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Hewlett Packard, D-76337 Waldbronn, Germany
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Abstract |
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In recent years, hapten-reactive T cells have regained considerable attention reflecting both their involvement in chemical- or drug-induced allergic disorders as well as their application in the analysis of general features of TCRantigen interactions (1–5). The MHC-restricted contacts between haptens such as TNP and the corresponding TCR have been shown to closely reflect those between TCR and nominal peptide antigens (4–7). T cell–antigenic epitopes were found to represent MHC-associated, haptenmodified peptides and several studies indicate that haptens, like peptides, may be contacted by the CDR3 loops of the specific TCR (5, 8). These findings opened a possibility to examine a novel interaction of T cells with antigen-presenting cells, recently described for T cells directed at nominal peptide antigens. Several groups have reported on the phenomenon of clonal T cell antagonism (9, 10), demonstrating that effector functions of these cells can be blocked by variants of the antigenic peptide. This phenomenon of theoretical as well as potentially practical importance has never been studied for T cells directed at hapten-conjugated peptides.
The TNP-specific, H-2Kb–restricted murine CTL used for these studies were induced from naive C57BL/6 spleen lymphocytes with synthetic peptides based on the sequence of the chicken OVA-derived peptide 257-264 (SIINFEKL, 07) (11) carrying either TNP or DNP substitutions on the
Thus, CTL reactive to 07TNP may serve as a readout system for antagonism by peptides differing from 07TNP either by amino acid exchanges in position 4 or by altered hapten substituents in position 7. Our findings demonstrate that both types of variant ligands, indeed, may result in an antagonistic blockade of TNP-specific responses. Moreover, we made use of this system to study the downregulation of antigen-specific TCR on mouse cells in response to agonistic versus antagonistic peptides.
Peptides.
Primary In Vitro Induction of CTL.
Cytotoxicity Assay.
Proliferation Assays.
Kb-stabilization on RMA-S Cells by Peptides.
FACS® Analysis of TCR Downregulation.
Amino acid sequences of all synthetic peptides employed in these experiments are listed in Table 1 with asterisks indicating the positioning of either DNP or TNP modifications. As mentioned in the Introduction, all peptides except for the controls M4L-TNP (17) and VSV (23) were derived from the OVA sequence 257-264 (short name 07) (11, 24). Three amino acid exchanges were introduced, i.e., 07(N4R), replacing the position 4 Asp by Arg and 07(N4G) with Gly replacing the Asp. Peptide O4DNP was derived by exchanging Asp with DNP-modified lysine. 
-amino group of Lys 263 (6). The reasons for selecting this particular carrier peptide were several: (a) peptide 07 binds with high affinity to the H-2Kb binding groove (11); (b) the molecular complex of Kb and peptide O7 has been analyzed by x-ray crystallography (13); (c) CTL reactive to the unmodified peptide 07 have been extensively studied with regard to antagonism by mutant peptides (10); and (d) CTL reacting to Kb-associated TNP-peptides modified in position 7 have been found to contact both the hapten in position 7 as well as the side chain of amino acid number 4 (6, 14).
Materials and Methods
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Abstract
Materials and Methods
Results
Discussion
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Animals and Cells.
C57BL/6 mice were provided by the specific pathogen-free breeding facility at the Max-Planck-Institute and used at 6–8 wk of age. Previously described cell lines employed in this study were: RMA and RMA-S T-lymphoma (15), the TNP-specific CTL clone III/1 (16, 17), the CTL clones 4G3 (18), and B3 (25), both specific for OVA257-264, and the OVAtransfected EL-4 line E.G7OVA (12).
Peptides were synthesized as described (17) using -N-TNP- or -N-DNP-lysine modifications (Neosystem, Strasbourg, France) to obtain haptenated derivatives. All peptides were purified by HPLC and purity was confirmed by sequencing and/ or mass spectrometry.
Hapten-peptide specific CTL were induced in vitro from C57BL/6 spleen cells as previously described (14). In brief, 4 x 105 naive spleen cells were cocultivated with 3 x 105 irradiated, syngeneic spleen cells, 1.25 µM peptide, and 7.5% ConA-induced rat spleen supernatant (CASN)1 as a source of IL-2 in 200-µl cultures in 96-well round bottom plates (Greiner, Nürtingen, Germany). RPMI 1640 medium was supplemented with 25 mM Hepes 10% FCS, 2 mM L-glutamine and 10 µM 2-mercaptoethanol. Cultures were restimulated and expanded weekly with fresh stimulators, peptide and CASN. Clones were generated by limiting dilution and subcloning at 0.3 cells/well.
Standard 4 h chromium release assays were performed as described (17), using 2 x 103 51Cr-labeled RMA target cells per well and graded effector/target ratios. Supernatants from targets in 1 N HCl served as 100% lysis controls, and spontaneous Cr release from targets in medium alone was set at 0%. Targets were pulsed with peptides during the labeling with 51Cr. For peptide-inhibition assays 51Cr-labeled targets were added to mixtures of antigenic peptides at suboptimal concentrations with serial dilutions of inhibitory peptides in a total volume of 150 µl. After 1 h at 37°C, CTL (4–5 d after restimulation) were added in 50 µl at an effector/target ratio of 3:1. Plates were centrifuged at 40 g, incubated for 4 h at 37°C, and spun again 5 min at 240 g. Released radioactivity was determined in 100 µl supernatant in a 
radiation counter. All data represent means of triplicates with SD <10%.
Peptides were mixed in 96-well roundbottom plates with 3 x 104 CTL (7–8 d after last stimulation) and 1.2 x 105 syngeneic, irradiated spleen cells in 200 µl supplemented RPMI medium containing 1% CASN. After 48 h, [3H]thymidine (0.5 µCi/well) was added, the cells harvested 14 h later on GF/A filters, and radioactivity determined in a scintillationfree β-counter (Dunn, Asbach, Germany). Peptide inhibition of T cell proliferation was determined by adding graded amounts of the peptides to be tested to a fixed but suboptimal concentration of the antigenic peptide before the addition of CTL and irradiated spleen cells as above.
Stabilization of Kb on RMA-S cells was measured as described previously (17, 19). In short, after 36 h at 26°C 1.2 x 105 RMA-S cells/well were mixed with graded concentrations of peptides in 96-well roundbottom plates, and 30 min later transferred to 37°C. After a 3-h incubation, cells were washed, labeled with the Kb-specific mAb Y3 (20), and stained with FITC-conjugated goat anti–mouse Ig (DIANOVA, Hamburg, Germany).
RMA cells (1.2 x 105) were mixed in 150 µl RPMI medium in round-bottom microtiter plates with graded concentrations of the peptides to be assayed, and incubated 1 h at 37°C. CTL, 7–10 d after the last restimulation, were then added at an effector/target ratio of 2:1 in 50 µl. After centrifugation for 5 min at 40 g, and subsequent incubation for 4 h at 37°C, cells were washed twice in 0.5 mM EDTA/PBS to disrupt conjugates. Cells were labeled either with mAb 145-2C11 (21), specific for CD3 or in the case of clone E8 with mAb RR3-16, specific for V
3.2 (22), and stained with FITC anti-hamster or -mouse Ig, respectively. For flow cytometric analyses in a FACScan® instrument, RMA cells (negative for V3.2) were gated out using FSC/SSC parameters and backgating on fluorescein-labeled cells. In some experiments the clone cells were double-stained using a CD8-specific, PE-labeled antibody (53-6.7; PharMingen, San Diego, CA) and FITC-labeled V3.2- or CD3-specific antibodies (RR3-16 and 145-2C11; PharMingen).
Results
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Abstract
Materials and Methods
Results
Discussion
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Synthetic Peptides and Hapten-specific CTL Clones.
The hapten-specific, H-2Kb–restricted CTL clones described here have been obtained by primary in vitro induction of C57BL/6 spleen lymphocytes with synthetic peptides.
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Peptide Binding to H-2Kb.
Since we aimed at analyzing the effect of a variant peptide on the effector functions of CTL clones in the presence of the antigen on the same antigen presenting cell, we had to distinguish between peptide–TCR interaction-related events and mere competition of these peptides for MHC binding. Therefore, all peptides were tested for binding to Kb. As shown in Fig. 2 A, all peptides stabilized empty Kb-molecules on RMA-S cells at half-maximal concentrations of 
5 nM. A second test was based on competitive inhibition of the Kb-restricted CTL clone III/1 (6, 16) that was specific for peptide M4L-TNP, but did not react to any of the 07-derived peptides. Admixture of graded concentrations of competitive peptides to a constant amount of M4L-TNP resulted in dose- dependent inhibition of target cell lysis by clone III/1. As shown in Fig. 2 B, again all peptides displayed identical competitive inhibitory efficacy, strongly suggesting that neither changes of amino acid 4 nor the modification of Lys in position 7 with DNP or TNP affected the affinity of peptide 07 for the Kb-binding groove.
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The variation of antagonistic, agonistic, and indifferent properties of the various peptides in relation to different CTL clones suggests that even under completely antagonizing conditions for one clone, the antigenic determinants on the same target may still be recognized by other CTL of corresponding specificities. As an example, admixture of 0.1–1 µM 07(N4R)DNP to 0.07 µM of the agonist 07(N4G) TNP totally abrogated specific lysis by clone G9a (Fig. 3 B), but did not at all affect the reactivity of clones E8 or H12b (Fig. 3, A and C).
In variance with other studies on peptide antagonism for CD8+ T cells (10, 25), in the above cited experiments agonist and potential antagonists were added simultaneously to the radio-labeled target cells. However, Fig. 4 demonstrates that the lysis of targets prepulsed with the agonist 07TNP was also inhibited by subsequent addition of peptides 07DNP or 07(N4G)DNP, but not of 07(N4R) or 07(N4G) demonstrating that the inhibitory capacity of the peptides is similar in both assays. The molar excess of inhibitor required for 50% inhibition in both types of experiments (10–100-fold) is comparable or even lower than that described by Jameson et al. (25) for the antagonistic inhibition of CTL specific for the unmodified peptide 07.
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These data confirm that in our experiments antagonistic and irrelevant peptides possess an absolutely identical potential to compete for the agonist. In addition, the absolute number of antigenic determinants on agonist-pulsed target cells is not decreased upon additional binding of antagonists. The inhibitory action of these molecules is, therefore, likely to involve active interference of antagonist–MHC complexes with TCR signaling.
TCR Is Engaged by Antagonistic Peptide–MHC Complexes.
To test for a possible engagement of TCR with antagonistic peptide–Kb complexes we employed cold target competition assays. Nonradioactive (cold) RMA targets loaded with various peptides were mixed with 51Cr-labeled (hot), 07TNP-pulsed targets and subjected to lysis by clone E8. As seen in Fig. 6, cold targets presenting either the agonist 07TNP or the antagonist 07DNP resulted in comparative inhibition of lysis. In contrast, targets pulsed with the irrelevant peptide 07(N4R) were indistinguishable from RMA cells without added peptide. Hence, the antagonistic epitope clearly interacts with the antagonized TCR. However, this epitope not only fails to initiate the relevant TCR signals for lysis and proliferation, but it even blocks their induction by TCR–antigen contacts with the correct determinants presented on the same cell.
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We, therefore, determined TCR expression on the TNPspecific clones E8, H12b, and G9a. We also analyzed the Kb-restricted CTL clone B3, induced against the OVApeptide SIINFEKL (25). For this clone the peptide O7(N4G) had been shown to be a potent antagonist (25). T cells were coincubated for 4 h with the Kb-expressing RMA target cells that were either untreated or preloaded with strong agonists or antagonists. A FACS® staining was performed either with anti CD3 mAb or with TCRV3.2specific mAb for clone E8. RMA targets could easily be gated out according to light scattering properties and did not require fluorescent labeling. As shown in Fig. 7 A in original FACS® histograms, all 4 clones revealed a significant loss of TCR only upon contact with the respective agonists, but not with antagonists. This proves (a) that the phenomenon of antigen-induced downregulation of TCR also applies to mouse T cells, and (b) that TCR downregulation occurs in reactions with hapten as well as to nominal peptide antigens (clone B3).
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In a further experiment we investigated the effect of antagonists on the agonist-mediated downregulation of TCR. Cells of clone E8 were exposed to RMA cells in the presence of a fixed concentration of the antigen O7TNP, which resulted in 40% reduction of TCR surface-expression (Fig. 7 C). Increasing concentrations of the antagonist 07DNP, but not of the irrelevant peptides O7(N4R) and O7(N4G) gradually inhibited the agonist-induced downregulation of TCR. To exclude an unspecific effect of DNP, peptide O4DNP (Table 1) was included as a further control. This peptide carries DNP in position 4 and is not recognized by clone E8, but stabilizes Kb-molecules on RMA-S cells even slightly better than O7DNP (data not shown). Also in this case, no inhibition of downregulation was observed. Moreover, also for a second clone (G9a) only the antagonist O7(N4R)DNP inhibited the loss of surface TCR induced by the agonist O7(N4G)TNP. The irrelevant peptide O7(N4R) had no effect (Fig. 7 D).
Antagonists Fail to Induce Anergy.
We also examined whether CTL, upon contact with antagonists, might be anergized in terms of a reduction of antigen-specific cytotoxicity or proliferative potential. E8 cells were incubated with BCECFAM (2', 7-bis-(carboxyethyl)-5(6')-carboxyfluorescein)- labeled RMA cells that were either untreated or preincubated with agonist, antagonist or irrelevant peptide. After a 4-h incubation the CTL were separated by FACS® sorting and mixed at a defined effector/target ratio with 51Cr-labeled RMA targets in the presence of titrated amounts of the antigen O7TNP (Fig. 8 A). In parallel we determined the downregulation of TCR on the CTL cells in these cultures (Fig. 8 B). The data revealed that, as in the above mentioned experiments (Fig. 7), the agonist-contact resulted in >50% loss of TCR. The reduced receptor density, in turn, was paralleled by a reduction of cytolytic activity. In contrast, preincubation with antagonist-treated target cells had no effect on TCR-expression as well as on the killing-potential of the CTL. Contact with antagonist–MHC complexes, thus, leaves the treated CTL functionally fully active. We also found (data not shown) the proliferative response of clone E8 to be unaffected by preincubation with antagonist-loaded target cells.
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In the present study we provide evidence that alterations of the amino acid subepitope in position 4 as well as of the hapten subepitope in position 7 resulted in peptides with potent antagonistic properties. Thus, removal of one of the three nitrogroups of the TNP-substituent in peptide O7TNP, resulting in the corresponding peptide O7DNP, conferred the strongest agonist for clone E8 into its most potent antagonist (see Figs. 1 A and 3 A). Antagonism in this context is understood as an inhibition of the effector function, i.e., agonist-mediated cytolysis, of a TNP-specific CTL clone by the simultaneous presence of agonistic and antagonistic peptides on the same target cell.
This finding has several interesting implications. On one hand it opens a way to study hapten antagonism in drug allergies. In these cases, e.g., in allergies to β-lactam antibiotics, various degrees of specificity or cross-reactivity between individual antibiotics have been observed (31), and numerous structural variations might permit the construction of potentially antagonistic ligands.
On the other hand, the use of hapten-peptide conjugates in studies of T cell antagonism has the advantage that these ligands, in contrast to most nominal peptide antigens, are detectable by hapten-specific antibodies even in their MHCassociated state (17). Employing this property we could show that (a) in our experiments antagonists did not differ from irrelevant peptides in their efficacy to compete with the agonist for binding to the Kb binding groove, and (b) that agonists as well as antagonists, once bound to Kb, could not be displaced from their binding sites even by addition of excessive amounts of high-affinity peptides (Fig. 5 B). In addition, saturation of surface expressed Kb molecules with external peptides did not prevent the subsequent loading of newly expressed Kb molecules with additional peptides on the same cell (Fig. 5 B).
The molecular basis of peptide antagonism is still poorly understood. T cell stimulation with some antagonists has been demonstrated to result in partial signaling and altered phosphorylation patterns of TCR associated proteins in CD4+ (9, 32) or CD8+ T cells (33). Other authors have discussed the relationship of antagonism and the induction of T cell anergy, but could not correlate TCR induced anergy to altered patterns of tyrosine phosphorylation (34). Although binding constants reported in the literature for various MHC-agonist-TCR complexes vary considerably (35–38), some models explain the phenomenon of TCR antagonism by postulating that defined differences of affinity between antagonist and agonist result in unfavorable kinetics for the activation of the T cell (39, 40). In support of these models, soluble TCR molecules of class I (41) and class II MHC–restricted T cells (29, 42) were found to bind with lower affinity to soluble complexes of MHC with antagonistic compared to agonistic peptides.
In this respect, the serial triggering model forwarded by the group of Lanzavecchia (26, 43) deserves particular interest. This model proposes the activation of large numbers of TCR by few MHC–agonist complexes, followed by a removal of the activated receptors from the cell surface. The kinetics of this downregulation of TCR is believed to be controlled by the affinity-dependent persistence of MHCpeptide-TCR complexes in the contact regions between APC and T cells. Optimal T cell activation would be characterized by a complete triggering of an optimal threshold number of TCR, and higher or lower TCR affinities might result in less efficient or even antagonistic signals. TCR– antagonist contacts of presumably lower affinity have been discussed by the authors as possibly resulting in an exhaustion of functional receptors below a critical threshold (26).
However, the activation-related downregulation of TCR has so far been demonstrated only for human T cells. In contrast, the effect of T cell antagonists has mostly been investigated in mouse systems. Our data now demonstrate that antigen-induced internalization of TCR is also observed for mouse T cells specific for haptenated as well as for nominal peptide antigens and, hence, represents a general phenomenon. Moreover, the extent of downregulation related in a dose-dependent fashion to the antigenicity of individual TNP-peptides (Fig. 7, A and B). In contrast, antagonistic peptides were indistinguishable from irrelevant peptides in failing to induce any downregulation of TCR (Fig. 7, A and B).
In fact, when added together with the agonist, only antagonists but not irrelevant Kb-binding peptides, blocked the removal of TCR (Fig. 7, C and D). The fact that no competition was observed even when irrelevant competitors were added at a 100–1,000-fold excess is reminiscent of lysis-inhibition data in Fig. 3. This implies that antagonists specifically interact with the TCR (also shown by cold target competition in Fig. 6) and actively inhibit TCR removal from the surface of the T cell. However, antagonists do not block T cell reactivity via a removal of available TCR.
It remains to be seen whether the process of internalization of activated TCR is a necessary part of the signaling cascade or rather indicates the removal of used receptors from the system. This leaves at least two possibilities to explain the action of antagonists: (a) Their interaction with TCR results in irreversibly wrong phosphorylation patterns of TCR-associated proteins and a lack of signals for internalization. The T cell surface would thus be largely occupied by non-reactive, though still antigen-binding, TCR. (b) The low stability of TCR–antagonist complexes allows for either no or incomplete phosphorylation, which, by later contact with the agonist, might in fact be completed. However, in this case MHC–antagonist complexes due to their molar excess on the APC as well as to their shorter contacts with specific TCR (26) might simply compete out the agonist–MHC complexes on the same APC. A continual interruption of the serial triggering mechanism either delaying or preventing a threshold for activation to be reached would therefore result in reduced T cell activity. The latter model would, in a way, compare to the cold target competition experiment (Fig. 6) except that the two competing structures are located on the surface of the same target cell.
Our data (Fig. 8) reveal that preincubation of CTL with antagonist-loaded target cells fails to induce any reduction of the cytolytic activity or of the proliferative response.
Therefore, we favor a scenario in which on the one hand, the TCR is not functionally inactivated by the contact with the antagonist while on the other hand, the serial triggering of many TCR, a prerequisite for full activation of the T cell, is interrupted.
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Acknowledgments |
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Submitted: 17 December 1996
Revised: 6 March 1997
1 Abbreviation used in this paper: CASN, ConA-induced rat spleen supernatant.
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References |
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