Neuroimmunology Laboratory, Department of Neurology,
1 Institute for Cell Biology, Department of Immunology and
2 Institute of Organic Chemistry, University of Tübingen, 72076 Tübingen, Germany
3 Immunology Division, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK
Correspondence to: W. Wienhold
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Abstract |
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Keywords: DRB1*0301, peptide binding, salt bridge, T cell epitopes, T cell repertoire, TCR contact residues
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Introduction |
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Regardless whether the T cell activation is initiated by conformational changes or by cross-linking of multiple TCR complexes on the surface of the T cell, it is the quality of these interactions that affects the quality of the T cell response. It has been shown that the affinities of a TCR bound to a MHC class II molecule with agonistic or antagonistic peptide ligands strictly correlate with the responsiveness of the T cell (5). In addition, modest variations in the affinity of MHC class I molecules with positively or negatively selecting ligands for the same TCR are capable of triggering different signals (6). A strong T cell response is usually seen when immunodominant peptides are bound to the restricting MHC. The phenomenon of immunodominance in the class II system is mainly thought to be caused by successful peptide competition for MHC class II binding in the loading compartment of APC (7). Alternatively, immunodominance could be explained by different antigen-processing mechanisms in different APC as it has been recently reported (8). To explore another attractive possibility, we investigated the structural features of the interaction of immunodominant epitopes derived from the model antigen tetanus toxin (TT) with their corresponding TCR. These epitopes triggered strong responses in several TT-specific T cell clones from DRB1*0301/DRB3*0101 donors. To understand how the hierarchy of immunodominance within a pool of similarly active antigenic peptide derived from the same protein may be established, we identified the residues in each peptide that interact with the corresponding TCR. Based on the previously reported crystal structure data of the DRB1*0301 complexed with the CLIP peptide (9) we predicted that, within the nonamer core segments fitting the groove, peptide residues at positions (P) P2, P5 and P8 projected out of the MHC class II binding groove facing the TCR. Hence, we focused our attention on these residues in examining the structural basis of antigen recognition of a panel of dominant TT epitopes. In these cases, we present evidence for a critical role of residue P8 of the nonamer core segment for the stimulation of TT-specific T cells. Moreover, the majority of TCR examined showed a strong interaction between a particular CDR3ß residue (in our case no. 3) and the corresponding residue at P8 of the peptide. In the context of the DRB1*0301/DRB3*0101 haplotype, this interaction enables the stimulation of at least four different synonymous TCR by the major T cell epitope TT(12721284).
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Methods |
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T cell clones
TT-specific T cells were cloned by limiting dilution in microtiter plates using 0.3 cells/well, together with 2x104 irradiated autologous PBMC preincubated with TT in complete medium containing 20 U/ml of IL-2 as described (10) and maintained in culture by periodic re-stimulation with autologous APC and TT. The T cell clones Pil-1, Pil-5, Pil-6 and Pil-36 [TT(12721284)], Pil-2 and Pil-33 [TT(10611075)] and Pil-45 [TT(279296)] were obtained from the same DRB1*0301/DRB3*0101 homozygous donor (Pil).
Antigens
Native TT was obtained from Calbiochem (La Jolla, CA) and used in 0.0110 µg/ml final concentrations. This holotoxin preparation is no longer distributed by Calbiochem.
The cDNA encoding amino acids 8651315 of TT (TT C-fragment, kindly provided by H. Niemann) was cloned and engineered to contain a C-terminal histidine-tag, which allows metallo-affinity chromatography on Ni2+ resins. Single colonies of transformed Escherichia coli M15 were grown in 500 ml 2xYT medium with 50 µM ampicillin and 25 µM kanamycin to an OD600 of 0.61.0, and were induced with 0.5 mM isopropyl-thiogalacto-pyranoside. After 24 h, the bacteria were harvested by centrifugation (3000 g for 20 min at 4°C). The pellet was resuspended in 50 mM phosphate buffer, 300 mM NaCl and 0.1 mM PMSF, pH 8.0. Bacteria were lysed in the same buffer containing 600 mM NaCl by three cycles of freezing and thawing, followed by three cycles of sonification with a Branson sonifier at 4°C for 30 s each. The lysate was centrifuged at 36,000 g and the supernatant passed over 1 ml Ni-NTA agarose (Qiagen, Hilden, Germany). The beads were washed with lysis buffer at pH 8.0 and 6.2, and eluted with 50 mM NaH2PO4, pH 4.5.
Peptides
Peptides (Table 1) were synthesized in an automated peptide synthesizer 432A (Applied Biosystems, Weiterstadt, Germany) following the Fmoc/tBu strategy, and analyzed by HPLC (System Gold; Beckmann Instruments, München, Germany) and MALDI-TOF mass spectrometry (G2025A; HewlettPackard, Waldbronn, Germany). Peptides showing a purity of <80% were purified by preparative HPLC. N-terminal biotinylation of apoB100(28772894) was carried out by five coupling steps using Fmoc-ß-alanine, Fmoc-
-aminocaproic acid (Ahx), Fmoc-
-lysine and Dmtr-biotin to form biotin-
-Lys-ß-Ala-Ahx-ß-Ala-[apoB100(28772894)].
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Peptide binding assay
Purified DRB1*0301 molecules (100 nM) were incubated at 37°C with 2 µM of the biotinylated, high-affinity DR17 ligand apoB(28772894) with or without competitor peptide in 96-well microtiter plates (Greiner, Nürtingen, Germany) in binding buffer (50 µl) containing 2 mM EDTA, 0.01% azide, 0.1 mM PMSF and 0.1% NP-40 adjusted to pH 5.0 by 1 M citrate. After 72 h, the neutralized sample of MHCpeptide complexes was separated from peptide excess by immunoprecipitation, and detected by successive incubation at 20°C with streptavidin (2.5 µg/ml) (Dianova, Hamburg, Germany) and bio-tinylated peroxidase (100 ng/ml; Dianova) for 45 min respectively, followed by incubation with ABTS (1 mg/ml) (Boehringer Mannheim, Mannheim, Germany). The absorbance at 405 nM was measured by an ELISA reader (Multiskan Plus; Titertek, Meckenheim, Germany) and non-specific signals (quadruplicates, typically <15% of maximal absorbance) were subtracted from the data.
Measurement of MHC class II binding of peptide variants at P8 was performed using the europium fluoroimmunoassay in a Wallac 1420 Victor multilabel counter (Wallac-ADL, Freiburg, Germany) as described (14).
Cloning and sequence determination of TCR
Cloning of the 5' end of TCR cDNAs, including the CDR3 and a part of the constant region, was performed by ligation-anchored PCR as described elsewhere (15). Briefly, total RNA was isolated using RNAzol (Cinna/Biotecx, Houston, TX) or TRIzol Reagent (Gibco BRL) and cDNA was synthesized from 24 µg of RNA using MMLV reverse transcriptase (Promega, Madison, WI) and oligo(dT) (Pharmacia, Freiburg, Germany) as primer. After first-strand cDNA synthesis, the RNA template was removed by treatment with sodium hydroxide (0.3 M) at 50°C for 30 min. Following neutralization with acetic acid and purification of single-strand cDNA by ammonium acetate precipitation, the purified cDNA was ligated to a 3'-NH2 blocked and 5'-phosphorylated anchor oligonucleotide (15) by T4 RNA ligase (New England Biolabs, Beverly, MA). The anchored cDNA was amplified by a first PCR reaction using a primer complementary to the anchor (F8/26) and one from the TCRA or TCRB constant region, near the 3' end. These primers were used at a final concentration of 0.06 µM (16). The following conditions were used for the first PCR: 94°C for 4 min, for 1 cycle; 94°C for 40 s, 56°C for 40 s, 72°C for 90 s, for 14 cycles; and 72°C for 10 min, for 1 cycle.
The diluted first-round PCR products were used as a template (final dilution 1:1001:400) for a nested PCR with same forward primer and inside reverse primers from the TCRA or TCRB constant region. The following conditions were used for the second, nested PCR: 94°C for 4 min, for 1 cycle; 94°C for 40 s, 56°C for 40 s, 72°C for 90 s, for 35 cycles; and 72°C for 10 min, for 1 cycle. Primer sequences were as follows (anchor: CTGCATCTATCTAATGCTCCTCTCGCTACCTGCTCACTCTGCGTGACATC; forward primer: F8/26: CGCAGA- GTGAGCAGGTAGC; reverse primers: RA-1: GAGGAAGGAGCGAGGGAG; RA-2: GTACACGGCAGGGTCAGG; RB-1:CAGGCAGTATCTGGAGTCATTG; RB-2: TCTGATGGCT- CAAACACAGC). PCR amplification of TCR cDNA using a panel of V and Vß subfamily-specific primers was performed as previously described (17). PCR products were polished by Pfu DNA polymerase (Stratagene, La Jolla, CA) for blunt-end ligation into a SmaI-digested pBluescript KS+ vector (Stratagene). Sequence analysis was performed using the AmpliTaq FS dye deoxy terminator cycle sequencing kit (Applied Biosystems, Foster City, California) and read in a PE-ABI Prism 310 automated DNA sequencer.
The entire 5' ends of TCR cDNAs from TT-specific T cell clones, except the ß chain of TCR from clone Pil-6, were sequenced after cloning of the 5'-anchored PCR products. The TCR ß sequence of clone Pil-6 was obtained after using the Vß subfamily-specific primer panel described above.
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Results |
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Structural prediction of antigenic peptide conformation bound to DRB1*0301 and DRB3*0101identification of potential TCR contact residues
To further investigate the structural basis of the strong T cell responses to those TT-derived antigenic peptides in the context of DRB1*0301/DRB3*0101, we analyzed their theoretical conformation based on the crystal structure data of the DRB1*0301CLIP peptide complex (9). Interestingly, this class II molecule was purified from a B lymphoblastoid cell line (9.5.3) which effectively presented TT(12741285) to a TT-specific T cell line (24). Although it remains open whether in this case the TT(12721284) is DRB1*0301 or DRB3*0101 restricted, or both (19,20), recent studies revealed a nearly identical peptide binding motif for both alleles which arise from a gene conversion event (23,25). Thus, with regard to the conformation of the three-dimensional structure of the peptide and the position of TCR-contacting residues, it is irrelevant whether this peptide is bound to DRB1*0301 or DRB3*0101. Crystallographic studies of other peptideMHC class II complexes suggested a common polyproline II-like conformation of the bound antigenic peptides (26,27). With regard to the highly efficient binding of the epitopes TT(279296) and TT(10611075) peptides to DRB1*0301 (Table 1), it can be argued that these peptides compete very efficiently for binding to the DRB1*0301 heterodimer in the antigen loading compartments of APC. Both peptides should therefore be presented preferentially on the DRB1*0301 molecule. Analysis of the conformation of CLIP bound to DRB1*0301 allowed a predicted alignment of the three TT-derived epitopes (Fig. 4
). Isoleucine at the N-terminal position 1 in all TT peptides should contact the non-polar pocket 1 of DRB1*0301 whereas the C-terminal leucine of TT(279296) and TT(10611075) and the Isoleucine of TT(12721284) may be deeply buried in pocket 9. Slight movements of DRB1*0301 side chain ß74R in pocket 4 should allow the fitting of the DRB1*0301-allele specific anchor residue aspartate in this pocket (9). Finally, the anchor residue P6, which is lysine in TT(279296) and asparagine in TT(10611075) and TT(12721284), correlates well to the known DRB1*0301 binding motif as well as to the negatively charged binding pocket 6. Knowing the anchor residues of the TT-derived peptides and the X-ray crystal structure of the CLIPDRB1*0301 complex we were also able to predict potential TCR contact residues. Up to date all published crystallographic data of MHC class IIpeptide complexes reveal a highly conserved conformation of the peptides in the binding groove (26,27). Thus, we suggest that the residues P1, P2, P5 and P8 should be primarily exposed to the TCR (Fig. 4
).
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As shown in Fig. 5, TT(12721284)-specific T cell clones Pil-1, Pil-36, Pil-4 and Pil-6 were highly responsive to the wild-type peptide as well as to the previously described shorter variant TT(12731284) (20), whereas the exchange of the negatively charged residue aspartate at TT1282, e.g. for leucine, clearly reduced T cell proliferation activity in a dose-dependent fashion. As expected, the most prominent effect was observed by introducing an inversely charged residue (lysine for aspartate) which further reduced the T cell response compared to the P8 D
L variant. Apart from Pil-4 clone, which showed a strong preference for the peptide analogue P8 D
E, the alteration from P8 aspartate to glutamate caused a more moderate reduction of T cell proliferation. It is interesting to note that clones Pil-4 and Pil-36, in which the TCR differ only in their CDR3
and CDR3ß usage, responded with a marked difference to the P8 D
E exchange (see Fig. 5
). In this case, the exchange of D
E apparently created a superagonistic peptide for Pil-4 cells with an increase of activity of at least one order of magnitude.
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Furthermore, we examined whether the described alterations at P8 had an influence on the pattern of the cytokine production of the T cells, possibly by creating altered peptide ligands (33,34). Secretion of IFN-, IL-10 and IL-4 was indistinguishable in the original peptide and the P8 analogues (data not shown).
These data clearly confirm the hypothesis of a strong interaction between the aspartate at P8 of the peptide core and the N-terminal arginine of the TCR CDR3ß loop in five different receptorligand pairs. We demonstrate the critical role of this `fixation point' especially in the case of the major immunodominant TT-derived peptide TT(12721284) which results in the recruitment of a broader T cell repertoire specific for this epitope.
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Discussion |
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Several aspects considered to be involved in immunodominance (37) are investigated.
Antigen processing of antigenspredetermination of immunodominance
As a fundamental prerequisite of the T cell response, internalized protein antigens must be proteolytically cleaved to give rise to the epitopes that will bind to MHC class II. Previous in vitro processing studies of TT (36) confirmed that the TT-derived epitopes TT(10611075) (described here for the first time) and TT(12721284) were not destroyed after incubation of the TT C-fragment with asparaginyl endopeptidase. Remarkably, Hewitt et al. demonstrated that the TT(12721284) epitope was flanked by cathepsin E and D cleavage sites (35). Since the sequence of the epitope TT(279296) is not included in the TT C-fragment, there were no data available on the cleavage sites of asparaginyl endopeptidase, cathepsin E or D in this region. Furthermore, only one short peptide [TT(951954)] was recovered from the N-terminal half of the TT C-fragment (residues 865-1114) which may represent the inability of both cathepsins to cleave this part of the molecule (35). Despite the lack of information about the proteolytic cleavage sites around the T cell epitopes TT(279296) and TT(10611075), we demonstrated that all three epitopes are generated during the processing in autologous APC and form T cell epitopes.
Peptide competition for class II presentation?
The second proposed requirement concerning immunodominance of a given peptide is the binding to the MHC molecule. Thus, if such peptides were generated from antigen degradation they should efficiently compete for MHC binding in the loading compartment (7,38). If this hypothesis is valid we should observe a good correlation between the immunogenicity of TT-derived epitopes and their binding capacity to purified DRB1*0301 molecules, but we were not able to confirm such a correlation in all cases. The strongest T cell response was found with TT(12721284) (Fig. 5 and data not shown), which binds only moderately to DRB1*0301 (Table 1
). Thus, other mechanisms like peptide editing by HLA-DM may have an influence on the presentation of T cell epitopes. However, we could not examine whether DM sensitivity is different in TT(12721284) bound to DRB3*0101 or DRB1*0301. A recent study demonstrated cross-presentation of antigenic peptides by DR52a and DR17 molecules (23) since the binding motifs of both alleles were found to be almost identical (23,25). In this regard, we favor the possibility that TT(12731284) or TT(12721284) is able to bind to both MHC alleles and it primarily depends on the TCR structure which complex is preferentially recognized. Such a promiscuous binding of a given peptide would clearly broaden the repertoire of specific T cells and therefore contribute to immunodominance.
A structural view on the T cell recognition of immunodominant epitopes
To investigate another parameter involved in immunodominance, we analyzed the physical interaction between the TCR and the TT-derived peptides. Based on the X-ray crystal structure data of the DRB1*0301CLIP complex (9) we predicted the conformation of the bound peptides and potential TCR contact residues. The currently known conformations of MHC class II-bound peptides are remarkably similar (26,27). Since there is growing evidence that DRB3*0101 and DRB1*0301 binding peptides use identical or very similar anchor residues (23,25), we can assume that the potential TCR contact residues of TT(12721284) are identical if the peptide is bound to DRB3*0101 or DRB1*0301.
The sequence analysis of the TCR heterodimers recognizing the TT-derived peptides on the DRB3*0101/DRB1*0301 background revealed two major characteristics of our studies. First, the immunodominant epitope TT(12721284) was capable of stimulating at least four different T cell clones (in the context of the autologous DRB3*0101/DRB1*0301 MHC restriction) derived from the same donor. Second, TT(279296) and TT(12721284), which share an aspartate at the TCR contact site P8, were recognized by TCR with an arginine at the third position of the CDR3ß loop, suggesting a potential salt bridge between these corresponding residues. Further support for this idea was the observation that T cell clones Pil-2 and Pil-33 were not able to respond to TT(12721284) despite the fact that they express the same TCR chain as the TT(12721284)-specific clone Pil-4 but differ in their CDR3
sequence (Table 2
).
The phenomenon of different TCR recognizing the same MHCantigen complex is known as synonymous TCR (39) and it is based on the determination of X-ray crystal structures of two TCRMHCpeptide complexes where two different TCR (derived from T cell clones of two individuals) recognized the same MHCpeptide complex (40). Additionally, it was recently shown that the recognition of a HTLV-I Tax peptide bound to HLA-A2 by two human TCR is also possible if both T cell clones differ completely in their fine specificity for all contacting peptide residues (41). In the case of the HTLV-I Tax peptide, the stimulation of different T cell clones by a single MHCpeptide complex is facilitated by a high degree of degeneracy of the TCR. Since we raised four synonymous TT(12721284)-specific T cell clones by re-stimulating with TT, this high frequency of clones derived from a single donor may reflect the high potency of this dominant epitope to stimulate T cells. Furthermore we observed a similar dominant T cell response to TT(12721284) among a series of other DRB1*0301-positive donors (data not shown) compared to other less prominent T cell epitopes of TT. Previously described T cell responses against TT-derived peptides with the same nonamer core, i.e. TT(12731284) (20) and TT(12741284) (24), also support the importance of this immunodominant epitope within the panel of TT-derived antigenic peptides.
We confirmed the strong interaction between aspartate at P8 of TT(279296) and TT(12721284) and arginine of the TCR CDR3ß loop by single amino acid alterations of the antigenic peptides. The hierarchy of alterations at P8 paralleled closely the expected degree of T cell response regarding the postulated ionic interaction. Thus, strong reduction or even complete abrogation of T cell response was achieved by inversion of the charge in residue at P8. Two surprising exceptions were obtained after stimulation with the conservative exchange of aspartate to glutamate at P8. The TT(12721284)-specific T cell clone Pil-4 responded significantly stronger to glutamate at P8, whereas the TT(279296)-specific clone Pil-45 failed to recognize the modified peptide. Interestingly, the TT(12721284)-specific clones Pil-4 and Pil-36 use the same TCR sequences with the exception of the CDR3 and CDR3ß loops. Regarding the CDR3 sequences, one possible explanation could be the variable length of the CDR3ß loops of both T cell clones. It has been reported previously (4,32,42,43) that CDR3ß may interact with the peptide segment P5 to P8. Such a simultaneous interaction between the CDR3ß loop and the antigenic peptide residues P5 and P8 is possible either if the orientation of the TCRclass II interaction is orthogonal as recently reported (44) or if it is diagonal as shown previously in the TCRpMHC class I crystals (4,40,42). In this more recent study (44), the crystal structure analysis of the scDC (mouse TCR)CA/I-Ak complex showed that the CDR3ß loop interacts only with the P8 glutamine side chain of conalbumin (CA) peptide. Detailed analysis of this crystal structure revealed that it is probable that CDR3ß residues could interact with P5 and P8 simultaneously in other TCRpMHC class II complexes (44). This suggests that the smaller loop of clone Pil-4 may prefer the larger residue volume of glutamate at the relative position P8, thus enabling the smaller CDR3ß loop to contact the antigen residues P5 and P8 simultaneously, whereas aspartate at P8 fails to interact with arginine in CDR3ß to the same extent as with glutamate at P8. In contrast to the other TT(12721284)-responding T cell clones Pil-1, Pil-6 and Pil-36, the strong response of Pil-4 to the P8 D
E variant may also reflect different peptides involved in positive selection during T cell development in the thymus. On the other hand, the abrogated response of Pil-45 against the D
E variant of TT(279296) is probably caused by steric hindrance due to the increased bulkiness of the residue at P8.
Taken together, our data show that a particular strong interaction between a TCR contact site of the antigenic peptide and a corresponding residue of the CDR3ß segment of the TCR may contribute to immunodominance. Such strong contacts could act as a `fixation point' which facilitates the recognition of a prominent peptide epitope by a broader repertoire of specific T cells expressing synonymous TCR.
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Acknowledgments |
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Abbreviations |
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APC antigen-presenting cell |
CA conalbumin |
CDR complementarity-determining region |
PBMC peripheral blood mononuclear cell |
TT tetanus toxin |
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Notes |
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Received 16 December 1999, accepted 7 February 2000.
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References |
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