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
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Abstract |
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Keywords: antigen processing, antigen presentation, epitope, gene vaccination, MHC
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Introduction |
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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 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
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 IIpeptide 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 Iiantigen 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 IIpeptide 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.
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Methods |
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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 EpsteinBarr 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 105 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 26 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).
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Results |
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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. 2A) and proliferation (Fig. 2B
). 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. 3A, 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. 3A
, 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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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. 3AC). DR1-restricted polyclonal T cells (Fig. 3A
) or T cell clones (Fig. 3C
) responded only to the MATA synthetic peptide, whereas DR4-restricted polyclonal T cells responded only to the MATT synthetic peptide (Fig. 3B
). 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. 3A
) or the DR1-restricted T cell clone (Fig. 3C
) and polyclonal DR4-restricted T cells failed to secrete IL-2 upon challenge with synthetic MATA peptide (Fig. 3B
). 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. 4). 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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Discussion |
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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 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 IIpeptide 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. 4
) 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 IIpeptide 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. 2). 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 Iiantigen 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.
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Acknowledgments |
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Abbreviations |
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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 IiMAT fusion protein |
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Notes |
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Received 10 April 2000, accepted 28 July 2000.
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References |
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