Peptide analogues of a T-cell epitope of ricin toxin A-chain prevent agonist-mediated human T-cell response

D. Castelletti and M. Colombatti

Section of Immunology, Department of Pathology, University of Verona, c/o Policlinico ‘G.B. Rossi’, L.go L.A. Scuro 10, I-37134 Verona, Italy

Correspondence to: M. Colombatti; E-mail: marco.colombatti{at}univr.it


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results and Discussion
 References
 
The clinical efficacy of immunotoxins (IT) containing ricin toxin A-chain (RTA) can be drastically reduced by anti-toxin-neutralizing antibodies developed by patients. Strategies aimed at epitope-specific modulation of the immune response must be therefore set up to broaden the clinical applicability of RTA-based IT. Prevention or reduction of humoral immune responses against RTA could be achieved by peptide-based down-modulating strategies. Peptide analogues were investigated as candidate antagonist altered peptide ligands (APL) considering the sequence of a previously identified dominant T-cell epitope of RTA (i.e. I175–E185) presented in the context of the HLA-DRB1*03011 allele. Alanine-substituted peptides provided information on the role of individual residues of the wild-type peptide and allowed to identify one antagonist APL corresponding to the double-mutant peptide E177A/A178D. The analogue E177A/A178D not only prevented the agonist from stimulating anti-RTA human T-cell clones but also failed to induce down-regulation of surface-expressed TCR, thus suggesting its possible use for in vivo immune modulation of anti-RTA responses.

Keywords: altered peptide ligands, antagonists, immune modulation, ricin toxin A-chain, T-cell epitopes


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results and Discussion
 References
 
Potent cytotoxic reagents [immunotoxins (IT)] are obtained by conjugating cell-selective vehicle molecules (e.g. antibodies, ligands, growth factors) to the enzymatic polypeptide ricin toxin A-chain (RTA). RTA-IT yielded promising results in the treatment of human diseases, particularly hematological neoplasias (1, 2). However, injection of RTA-IT often induces the development of high-titer anti-toxin antibodies, affecting IT efficacy and strongly limiting a more general application of RTA-IT in vivo (3, 4). Development of antibodies to the targeting molecules can be circumvented by using reagents of human origin. Prevention or modulation of anti-RTA immune response will instead require a more complex intervention.

Strategies to prevent or reduce the anti-RTA immune response were extensively evaluated in animal models by treating mice with cyclophosphamide (5) or with other immunosuppressive agents (6). These approaches, however, may not be directly applicable in humans because immunosuppressive drugs can increase the risk of concomitant infections and malignancies. To overcome these limitations, development of less aggressive and more selective methods to suppress or reduce the host immune response against RTA would be advisable. The observation that human anti-ricin antibodies belong to the IgG class indicates that RTA is a thymus-dependent antigen able to induce a secondary immune response involving both T cells and B cells (7). Manipulating the response of potentially reactive T cells using altered peptide ligands (APL) represents a very attractive approach (8). Once immunodominant T-cell epitopes are identified, down-modulation of the immune response can be achieved using APL (9). Antagonist peptide analogues are able to inhibit the agonist-mediated T-cell activation and therefore the downstream events leading to a humoral immune response (10). Functional inhibition of T-cell responses by APL has been described in animal models of autoimmune diseases (11, 12) and encouraging results have been obtained also in humans (13, 14).

We have previously described a set of human RTA-specific T-cell clones, obtained by in vitro priming of peripheral blood cells from healthy donors as a source of responder T cells (7). Clonal T cells recognized a minimal T-cell epitope spanning the sequence I175–E185 of RTA (7) by preferentially engaging TCR-V{alpha}1 and TCR-Vß8 chains. The epitope I175–E185 is presented to anti-RTA T-cell clones in association with HLA-DRB1*11011 and HLA-DRB1*0301 molecules, which are widely expressed in the Caucasian population (15).

We therefore set out to investigate the reactivity and the modulating properties of alanine-substituted analogues of I175–E185 considering the proliferative response of anti-RTA T-cell clones. By this approach we identified one candidate epitope-derived APL with antagonist properties, which could become a suitable tool to prevent/reduce anti-RTA responses.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results and Discussion
 References
 
Chemicals and reagents
All the chemicals were purchased from Sigma (St Louis, MO, USA). Peptides based on the sequence of the RTA and point mutant peptides were synthesized by an Applied Biosystem (Monza, Italy) automated synthesizer on solid phase (16). Purity was assessed by HPLC and mass spectrometry and was found to be >90%.

RTA-specific T-cell clones
Antigen-specific T-cell clones were obtained by in vitro priming of PBMC obtained from a normal donor (donor M.C.) with no history of previous sensitization to RTA and no evidence of anti-RTA antibodies in the serum. The RTA-specific T-cell clones used in the present study were partly characterized previously (7), and those retaining a higher proliferative potential were selected for further studies. Here T-cell clones MC44, MC69, MC37 and MC52 were used. All the T-cell clones showed comparable proliferation in response to the stimulating peptide I175–E185, expressed CD4 antigen and recognized the RTA peptide I175–E185 in the context of the MHC class II allele HLA-DRB1*0301 utilizing V{alpha}1 and Vß8 TCR chains (7). Use of different T-cell clones in different sets of experiments is due to the limited availability of clonal responder T cells in some instances.

B-lymphoblastoid cell lines
An autologous B-lymphoblastoid cell line (B-LCL) was obtained from a depleted PBMC population from the donor M.C. as reported elsewhere (7). Briefly, freshly isolated PBMC were depleted of T cells and infected with an EBV-containing supernatant of the marmoset cell line B95-8 (American Tissue Culture Collection, Rockville, MD, USA) and grown under standard culture conditions (see below).

Two EBV-transformed B-LCL (from European Collection for Biomedical Research, Southampton, UK), homozygous for the indicated human MHC class II alleles, were used as antigen-presenting cells (APC) in proliferation assays. WT49, expressing HLA-DRB1*0301, and SWEIG, expressing HLA-DRB1*11011, were chosen based on the HLA typing of the PBMC used as source of anti-RTA T-cell clones (17, 18). Nomenclature of HLA-DR alleles is reported by Robinson et al. (19).

Cell culture conditions
T-cell clones as well as B-LCL were grown under standard culture conditions, i.e. at 37°C in a 5% CO2 atmosphere, in RPMI-1640 medium (Life Technology, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Boehringer, Mannheim, Germany), 2 mM L-glutamine (Seromed, Berlin, Germany) and antibiotics (penicillin–streptomycin, 400 U ml–1). T-cell clones required the addition of 100 U ml–1 of human recombinant IL-2 (rIL-2) (Chiron, Siena, Italy) and periodic stimulation with either 1% PHA (Life Technology) or 25–50 µg of heat-denatured native RTA (dRTA) in the presence of irradiated (4000 rad) autologous PBMC.

Proliferation assay
RTA-specific T cells were co-cultured with either autologous or homozygous B-LCL as APC to measure antigen-induced T-cell proliferation. T cells were plated at 1–2 x 105 cells per well in 96-well culture plates (Greiner, Longwood, FL, USA), following exhaustive washings to eliminate rIL-2 from cell cultures and overnight incubation in the absence of rIL-2. APC were irradiated (4000–6000 rad) by exposure to a source of 137Cs radiations and plated 1 : 2 with respect to responder T cells. When a pre-pulsing step was required, irradiated APC were incubated for 2–4 h or overnight in the presence of dRTA or RTA-derived peptides and washed twice with RPMI-1640 medium before co-culture with T cells. After 3–4 days, T-cell proliferation was assessed by addition of 0.5 µCi of tritiated thymidine ([3H]TdR) (Amersham, Amersham, UK) to the culture and incubation for further 16 h. Following cell harvesting, washing and drying onto glass fiber filters (using a cell harvester, Dynatech, Haverhill, MA, USA), the radioactivity incorporated by the cells was measured in a ß-spectrometer (Wallac 2406). The counts per minute (c.p.m.) values obtained were used to calculate the stimulation index (SI), which is defined as the mean c.p.m. of triplicate or duplicate antigen-stimulated samples divided by the mean c.p.m. of control cultures without stimulating antigen (i.e. the mock-treated controls). Proliferation was considered significant when SI ≥ 2. Values of [3H]TdR incorporation in control samples were usually <500 c.p.m.

Inhibition of antigen processing
T-cell proliferation assays were performed essentially as described above, by adding 1 x 105 cells per well from a T-cell clone to 2 x 105 cells per well of pre-pulsed WT49 cells, which were either irradiated or fixed after antigen pulsing. The irradiated WT49 cells were used as positive control of competent APC, whereas inhibition of intracellular processing of antigen was achieved by treating WT49 cells as follows: after extensive washings, cells were re-suspended in 100 µl of complete medium supplemented with 80 µM chloroquine. After 10 min incubation at room temperature, peptides (10 µg ml–1) were added and cells were incubated for further 4 h at 37°C. Excess antigen was removed by washing the cells twice in complete medium and once in PBS. Cells were then fixed with 0.25% glutaraldehyde for 60 s and fixation was blocked by adding 0.2 M glycine for 10 min.

TCR antagonism assay
Antagonist activity of two RTA-based peptides (E177A and E177A/A178D) was assayed by proliferative assays, in which the APC were pre-pulsed with the agonist peptide I175–E185, essentially by following the method described by Frasca et al. (8). Autologous B-LCL cells were pre-pulsed overnight with a fixed dose of I175–E185, washed and then incubated for further 5 h with either E177A or E177A/A178D at various concentrations. In parallel, equal amounts of the same antagonist peptides alone were incubated overnight with the autologous B-LCL. The peptide I175–E185 was used at 10 µg ml–1, corresponding to the concentration resulting in 50% of maximum T-cell stimulation, according to dose–response experiments performed previously. Following incubations with peptides, the APC were irradiated (6000 rad) and added to T-cell clones in 96-well round-bottom microplates (5 x 104 cells per well). After 3 days, T-cell proliferation was measured by following the standard protocol described above. The data are presented as percent inhibition of T-cell proliferation with respect to the maximum c.p.m. values obtained with the stimulating peptide alone or in the absence of other antigenic stimuli.

Down-modulation of TCR expression
Down-modulation of TCR expression was evaluated by measuring the surface expression of the CD3 co-receptor by T-cell clones, pre-incubated separately with RTA-based peptides (L161–T190, I172–M188, I175–E185) and with mutants of the I175–E185 peptide (E177A and E177A/A178D). The assay was performed essentially as described elsewhere (20). Briefly, autologous B-LCL cells (2 x 105 cells per well) were re-suspended in 100 µl of 5% FBS-containing RPMI-1640 medium and pulsed for 3 h with peptides (at 50 µg ml–1 concentration). A mock-treated sample was also included as control. Following three washings with medium to eliminate unbound peptides, pre-pulsed B-LCL cells were mixed with resting T cells (1 x 105 cells per well) in round-bottom microplates and centrifuged to allow the formation of conjugates at 37°C. After 4 h incubation, cells were re-suspended and washed in PBS containing 0.5 mM EDTA to break the conjugates. Cells were then stained with a PE-conjugated anti-CD3 mAb (Becton-Dickinson, San Diego, CA, USA) and the CD3-associated fluorescence was analyzed with a FACScan (Coulter, Hialean, FL, USA). A sample stained with an irrelevant PE-labeled mAb (Becton-Dickinson) was considered as the negative control. Expression of cell-surface CD3 was evaluated by considering the mean fluorescence intensity. Data are presented as percent TCR down-regulation (i.e. reduction of CD3 surface expression) with respect to the maximum CD3 level (100% expression) measured in T-cell clones that were incubated with non-pulsed APC.


    Results and Discussion
 Top
 Abstract
 Introduction
 Methods
 Results and Discussion
 References
 
In the present study, the T-cell epitope I175–E185 was investigated in detail by assaying single-substituted analogues for their ability to induce and modulate RTA-specific T-cell activation. A set of high-responder human anti-RTA T-cell clones were used which recognized the relevant epitope in the context of the widely distributed MHC class II allele HLA-DRB1*0301. Ricin is a member of the ribosome-inactivating proteins (RIP) family which also includes a number of single-chain RIP-I, such as dianthin, momordin, saporin, pokeweed anti-viral protein and gelonin. The enzymatic subunit of ricin (RTA) and toxins belonging to the RIP-I group are all of similar size, all carry out the same N-glycosidation reaction and show a high degree of sequence similarity (21, 22). The T-cell clones used by us recognize a region of RTA containing the enzymatic site of action of the toxin which also shows a high degree of similarity with domains belonging to other RIP-I (7). However, they do not recognize a panel of RIP-I showing stretches similar but not identical to fragment L161–T190 (7).

Role of individual amino acid residues in T-cell activation
It was reported that the most striking effects on the binding of peptides to the HLA-DRB1*0301 allele (23) are caused by hydrophobic and aromatic residues in position 1 (i.e. the primary anchor) and position 2 within the nonapeptide core that typically characterizes class II-restricted T-cell epitopes. The role of isoleucine 175 as the primary anchor of the RTA epitope I175–E185 was indeed confirmed on the basis of several experimental findings (7). Peptide positions 4, 6, 7 and 9 of the HLA-DRB1*0301-specific binding motif are usually defined as secondary anchors devoted to peptide stabilization, whereas there is typically a TCR-contact residue in position 3. Anchor IV (in position 4) is dominated by an aspartic acid; positions 5 and 6 have preference for basic and hydrophilic amino acids and position 7 for hydrophobic and aromatic side chains (23). Finally, positions 8 and 9 are occupied by structurally different residues (23). Based on this background information, we observed that the sequence of the epitope I175–E185 satisfies the main chemical restrictions for a productive presentation by HLA-DRB1*0301 molecules, with the exception of position 4 that is occupied by alanine. According to this, the region I175–Y183 is likely to represent the nonapeptide core of the epitope I175–E185, fitting the MHC groove and including most of the crucial residues involved in interactions with both TCR and MHC. To evaluate the relative contribution of individual amino acids of the epitope I175–E185, the reactivity of single-substituted peptides was evaluated in proliferation assays. The contribution of the amino acid side chains in either MHC binding or TCR engagement was abrogated by replacing each peptide position with an alanine.

Table 1 shows the sequence and the stimulation effect of the alanine-substituted peptides (used at the fixed concentration of 20 µg ml–1) in comparison with the wild-type epitope I175–E185. The same set of mutated peptides was also assayed in dose–response experiments using different T-cell clones. Figure 1 shows the results obtained with one representative T-cell clone (MC44). Abrogation of the epitope's reactivity provided by almost every substitution within the sequence I175–Y183 strongly supported its identification as the nonapeptide core of the epitope. In fact, only the mutation S176A in peptide position 2 did not affect the stimulatory efficiency of the epitope (Fig. 1) and can be therefore considered an ‘indifferent substitution’ (23). A slight stimulatory effect was observed only when the mutated peptides substituted in R180, F181 and Y183 were used at very high doses (Fig. 1). Substitution of side residues I184 and E185 led only to a partial reduction of T-cell stimulation (Table 1 and Fig. 1), confirming their stabilizing effect in anchoring the peptide.


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Table 1. Effect of single-substituted peptides on T-cell proliferationa

 


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Fig. 1. The wild-type epitope and mutated peptides show different stimulation effects in dose–response proliferation assays. The wild-type epitope (I175–E185) and mutated peptides (I175A, S176A, E177A, R180A, F181A, Q182A, Y183A, I184A, E185A) are compared for their dose-dependent stimulation potential. Cells of the T-cell clone MC44 were used as a source of responder cells.

 
Although pI175–E185 represents the minimal epitope identified, peptides of different lengths can also fit into an MHC class II peptide-binding groove. Naturally processed peptides could be longer than the minimal epitope identified by us. To investigate if RTA peptides of larger size can induce T-cell activation without modifications or if they need trimming to be presented, the 30-mer peptide L161–T190 and the 15-mer peptide C171–E185 were tested in proliferation assays under conditions impairing intracellular processing using B-LCL WT49 cells as APC. As a control peptide I175–E185 was used. WT49 cells, either untreated or fixed, retained the ability to present all three RTA-based peptides (data not shown). Therefore, also larger peptides could be presented without further processing.

Peptide analogues that failed to directly stimulate T-cell clones (i.e. E177A, R180A, F181A, Q182A and Y183A) were used as competitors of the native peptide I175–E185. Reduced T-cell stimulation achieved by the agonist peptide in the presence of 20-fold molar excess of each mutant was taken as a good indicator of the ability of a mutant to interfere with the sequence of events leading to T-cell activation and proliferation. Under these conditions, only the mutant E177A competed out the epitope I175–E185 and prevented the agonist-mediated T-cell stimulation by providing a 15 and 40% inhibition when the B-LCL WT49 (homozygous for HLA-DRB1*0301) and the autologous B-LCL cell lines were used as APC, respectively (data not shown). The analogues failing to inhibit T-cell stimulation induced by a wild-type peptide are considered to contain residues involved in MHC class II binding, whereas peptides retaining a significant inhibitory ability are considered to be altered in correspondence to TCR-contact residues (24). Thus, the amino acid in position 3 (E177) of our epitope I175–E185 may play a crucial role as a TCR-contact residue.

In Fig. 2, dose–response curves obtained on stimulating T-cell clone MC69 with the mutant E177A alone were compared with results obtained when the mutant was added to WT49 cells in combination with fixed amounts of the wild-type peptide I175–E185. A sub-optimal (2.5 µg ml–1, panel A) concentration and a stimulating (10 µg ml–1, panel B) concentration of the I175–E185 peptide were used. For comparison, the other alanine-substituted peptides were also assayed in the presence of a sub-optimal dose of I175–E185 (2.5 µg ml–1). Use of sub-optimal doses of wild-type peptide allowed to maximize the possible displacing effect of a competitor ligand. Three main profiles of the competitive effect of the mutants emerged from our results (represented in Fig. 3, panels A and B). Peptides S176A (shown as an example in panel A), I184A and E185A stimulated anti-RTA T-cell clone MC69 with comparable efficiency in the presence or absence of the wild-type I175–E185. Peptides R180A, F181A, Q182A and Y183A (the latter reported in panel B) failed to activate T-cell clones (except at very high concentrations) if tested alone, whereas they revealed a synergic effect on T-cell activation when combined with the wild-type epitope. This finding could be explained as the recruitment of a threshold number of TCR leading to a productive T-cell activation only when both wild-type and mutant peptides are present, which is not obtained in the presence of the mutants alone or in the presence of a sub-optimal concentration of the agonist peptide (25). Finally, E177A was the only mutant which was unable to activate T cells or to improve the epitope's reactivity at each concentration assayed (Fig. 2, panel A). More interestingly, however, E177A inhibited the epitope-mediated T-cell stimulation when the wild-type I175–E185 was added at the concentration of 10 µg ml–1, corresponding to a 50% stimulation (Fig. 2, panel B).



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Fig. 2. E177A single-substituted mutant competes with the wild-type epitope in T-cell activation assays. WT49 cells were pre-pulsed with the indicated amounts of the mutant peptide E177A and then co-cultured with anti-RTA clonal T cells (MC69) in the presence (filled circles) or in the absence (empty circles) of 2.5 µg ml–1 of wild-type peptide I175–E185 (panel A). In panel B are shown dose–response curves of the wild-type peptide I175–E185 (filled triangles) and of the E177A mutant, alone (empty circles) or in the presence of 10 µg ml–1 stimulating I175–E185 (filled circles). At the end of the co-culture period, T-cell proliferation was measured by evaluating the incorporation of [3H]TdR (see also Methods). wt = wild type.

 


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Fig. 3. Effect of single-substituted mutants on T-cell proliferation. WT49 cells were pre-pulsed with the indicated amounts of the mutant peptides S176A (panel A) or Y183A (panel B) taken as examples. Proliferative response of clonal T cells (MC69), after incubation with the pre-pulsed WT49 cells either in the presence (filled circles) or in the absence (empty circles) of 2.5 µg ml–1 of wild-type peptide I175–E185, was measured by evaluating the incorporation of [3H]TdR (see also Methods).

 
E177A and E177A/A178D as antagonist peptides
Taking into account the restrictions set forth for a suitable binding of the peptide to HLA-DRB1*0301 molecules (23), the residue A178 of the epitope I175–E185 was replaced with aspartic acid, allowing to obtain a peptide (A178D) endowed with greater stimulatory ability than the wild-type peptide. The mutant activated all T-cell clones assayed ~40% more effectively than I175–E185 (data not shown), possibly because of an improved peptide anchoring to MHC (21). The mutation A178D was then combined with the substitution of E177 in position 3, thus obtaining the double-mutant E177A/A178D. Comparable to the peptide E177A, the double-mutant E177A/A178D failed to activate anti-RTA T-cell clones due to the substitution at a TCR-contact site (E177).

We then evaluated the ability of E177A and of E177A/A178D to interfere with agonist-mediated T-cell activation and therefore to act as antagonists of the wild-type peptide. Figure 4 shows the results of two representative TCR antagonism assays performed with the anti-RTA T-cell clones MC37 and MC52. After pre-pulsing the autologous B-LCL with a stimulating concentration of the I175–E185 peptide (10 µg ml–1), APC were washed to remove unbound peptide. The subsequent incubation in the presence of competitor peptide, either E177A or E177A/A178D, allowed the bound epitope to be displaced on the surface of APC. T-cell activation was finally measured in a proliferation assay after addition of responder clonal T cells. Results illustrated in Fig. 4 demonstrated that the double mutant behaved as a more effective competitor in displacing the native I175–E185 epitope than E177A. Thus, E177A/A178D can act as an efficacious antagonist APL and might be considered as a suitable tool for negatively interfering with agonist-induced T-cell activation also in vivo.



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Fig. 4. Peptides E177A and E177A/A178D antagonize the T-cell stimulation induced by the wild-type epitope. The antagonistic effects of mutants E177A and E177A/A178D were compared in antagonism assays using the anti-RTA T-cell clones MC52 (filled symbols) and MC37 (empty symbols). Stimulation with the native I175–E185 alone produced SI of 13.8 and 28.5 in MC52 and MC37, respectively. These values were taken as 100% to calculate the percent inhibition of proliferation of T cells stimulated with autologous B-LCL cells pre-pulsed with E177A (squares) or with E177A/A178D (triangles) displacing 10 µg ml–1 I175–E185. Percent inhibition is reported on the y-axis.

 
TCR down-modulation analysis
Peptides displaying antagonist features not only fail to activate T cells but they also prevent the generation of a TCR-mediated activation signal by agonist peptides (26). It has been reported that antagonist APL fail to induce down-regulation of surface-expressed TCR at concentrations much higher than those required for inducing the partial tyrosine phosphorylation of CD3-{zeta} chains (27). These observations indicate that antagonist peptides can interfere with the very initial steps of the T-cell activation, i.e. at the level of TCR engagement (28). The effect of antagonist APL can be therefore ascribed to competitive recruitment of TCR and subsequent prevention of TCR oligomerization (29) and productive engagement by agonist peptides (30).

The effect of E177A and E177A/A178D on surface-expressed TCR was compared with the effect induced by three stimulating peptides L161–T190, I172–M188 and I175–E185 (of 30-mer, 17-mer and 11-mer residues, respectively, all containing the minimal epitope I175–E185) using T-cell clone MC52. Autologous B-LCL cells were pre-pulsed with each peptide and co-cultured with anti-RTA T-cell clones to allow the formation of cell–cell conjugates. TCR expression on the T-cell surface was then evaluated by CD3 fluorescent staining and cytofluorometric analysis. As shown in Fig. 5, the treatment with each agonist peptide resulted in a marked decrease in the surface expression of the TCR/CD3 complex to 25–50% of normal levels, whereas treatment with the antagonists (E177A or E177A/A178D) failed to induce any appreciable TCR down-modulation. The lack of a productive TCR engagement by the antagonists, both displaying the E177A substitution, can be therefore hypothesized as a possible mechanism leading to the inhibition of the agonist-induced T-cell stimulation. The total absence of TCR down-regulation observed in the presence of the double-mutant E177A/A178D might be ascribed to a superior ability to displace binding of the agonist as compared with the single-mutant E177A due to the effect of the A178D substitution which enhances the interaction with MHC class II molecules (23). Small changes in the sequence of a T-cell epitope may result in the loss of activation signals in T cells and in the inhibition of agonist-induced T-cell responses (8). Antagonist APL can therefore be considered as suitable reagents for immunological manipulations, such as suppression of humoral responses against molecules of therapeutic relevance. The observation that the epitope I175–E185 maps immediately downstream to a linear B-cell determinant (i.e. L161–I175) (31) allows to define a highly immunogenic region of RTA (corresponding to the stretch L161–E185). It has been reported that the induction of antibodies to a given sequence could in some cases depend on the existence of Th cells specific for a stretch proximal to the antibody epitope. This so-called ‘T–B reciprocity’ (32) could thus explain the high antigenic potential of a limited RTA domain containing both a T-cell and a B-cell epitope. This RTA region is therefore an ideal target for rational antigen modifications aiming at preventing/reducing the triggering of an anti-RTA immune response. Indeed, it was shown in animal models (33, 34) that inhibiton of immune response to an immunodominant epitope prevents spreading of immune recognition and response to secondary/sub-dominant epitopes. Moreover, single conservative amino acid substitutions prevented the induction of responses to both the immunodominant and sub-dominant epitopes, which corresponded to the loss of response to the whole protein molecule (35). It could thus be hypothesized that in vivo application of the APL described by us might result in failure to respond to injected RTA. This of course must be verified in appropriate in vivo models.



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Fig. 5. Antagonist peptides E177A and E177A/A178D fail to induce TCR internalization. The effect of agonist peptides of different lengths (L161–T190, I172–M188, I175–E185) and of the mutants E177A and E177A/A178D on the down-modulation of the TCR was compared. Autologous B-LCL cells were pre-pulsed separately with 50 µg ml–1 of each peptide and added to T cells (T-cell clone MC52). TCR down-modulation was measured by evaluating the surface expression of CD3 of stimulated T lymphocytes by flow cytofluorometry with respect to a control sample (non-pulsed APC).

 
We have here demonstrated that APL are able to prevent the native epitope from activating RTA-specific T-cell clones in vitro, in particular the whole cascade of biochemical signals that normally result in T-cell proliferation. Thus, treatment strategies exploiting the properties of the antagonist peptide discovered by us could also be envisaged; for example, the inoculation of the APL before or at the same time of a treatment cycle with RTA-based IT may prevent the development of potentially anti-RTA reactive T cells and allow more effective treatment regimens. In vivo confirmation of such a hypothesis is of course warranted. The MHC class II allele which is the target of the inhibitory effect of the APL described by us has a broad distribution in the Caucasian population (15); thus, a considerable number of subjects could in principle benefit from such immune-modulating strategies.


    Acknowledgements
 
We are grateful to C. Servis (Institut de Biochimie, University of Lausanne, Switzerland) for the synthesis of peptides used in this paper and for useful suggestions. This work was supported in part by Associazione Italiana per la Ricerca sul Cancro, by Fondazione Cassa di Risparmio di VR-VI-BL-AN, Bando 2001 ‘Ambiente e Sviluppo Sostenibile’, and by Ministero dell'Istruzione, dell'Università e della Ricerca (Cofin 40%, 2003).


    Abbreviations
 
APC   antigen-presenting cells
APL   altered peptide ligands
B-LCL   B-lymphoblastoid cell line
c.p.m.   counts per minute
dRTA   heat-denatured native RTA
FBS   fetal bovine serum
[3H]TdR   tritiated thymidine
IT   immunotoxins
rIL-2   recombinant IL-2
RIP   ribosome-inactivating proteins
RTA   ricin toxin A-chain
SI   stimulation index

    Notes
 
Transmitting editor: L. Moretta

Received 21 June 2004, accepted 11 January 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results and Discussion
 References
 

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