Department of Immunohaematology and Blood Bank, Leiden University Medical Center, University Medical Center, Post Box 9600, 2300 RC, Leiden, The Netherlands
Correspondence to: F. Koning
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Abstract |
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Keywords: agonist, alloreactive T cell, antagonist, peptide library
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Introduction |
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For antigen-specific T cells, down-regulation of the response can be achieved using altered peptide ligands (APL) (4). Subtle changes in the target peptide can transform an agonist into an antagonist, that inhibits antigen-specific proliferation or cytolysis when present in concentrations that are 10- to 1000-fold higher than the agonist. The exact mechanism responsible for this down-regulatory effect is still subject to investigation.
Evidence has accumulated that the response of alloreactive T cells can also be dependent on peptides bound to the allogeneic MHC (512). APL may thus be able to modulate allorecognition. In order to design APL for alloreactive T cells, however, the sequence of the stimulatory peptide ligand has to be known, which is usually not the case (12,13).
The development of random synthetic peptide libraries has been useful in the search of ligand binding peptides (14,15). In particular, peptide libraries containing motifs that enrich for HLA-binding peptides, so-called dedicated libraries, provide an important tool in the search for peptides agonists (16). In the present study we investigated whether such a dedicated synthetic peptide library can be used to find agonists for alloreactive T cells. In this way, elaborate, and possibly futile, peptide elutions for the identification of the original allopeptide are circumvented. The library was screened with alloreactive T cells that are directed against HLA-DR3. Using the peptide loading deficient DM-mutant EpsteinBarr virus-transformed B lymphoblastoid cell line (EBV-BLCL) 7.9.6 and its unmutated progenitor 8.1.6, it was determined that these T cells are dependent on yet unidentified DR3-bound peptides for their response (12). Next, putative T cell stimulatory residues present in the library-derived agonist peptide were defined and substituted. The resulting APL were tested for their capacity to suppress the alloresponse.
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Methods |
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DM-mutant and progenitor cell line
DM-mutant EBV-BLCL cell line 7.9.6 and progenitor cell line 8.1.6 (17) were a kind gift of Dr E. Mellins. Phenotype of the HLA class II molecules of the cell lines is DRß1*0301; DQ1*0501; DQß1*0201; DPß1*0401/0401. The cell surface expression of the HLA-DR molecules by 7.9.6 and 8.1.6 is similar as determined by FACScan analysis using anti-DR mAb B8.11.2 (18).
Synthesis of peptides and peptide libraries
Peptides were synthesized by solid-phase strategies as described earlier (19). The purity of the peptides was determined by analytical reversed-phase HPLC and proved to be at least 80%. The integrity of the peptides was determined by laser desorption time-of-flight mass spectrometry on a lasermat mass spectrometer (Finnigan MAT, UK). The peptide library was synthesized using hybrid TentagelH-AM resin (Rapp, Tübingen, Germany) (20). The library consists of 4x106 14-mer peptides that contain an HLA-DR3-binding motif (21), which is integrated as follows: XXXZXXDXXXXXXX. Position Z, the first anchor, contains an aromatic or aliphatic residue: L, I, M, V, A, Y or F. All peptides contain a D at position 7 as second anchor. Xs are random positions containing one of the natural amino acids (C was omitted for synthetic reasons). The hybrid resin beads (particle size 90 µm, loading 100 pmol) consists of 84 pmol peptide coupled via an acid-labile linker and 16 pmol coupled via an acid-stable linker. The acid-labile linker allows partial cleavage of peptide for repeated screening of the library. The acid-stable portion of peptide was used to determine the amino acid sequence of the peptide by Edman degradation.
Proliferation assays
Responder cells at 10,000/well were co-cultured with 100,000 irradiated PBL (3000 rad) or 20,000 mitomycin-treated EBV-BLCL in flat-bottom microtiter plates. Proliferation against peptide pools was assayed in a total volume of 50 µl culture medium. All other assays were done in a total volume of 150 µl. After 48 h of incubation (37°C, 5% CO2) the cultures were pulsed with 50 µl [3H]thymidine (10 µCi/ml) and harvested 1416 h later. [3H]Thymidine incorporation was then measured by liquid scintillation counting. The indicated c.p.m. represent median values of triplicate cultures and the SDs were <15% of the mean.
Screening of the synthetic peptide library
The library was divided into 192 pools of ~20,000 beads. Part of the peptide coupled via the acid-labile linker was released from the beads according to the method described by Hiemstra et al. (20). Each peptide pool was dissolved in 5 µl DMSO and 200 µl 30 mM phosphate buffer, pH 7.5. The T cell stimulatory capacity of each peptide pool was tested by adding 2 µl of the pool to DM-mutant cell line 7.9.6 and T cell clone at the initiation of the proliferation assay. Peptide pools were screened in a total volume of 50 µl, resulting in an individual peptide concentration of ~6 nM during the assay. All pools were screened in monoplo and pools that stimulated were rescreened in triplo (first screening). Next, beads of active pools were subdivided into pools of ~70 beads. Again acid-labile-coupled peptide was released and tested as described above (second screening). For the third round the acid-labile peptide on the beads was tested individually. Finally, peptide sequences were determined by manual application of single beads to a cartridge and subsequent sequencing of the acid-stable peptide using a Hewlett-Packard G1005A protein sequencer.
Antagonist assays
APL were tested for antagonistic properties by addition of peptide at the initiation of the assay in concentrations ranging from 0.1 to 10 µM.
The DR3-peptide binding assays
HLA-DR3 molecules were isolated as described by Geluk et al. (22). The DR preparation was titered in the presence of 100 fmol standard peptide to determine the DR concentration necessary to bind 1020% of the total fluorescent signal. All subsequent inhibition assays were then performed at this concentration. Peptides, of which the DR3-binding capacity was to be determined, were added to DR3 molecules simultaneously with the standard fluorescence-labeled peptide, HSP65 p313. The DRpeptide complexes were separated from free peptide by gel filtration on a Synchropak GPC 100 column (250 mmx4.6 mm; Synchrom, Lafayette, IN). Fluorescent emission was measured at 528 nm on a Jasco FP-920 fluorescence detector (B & L Systems, Zoetermeer, The Netherlands). The percentage of labeled peptide bound was calculated as the amount of fluorescence bound to MHC divided by total fluorescence. The concentration of peptide inhibitor yielding 50% inhibition (IC50) was deduced from the doseresponse curve.
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Results |
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Selection of peptide-specific alloreactive T cell clones for screening of the peptide library
Previously we have raised alloreactive T cells clones against HLA-DR3, and tested these for their proliferative response against the DM-mutant EBV-BLCL 7.9.6 and its unmutated progenitor cell line 8.1.6 (12). Out of 64 HLA-DR3-specific T cell clones, 59 T cell clones were either completely (33 T cell clones) or partly (26 T cell clones) dependent on the presence of HLA-DM for their alloresponse (12). All 59 T cell clones are thus probably dependent on the presence of a DR3-bound peptide and may be used for screening a random synthetic 14-mer peptide library to find a DR3-restricted agonist.
As the concentration of individual peptides present in the synthetic 14-mer library is relatively low (6 nM) during screening, it is probably essential to use T cell clones with high-affinity TCR. We therefore selected four T cell clones that were able to recognize EBV-BLCL 8.1.6 at low stimulator cell concentrations and that were clearly peptide dependent as they discriminated well between stimulators 7.9.6 and 8.1.6 (results not shown).
Peptide library screening results
The four selected alloreactive T cell clones were used to screen the peptide library according to the protocol of repeated screening described in Methods. In the first screening round two T cell clones did not respond to any of the peptide pools. T cell clone 6210 responded to two peptide pools and T cell clone 6234 responded to 20 peptide pools. Further screening resulted in one positive pool for T cell clone 6210 in the second and third screening round. For T cell clone 6234 six positive pools were selected for further screening. The sequences of the stimulatory peptides were subsequently determined by Edman degradation (Table 1). In total, five stimulatory peptide sequences could be determined: one sequence for T cell clone 6210 and four sequences for T cell clone 6234. All sequences contain an aliphatic or aromatic residue (L, I, M, V, A, Y or F) on position 4 (first anchor) and an aspartic acid (D) on position 7 (second anchor) corresponding with the HLA-DR3-binding motif incorporated in the library. In three of the five sequences position 8 could not be determined. As in particular a histidine (H) or tryptophan (W) could not be excluded on this position, these amino acids were chosen to be incorporated on position 8 of the peptides. The five library-derived sequences were synthesized and tested for recognition by T cell clones 6210 and 6234. Sequence (1) and (2) as well as sequences (3), (4) and (5) containing the tryptophan on position 8 were stimulatory in the context of DM-mutant 7.9.6. The doseresponse curves are depicted in Fig. 1
. The peptides are all recognized at 6 nM, which is the concentration of individual peptide in the library.
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Only one agonist was found for T cell clone 6210, so here we evaluated the importance of the individual amino acid residues by testing alanine substitution analogs (Fig. 2). The peptide contains two DR3-binding motifs: the Y4-D8, which is incorporated in the library, and the V5-D9. As alanine substitutions of each of both aspartic acids result in abrogation of the response, the alanine-scan gives no insight into which of the motifs is used to compose the complex recognized by T cell clone 6210. Residues that least tolerate a change to alanine and that are not potential MHC binding residues, residues H9 and F11, were considered as potential TCR contact residues.
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Discussion |
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Nowadays peptide libraries are frequently used for the identification of T cell ligands. These library-derived ligands are almost always mimicry epitopes that barely resemble the natural epitope. Replacement studies on such agonists may eventually lead to a potential natural epitopes of the T cell (16). Importantly, for the present study the natural epitope was not of interest as an effective antagonist could be constructed directly on the basis of the mimicry epitope.
Two out of the four alloreactive T cell clones that were used for screening the library did not respond to peptide pools of the library. As the individual peptide concentration in the peptide library is only 6 nM, lack of response could be explained by this low screening concentration. On the other hand, the library consists of only 4x106 peptides out of the 2.2x1021 possible sequences. It is therefore probable that for these T cell clones no proper agonists were present in the peptide library. If this is indeed the case it implies that T cell clones 5922 and 6221 allow relatively little variation in the peptide that is recognized in the context of HLA-DR3.
Comparison of the number of pools that induce a response of T cell clones 6210 and 6234 in the first round of screening illustrates that alloreactive T cell clones may indeed differ in their allowance for sequence variation of the allopeptide. While T cell clone 6234 responded to 20 out of 192 peptide pools, T cell clone 6210 recognized only two peptide pools. T cell clone 6234 thus displays a more degenerate recognition of peptide. This degeneracy may be the reason why, in contrast to T cell clone 6210, no peptide antagonist could be found for T cell clone 6234.
Three APL induced down-regulation of the response of T cell clone 6210. APL in which the histidine on position 9 was substituted for a glutamine (Q) or a methionine (M) most efficiently suppressed the response against the library-derived agonist, while changing the histidine to an alanine (A) resulted in a less effective inhibitor (Fig. 3). To rule out that this suppression of the T cell response was due to competition for binding to the HLA-DR3 molecule, two unrelated HLA-DR3-binding peptides were also tested for their inhibitory capacity. Peptide 313 of the HSP65 molecule is known as a strong binder to HLA-DR3 (22) and agonist 5 induced a DR3-restricted response by T cell clone 6234. Neither of these peptides could inhibit the response of T cell clone 6210. In addition we show that the binding capacity of all APL to HLA-DR3 is comparable to that of HSP65 p313. This indicates that down-regulation of the T cell response is due to the antagonistic properties of the APL and not to competition.
Previous reports have demonstrated that also in the murine system the MHC class II-specific alloresponse can be down-regulated (24,25). Both studies concern well-defined systems of self-restricted antigen-specific T cells that cross-react with an alloepitope. In the first study (24) the helix of the self-MHC was mutated, resulting in the transformation of an agonistic MHCpeptide complex into an antagonistic complex. In the second study (25) alloreactivity was down-regulated by an antagonist peptide that operated in the context of the self-restricted determinant. So here both the self-MHC molecule as well as the allogeneic MHC molecule had to be present on the cell surface of the APC to demonstrate the antagonistic effect of the APL. Our results show that an APL that is recognized directly via the allogeneic MHC can also accomplish antagonism of alloreactivity. Importantly, little has to be known about the alloreactive T cell clone with respect to its fine specificity.
In conclusion, we find that APL can antagonize alloreactive T cell clones. Yet the degree of peptide specificity of the T cell clone may dictate whether its response can be antagonized by a peptide: the more degenerate the peptide recognition of an alloreactive T cell clone, the more difficult it may be to find a peptide antagonist. For alloreactive T cells, degenerate peptide recognition by a TCR can reflect an increased specificity for allogeneic residues on the helix of the MHC molecule (26). Such T cell clones are thus more likely to be antagonized by altered MHC molecules than by APL. Therefore the usefulness of APL in the down-regulation of alloreactivity probably relies on the degree of peptide dependency of the individual T cell clones that constitute the response. Analysis of the peptide specificity of in vivo-generated alloreactive T cell clones will thus be necessary to determine whether peptide antagonists may effectively suppress the alloresponse in transplanted patients.
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Acknowledgments |
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Abbreviations |
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APL | altered peptide ligands |
EBV-BLCL | EpsteinBarr virus-transformed B lymphoblastoid cell line |
PBL | peripheral blood lymphocyte |
DMSO | dimethylsulfoxide |
TCGF | T cell growth factor |
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Notes |
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Received 29 October 1998, accepted 4 January 1999.
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Reference |
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