acknowledgments
 
This study was supported by a grant from the Sonderforschungsbereich 502, Teilprojekt T2 and by the Deutsche Forschungsgemeinschaft Ko 810/4. We thank Dr P. Gleeson for critical reading of the manuscript.


    Abbreviations
 
APC antigen-presenting cell
BLCL B-lymphoblastic cell line
CLIP class II-associated Ii peptides (CLIP)
HEL hen egg lysozyme
Ii invariant chain
MAT matrix protein from influenza A virus
PBL peripheral blood lymphocyte
rIiMAT recombinant Ii–MAT fusion protein

    Notes
 
Transmitting editor: D. Tarlinton

Received 10 April 2000, accepted 28 July 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Ziegler, K. and Unanue, E. R. 1981. Identification of a macrophage antigen-processing event required for I-region-restricted antigen presentation to T lymphocytes. J. Immunol. 127:1869.[Abstract/Free Full Text]
  2. Germain, R. N. and Margulies, D. H. 1993. The biochemistry and cell biology of antigen processing and presentation. Annu. Rev. Immunol. 11:403.[ISI][Medline]
  3. Stockinger, B., Pessara, U., Lin, R. H., Habicht, J., Grez, M. and Koch, N. 1989. A role of Ia-associated invariant chains in antigen processing and presentation. Cell 56:683.[ISI][Medline]
  4. Fling, P. S., Arp, B. and Pious, D. 1994. HLA-DMA and -DMB genes are both required for MHC class II/peptide complex formation in antigen-presenting cells. Nature 368:554.[ISI][Medline]
  5. Liljedahl, M., Winqvist, O., Surch, C. D., Wong, P., Ngo, K., Teyton, L., Peterson, P. A., Brunmark, A., Rudensky, A. Y., Fung-Leung, W.-P. and Karlsson L. 1998. Altered antigen presentation in mice lacking H2-O. Immunity 8:233.[ISI][Medline]
  6. Freisewinkel, I. M., Schenck, K. and Koch, N. 1993. The segment of invariant chain that is critical for association with MHC class II molecules contains the sequence of a peptide eluted from class II polypeptides. Proc. Natl Acad. Sci. USA 90:9703.[Abstract]
  7. Bakke, O. and Dobberstein, B. 1990. MHC class II-associated invariant chain contains a sorting signal for endosomal compartments. Cell 63:707.[ISI][Medline]
  8. Geuze, H. J. 1998. The role of endosomes and lysosomes in MHC class II functioning. Immunol. Today 19:282.[ISI][Medline]
  9. Mellman, I., Turley, S. J. and Steinman, R. M. 1998. Antigen processing for amateurs and professionals. Trends Cell. Biol. 8:231.[ISI][Medline]
  10. Rabinowitz, J. D., Vrljic, M., Kasson, P. M., Liang, M. N., Busch, R., Boniface, J. J., Davis, M. M. and McConnel H. M. 1998. Formation of a highly peptide-receptive state of class II MHC. Immunity 9:699.[ISI][Medline]
  11. van Ham, S. M., Grüneberg, U., Malcherek, G., Bröcker, I., Melms, A. and Trowsdale, J. 1996. Human histocompatibility leukocyte antigen (HLA)-DM edits peptides presented by HLA-DR according to their ligand binding motifs. J. Exp. Med. 184:2019.[Abstract]
  12. Chaux, P., Vantomme, V., Stroobant, V., Thielemans, K., Corthals, J., Luiten, R., Eggermont, A. M. M., Boon, T. and van der Bruggen, P. 1999. Identification of MAGE-3 epitopes presented by HLA-DR molecules of CD4+ T lymphocytes. J. Exp. Med. 189:767.[Abstract/Free Full Text]
  13. Fujii, S., Senju, S., Chen, Y.-Z., Ando, M., Matsushita, S. and Nishimura, Y. 1998. The CLIP-substituted invariant chain efficiently targets an antigenic peptide to HLA class II pathway in L cells. Hum. Immunol. 59:607.[ISI][Medline]
  14. Nakano, N., Rooke, R., Benoist, C. and Mathis, D. 1997. Positive selection of T cells induced by viral delivery of neopeptides to the thymus. Science 275:678.[Abstract/Free Full Text]
  15. Sanderson, S., Frauwirth, K. and Shastri, N. 1995. Expression of endogenous peptide–major histocompatibility complex class II complexes derived from invariant chain–antigen fusion proteins. Proc. Natl Acad. Sci. USA 92:7217.[Abstract]
  16. Wang, R.-F., Wang, X., Atwood, A. C., Toalian, S. L. and Rosenberg, S. A. 1999. Cloning genes encoding MHC class II-restricted antigens: Mutated CDC27 as a tumor antigen. Science 284:1351.[Abstract/Free Full Text]
  17. Siebenkotten, I. M., Carstens, C. and Koch, N. 1998. Identification of a sequence that mediates promiscuous binding of invariant chain to major histocompatibility complex class II allotypes. J. Immunol. 160:3355.[Abstract/Free Full Text]
  18. van Bergen, J., Schoenberger, S. P., Verreck, F., Amons, R., Offringa, R. and Koning, F. 1997. Efficient loading of HLA-DR with a T helper epitope by genetic exchange of CLIP. Proc. Natl Acad. Sci. USA 94:7499.[Abstract/Free Full Text]
  19. Okyama, H. and Berg, P. A. 1983. cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol. Cell. Biol. 3:280.[ISI][Medline]
  20. Rose, J. K., Buonocore, L. and Whitt, M. A. 1991. A new cationic liposome reagent mediating nearly quantitative transfection of animal cells. BioTechniques 10:520.[ISI][Medline]
  21. Wu, S., Gorski, J., Eckels, D. D. and Newton-Nash, D. K. 1996. T cell recognition of MHC class II-associated peptides is independent of peptide affinity for MHC and sodium dodecyl sulfate stability of the peptide/MHC complex. J. Immunol. 156:3815.[Abstract]
  22. Hammer, J., Valsasnini, P., Tolba, K., Bolin, D., Higelin, J., Takacs, B. and Sinigaglia, F. 1993. Promiscuous and allele-specific anchors in HLA-DR-binding peptides. Cell 74:197.[ISI][Medline]
  23. Gluzman, Y. 1981. SV40-transformed simian cells support the replication of early SV40 mutants. Cell 23:175.[ISI][Medline]
  24. Mutis, T., Schrama, E., Melief, C. F. and Goulmy, E. 1998. CD80-transfected acute myeloid leukemia cells induce primary allogeneic T-cell responses directed at patient specific minor histocompatibility antigens and leukemia-associated antigens. Blood 92:1677.[Abstract/Free Full Text]
  25. Marshall, K. W., Liu, A. F., Canales, J., Perahia, B., Jorgensen, B., Gantzos, R. D., Aguilar, B., Deveaux, B. and Rothbard, J. B. 1994. Role of the polymorphic residues in HLA-DR molecules in allele-specific binding of peptide ligands. J. Immunol. 94:4946.
  26. Kvist, S., Wiman, K., Claesson, L., Peterson, P. A. and Dobberstein, B. 1982. Membrane insertion and oligomeric assembly of HLA-DR histocompatibility antigens. Cell 29:61.[ISI][Medline]
  27. Busch, R., Cloutier, I., Sekaly, R.-P. and Hämmerling, G. J. 1996. Invariant chain protects class II histocompatibility antigens from binding intact polypeptides in the endoplasmic reticulum. EMBO J. 15:418.[Abstract]
  28. Hitzel, C., van Endert, P. and Koch, N. 1995. Acquisition of peptides by MHC class II polypeptides in the absence of the invariant chain. J. Immunol. 154:1048.[Abstract/Free Full Text]
  29. Pierre, P. and Mellman, I. 1998. Developmental regulation of invariant chain proteolysis controls MHC class II trafficking in mouse dendritic cells. Cell 93:1135.[ISI][Medline]
  30. Roche, P. A. and Cresswell, P. 1990. Invariant chain association with HLA-DR molecules inhibits immunogenic peptide binding. Nature 345:615.[ISI][Medline]
  31. Malcherek, G., Gnau, V., Jung, G., Rammensee, H.-G. and Melms, A. 1995. Supermotifs enable natural invariant chain-derived peptides to interact with many major histocompatibility complex-class II molecules. J. Exp. Med. 181:527.[Abstract]
  32. Denzin, L. K. and Cresswell, P. 1995. HLA-DM induces CLIP dissociation from MHC class II {alpha}ß dimers and facilitates peptide loading. Cell 82:155.[ISI][Medline]
  33. Natarajan, S. K., Assadi, M. and Sadegh-Nasseri, S. 1999. Stable peptide binding to MHC class II molecule is rapid and is determined by a receptive conformation shaped by prior association with low affinity peptides. J. Immunol. 162:4030.[Abstract/Free Full Text]
  34. Busch, R., Strang, G., Howland, K. and Rothbard, J. B. 1990. Degenerate binding of immunogenic peptides to HLA-DR proteins on B cell surfaces. Int. Immunol. 12:443.
  35. Sponaas, A. M., Carstens, C. and Koch, N. 1999. C-terminal extension of the MHC class II associated invariant chain by an antigenic sequence triggers activation of naive T cells. Gene Therapy 6:1826.[ISI][Medline]
  36. Viner, N. J., Nelson, C. A. and Unanue, E. R. 1995. Identification of a major I-Ek-restricted determinant of hen egg lysozyme: limitations of lymph node proliferation studies in defining immunodominance and crypticity Proc. Natl Acad. Sci. USA 92:2214.[Abstract]
  37. Viner, N. J., Nelson, C. A., Deck, B. and Unanue, E. R. 1996. Complexes generated by the binding of free peptides to class II MHC molecules are antigenically diverse compared by intracellular processing. J. Immunol. 156:2365.[Abstract]
  38. Sloan-Lancaster, J., Evavold, B. D. and Allen, P. M. 1993. Induction of T-cell anergy by altered T-cell-receptor ligand on live antigen-presenting cells. Nature 363:156.[ISI][Medline]