Definition of agonists and design of antagonists for alloreactive T cell clones using synthetic peptide libraries

H. Saskia de Koster, Corine J. Vermeulen, Hoebert S. Hiemstra, Reinout Amons, Jan W. Drijfhout and Frits Koning

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


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 
Alloreactive T cells form an important barrier for organ transplantation. To reduce the risk of rejection patients are given immunosuppressive drugs, which increase the chance of infection and the incidence of malignancies. It has been shown that a large proportion of alloreactive T cells specifically recognize peptides present in the groove of the allogeneic MHC molecule. This implies that it might be possible to modulate the alloresponse by peptides with antagonistic properties, thus preventing rejection without the side effects of general immunosuppression. Peptide antagonists can be designed on the basis of the original agonist, yet for alloreactive T cells these agonists are usually unknown. In this study we have used a dedicated synthetic peptide library to identify agonists for HLA-DR3-specific alloreactive T cell clones. Based on these agonists, altered peptide ligands (APL) were designed. Three APL could antagonize an alloreactive T cell clone in its response against the library-derived agonist as well as in its response against the original allodeterminant, HLA-DR3. This demonstrates that peptide libraries can be used to design antagonists for alloreactive T cells without knowledge about the nature of the actual allostimulatory peptide. Since the most potent agonists are selected, this strategy permits detection of potent antagonists. The results, however, also suggest that the degree of peptide dependency of alloreactive T cell clones may dictate whether a peptide antagonist can be found for such clones. Whether peptide antagonists will be valuable in the development of donor–patient-specific immunosuppression may therefore depend on the specificity of the in vivo-generated alloreactive T cells.

Keywords: agonist, alloreactive T cell, antagonist, peptide library


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 
Acute rejection of solid organ transplants correlates with a strong response of CD4+ T cells that are directed against the allogeneic MHC class II molecules present in the graft (1). Treatment with immunosuppressive drugs considerably improves graft survival, yet this therapy increases the risk of infections and malignancies (2). Therefore research has focused on the development of less aggressive methods to suppress the response of alloreactive T cells (3).

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 Epstein–Barr 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.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 
Generation of alloreactive T cell clones
The alloreactive T cell clones used for the assays have been described previously (12): 5x106 stimulator cells were irradiated (3000 rad) and incubated with 7x106 responder cells in 10 ml RPMI 1640 supplemented with 10% pooled human serum in 50 ml culture flasks at 37°C 5% CO2. After 10 days the cultures were re-stimulated with the original stimulator cells (3000 rad) at a ratio of 10 peripheral blood lymphocytes (PBL) per responder cell. The lines were cloned by limiting dilution to 0.3 cells/well in the presence of T cell growth factor (TCGF; Biotest Serum) and irradiated feeder cells: a mixture of freshly isolated PBL and the original stimulator cells. The T cell clones were expanded by serial exposure to TCGF and irradiated feeder cells, and stored at –70°C until testing.

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; DQ{alpha}1*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 14–16 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 10–20% 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 p3–13. The DR–peptide 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 dose–response curve.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 
The synthetic peptide library
The peptide library was synthesized on 4x106 resin beads, each bead containing one unique sequence. The library was constructed such that all library sequences contain an HLA-DR3-binding motif (21). In this way we enriched for HLA-DR3-binding-peptides and consequently for candidates that stimulate the T cell clones in the context of HLA-DR3.

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 1Go). 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 dose–response curves are depicted in Fig. 1Go. The peptides are all recognized at 6 nM, which is the concentration of individual peptide in the library.


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Table 1. Peptide sequences derived from the synthetic 14mer library
 



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Fig. 1. Dose–response curves of (a) T cell clone 6210 and (b) T cell clone 6234. The responses of the T cell clones against the library-derived sequences were measured in a proliferation assay.

 
Development of APL with antagonistic properties
The library-derived agonists were used as templates for the design of APL that could antagonize the response of the alloreactive T cell clones. For this purpose we focused on potential TCR contact residues of the peptides. For the agonists of T cell clone 6234, four potential TCR contact residues could be deduced by comparing the four library-derived sequences in order to identify conserved amino acids other than the incorporated DR3-binding motif. Similar residues were found on positions 5 (A), 6 (I or V), 8 (aromatic residue) and 12 (aromatic or aliphatic residue).

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. 2Go). 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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Fig. 2. Alanine scan of the library-derived agonist of T cell clone 6210. All residues were replaced by an alanine and the resulting peptides were tested for their stimulatory capacity in the presence of DM-mutant 7.9.6 in a proliferation assay.

 
Subsequently, single residues of the library-derived agonist sequences were altered and the APL tested for antagonistic properties. For T cell clone 6234, 24 APL were synthesized using the most potent agonist, sequence (2), as template. Nine of the 24 APL were non- or low stimulators and thus potential antagonists. Yet none of these nine APL could antagonize the response of T cell clone 6234 to the agonist (results not shown). For T cell clone 6210, 19 APL were synthesized of which seven were non-stimulatory. Replacement of the histidine (H) at position 9 of the agonist for T cell clone 6210 by a Q, A or M resulted in APL that could down-regulate the response against the library-derived agonist in the context of DM-mutant 7.9.6 at peptide concentrations of 1–10 µM (Fig. 3Go). Two other irrelevant HLA-DR3-binding peptides, HSP65 p3–13 (22) and one of the agonists found for T cell clone 6234 (peptide 5; Fig. 3Go), did not influence the response of T cell clone 6210. The latter result indicates that the antagonistic effect is not merely due to competition for binding.



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Fig. 3. The effect of APL on the peptide-specific response of T cell clone 6210. T cell clone 6210 was stimulated with DM-mutant 7.9.6 and 60 nM of synthetic agonist. APL were added in a concentration range.

 
These APL were then tested for down-regulation of the reactivity of T cell clone 6210 against allostimulatory DR3+ PBL (Fig. 4Go). Addition of the wild-type peptide resulted in an increase of the alloresponse. In contrast, all three APL that down-regulate the response against the library peptide also restrained the alloresponse in a dose-dependent fashion. The peptide containing the h -> Q substitution most effectively inhibited both the peptide-specific and the alloresponse of T cell clone 6210.



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Fig. 4. The effect of APL on the alloresponse of T cell clone 6210. T cell clone 6210 was stimulated with HLA-DR3+ PBL in the absence of synthetic peptide. APL were added in a concentration range. Peptide antagonists for alloreactive T cells

 
Comparison of the binding capacities of agonist and antagonists
To further exclude the possibility that down-regulation of the T cell responses is due to competition for binding to the HLA-DR3 molecule, the binding potentials of the library-derived agonists and of the APL to HLA-DR3 were determined using the direct binding assay (23). All APL display binding capacities that are equal or lower compared to the agonist and to control peptide HSP65 p3–13 peptide as reflected by their IC50 values (Table 2Go).


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Table 2. The binding capacities of library derived agonist (1), three antagonists and control peptide HSP65 p3–13 to HLA-DR3 as determined by a direct binding assay (23)
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 
Peptides are involved in the constitution of the alloepitopes recognized by the greater part of alloreactive T cells. Therefore, peptide antagonists should be capable of restraining the allospecific T cell response. Here we show that it is possible to (i) define agonists for alloreactive T cells using a peptide library and (ii) effectively suppress the response of an HLA-DR3-specific alloreactive T cell clone with an APL designed on the basis of the agonist.

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. 3Go). 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 3–13 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 p3–13. 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 {alpha} helix of the self-MHC was mutated, resulting in the transformation of an agonistic MHC–peptide 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 {alpha} 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.


    Acknowledgments
 
The authors thank W. Benckhuijsen for the synthesis of peptides, Dr E. Mellins for the generous gift of cell lines 7.9.6 and 8.1.6, Dr A. Geluk for the peptide-binding studies, and Professor F. Claas, Professor E. Goulmy and Dr B. O. Roep for reading the manuscript. This work was supported by the Dutch Kidney Foundation.


    Abbreviations
 
APLaltered peptide ligands
EBV-BLCLEpstein–Barr virus-transformed B lymphoblastoid cell line
PBLperipheral blood lymphocyte
DMSOdimethylsulfoxide
TCGFT cell growth factor

    Notes
 
Transmitting editor: E. Simpson

Received 29 October 1998, accepted 4 January 1999.


    Reference
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 

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