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Interaction of Sulfonamide Derivatives with the TCR of Sulfamethoxazole-Specific Human ß⁺ T Cell Clones¹

Institute of Immunology and Allergology, Inselspital, Bern, Switzerland

	Abstract

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Drugs like sulfamethoxazole (SMX) or lidocaine can be presented to specific human ß⁺ T cell clones (TCC) by undergoing a noncovalent association with MHC-peptide complexes on HLA-matched APCs. For a better understanding of the molecular basis of the recognition of such drugs by specific TCC, we investigated 1) the fine specificity of the recognizing TCR, 2) the dose-response relationship for the induction of proliferation or cytokine production, and 3) the mechanism of TCR triggering. For that purpose, we tested the reactivity of 11 SMX-specific CD4⁺ TCC and 2 SMX-specific CD8⁺ TCC to a panel of 13 different sulfonamide derivatives bearing the same core structure. Five of 13 clones recognized only SMX, while all other clones were responding to as many as 6 different compounds. Some of the compounds needed up to two orders of magnitude higher concentrations than SMX to stimulate TCC, thereby displaying features of weak agonists. Different clones showed clear differences in the minimal drug concentration required for the induction of a proliferative response. Therefore, weaker or stronger agonistic properties were not a characteristic of a given sulfonamide derivative but rather an intrinsic property of the reacting TCR. Finally, the number of down-regulated TCRs was a logarithmic function of the ligand concentration, implicating that specific T cells were activated by serial TCR engagement. Our data demonstrate that, despite the special way of presentation, nonpeptide Ag like drugs appear to interact with the TCR of specific T cells in a similar way as peptide Ags.

	Introduction

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The T cell involvement in drug allergic reactions is well established (for review see 1 . However, the molecular mechanism behind the recognition of small molecular compound like drugs by the TCR has not yet been fully elucidated. It has been thought for several years that drugs can be recognized by specific T cells only after covalent binding to serum or cellular proteins, which, after subsequent processing, are presented by MHC class I or II molecules (2, 3, 4, 5). This pathway of drug presentation requires chemically reactive compounds with the ability to covalently modify side chains of amino acids. However, recent studies from our group have shown that drugs like lidocaine or sulfamethoxazole (SMX),³ which are not chemically reactive per se, could be presented to specific T cells without the need of uptake, metabolism, and processing (6, 7, 8). Preincubation of APCs with these drugs did not result in any detectable responses. Therefore, we postulated that lidocaine and SMX were associating with MHC and/or peptide in a noncovalent way, forming a rather unstable MHC-peptide-drug complex.

For peptide-specific ß⁺ T cell clones (TCC), it is known that the TCR can recognize ligands that are slightly altered. The recognition of these altered peptide ligands (APL) can lead to dramatic functional consequences for the T cell (9). These altered responses include cytokine production in the absence of proliferation, differential cytokine production, anergy, and antagonism of the response to the wild-type Ag (10, 11, 12, 13). Therefore, we were interested if chemical modification of nonpeptide Ag like drugs can lead to "altered drug ligand"-like responses.

The sustained signaling necessary for T cell activation requires a continuous engagement of TCRs with MHC-peptide complexes on APCs (14). Over time, as many as 20,000 TCRs can be triggered by as few as 100 peptide-MHC complexes (15). Thus, the fast kinetics of a single TCR-ligand interaction seem to be a key feature allowing multiple TCR engagements by a single agonist. In addition, T cells appear to "count" the number of TCRs triggered and to respond by proliferation and cytokine production when a certain activation threshold is reached (16). Also, the serial triggering model of T cell activation offers a new kinetic explanation for the phenomenon of TCR antagonism and partial agonism. Ligands with lower than optimal stability may engage TCRs at higher rates compared with agonists, thus transducing partial signals at a very high rate (17, 18). This may result in the activation of different biological responses or in an efficient antagonism, possibly due to rapid spoiling of TCRs or rapid consumption of signaling components (19, 20).

It has been shown for murine trinitrophenyl-specific, class I-restricted CTL that the MHC-restricted contacts between the hapten and the corresponding TCR closely reflect those between TCR and nominal peptide Ags (21, 22, 23, 24). Previous reports from our laboratory demonstrated that noncovalently binding drugs can evoke a vigorous response in specific ß⁺ TCC (6, 7, 8, 25). Therefore, we reasoned that the unstable trimolecular MHC-peptide drug complex might interact with a given TCR similar to peptide Ags. To prove this hypothesis, we analyzed how subtle changes in drug molecules like SMX could alter their recognition by a corresponding TCR. For that purpose, we tested the reactivity of several SMX-specific TCC by using a panel of different sulfonamide derivatives. The obtained data indicate that the unstable trimolecular MHC-peptide-drug complex is interacting with a specific TCR in a similar way as nominal peptide Ags.

	Materials and Methods

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Culture media

Culture medium (CM) consisted of RPMI 1640 supplemented with 10% pooled heat-inactivated human albumin serum (Swiss Red Cross, Bern, Switzerland), 25 mM HEPES buffer, 2 mM L-glutamine (Seromed, Fakola, Basel, Switzerland), 25 µg/ml transferrin (Biotest, Dreieich, Germany), 10 mg/ml streptomycin, and 100 U/ml penicillin. The CM⁺, used to culture TCC, was enriched additionally with 50 U/ml rIL-2 (obtained from Dr. D. Wrann, Sandoz Research Institute, Vienna, Austria).

Drugs used for cell stimulation

All drugs (see Fig. 1) used in these experiments have been tested previously for the inhibitory response to mitogens (PHA) in nonallergic individuals. Stock solutions of each drug in RPMI 1640/0.05 M NaOH were always freshly prepared just before use. Except sulfisomidine and sulfamethazine (Aldrich Chemie, Buchs, Switzerland) and sulfadoxine (a gift from Dr. L. Heidecker, Hoffmann La Roche, Basel, Switzerland), all sulfonamide derivatives used in this study were purchased from Sigma (St. Louis, MO).

View larger version (15K): [in this window] [in a new window]   FIGURE 1. Chemical structures of the used sulfonamide derivatives. All sulfonamide derivatives used in this study are composed by a sulfanilamide core structure (R1) and a distinct heterocycle (R). The compounds are grouped according to the different heterocycles: 1) compounds with a five-ring-heterocycle, 2) pyridine derivative, 3) pyrimidine derivatives containing a guanidine partial structure, 4) pyrazine derivative, and 5) pyrimidine derivatives without a guanidine partial structure.

Freshly isolated PBMC from a SMX allergic donor (6) were stimulated with SMX (0.2–0.5 mg/ml) in CM at a cell density of 2 x 10⁶ per well in a 24-well plate (No. 3047; Falcon, Lincoln Park, NJ). CM⁺ was added after 7 days of culture. After 14 days, bulk cultures were restimulated with autologous irradiated (4000 rad) PBMC plus SMX (0.2 mg/ml) and tested for their specificity. Fourteen days later, specific T cell lines (TCL) were cloned by limiting dilution as described earlier (25). Growing TCC were expanded in CM⁺ and restimulated every 14 days with allogeneic irradiated (6000 rad) PBMC plus PHA (1 µg/ml) (Bacto; Difco, Detroit, MI).

Immunofluorescence and PCR-based TCR Vß analysis

Monoclonality of TCC used was proven either by staining with a panel of 21 different mAb recognizing different Vß gene products (Immunotech, Marseille, France) or by RT-PCR based TCR-oligotyping as described previously (3).

Proliferation assay

To determine the proliferation of the TCC to the different sulfonamide derivatives, 5 x 10⁴ TCC cells were incubated with 5 x 10³ irradiated autologous B lymphoblastoid cell line (B-LCL) in the presence of the indicated drug at different concentrations in 200 µl CM in a U-bottom microplate (Falcon No. 3077) for 48 h. Cultures were pulsed with [³H]thymidine (0.5 µCi) for the last 8 h, and cells were then harvested onto glass fiber disks and counted in a microplate beta-counter (Inotech Filter Counting System INB 384; Inotech, Dottikon, Switzerland).

Cytokine measurement

To detect cytokines produced after stimulation with the different compounds, supernatants of cells stimulated as described above were collected after 24 h and cytokines were quantified by a sandwich ELISA according to standard protocols (PharmMingen, San Diego, CA; 26 . Detection limits were 10 pg/ml.

Measurement of TCR down-regulation

TCR down-regulation induced by different compounds was determined as described previously (8, 15). Briefly, 2.5 x 10⁴ clone cells were mixed with 5 x 10⁴ autologous B-LCL cells in 200 µl CM in U-bottom microplates in the presence or absence of the drug at the indicated concentrations. The plates were centrifuged with 1200 rpm for 2 min to allow conjugate formation and then were incubated at 37°C. After 6 h, cells were resuspended, washed in PBS containing 0.5 mM EDTA to break the conjugates, and stained with anti-CD3 (UCHT-1; Dako, Zug, Switzerland) followed by a FITC-labeled goat anti-mouse Ig (Dako). The samples were analyzed on an EPICS profile II flow cytometer (Coulter Immunology, Hialeah, FL). The absolute numbers of CD3 molecules per cell was estimated by reference to a standard curve of beads coated with known amounts of mouse Ig according to the manufacturers instruction (Qifikit; Dako). Cytokine production was measured in the culture supernatant using an ELISA as described above.

	Results

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SMX-specific TCC and chemical structures of the used sulfonamide derivatives

In previous studies, we have shown that specific TCC can recognize per se nonreactive drugs like lidocaine or SMX presented in a labile association with MHC-peptide complexes (6, 7, 8). In most cases, the recognition of this trimolecular MHC-peptide-drug complex is HLA-allele restricted (6, 8), although exceptions have been described (7).

To extend our knowledge of the interaction of drugs with specific T cells, we analyzed the influence of slight alterations of the parent drug SMX on its recognition by a given TCR. For this purpose, we generated a panel of 11 SMX-specific CD4⁺ TCC and 2 SMX-specific CD8⁺ TCC from an allergic donor suffering from a hypersensitivity reaction to SMX. All clones recognized SMX in the context of the HLA-DRB1*1001 molecule or HLA-B44, respectively (Ref. 6, and data not shown). The clones were secreting a Th0-like cytokine pattern with high amounts of IL-5 (see Fig. 2, and data not shown). All TCC used were monoclonal; the Vß gene usage of the clones further used in this study is indicated in Table I.

View larger version (34K): [in this window] [in a new window]   FIGURE 2. Dose-response curves of the sulfonamide derivatives stimulating the broadly reactive TCC 8.15. The broadly reactive TCC 8.15 was incubated with various amounts of different stimulating sulfonamide derivatives in the presence of irradiated autologous B-LCL. A, Proliferative response measured after 48 h by [3H]thymidine incorporation. B–D, Cytokine production [IL-4, B; IL-5, C; and IFN-, D] measured after a culture period of 24 h by standard ELISA. Detection limit was 10 pg/ml. A representative of three independent experiments is shown.

All compounds described above stimulated a SMX-specific polyclonal TCL from the same donor by using autologous B-LCLs as APCs (data not shown).

Cross-reactivity pattern of SMX-specific TCC

To analyze the cross-reactivity pattern of the 13 clones, we used the above described panel of 13 different sulfonamide derivatives. TCC were stimulated in the continuous presence of 800 µM (corresponds to 200 µg/ml) of the respective sulfonamide derivative by using autologous B-LCL cells as APC. The different cross-reactivity pattern observed are summarized in Table I. The five TCC in group I, including the two CD8⁺ TCC, were highly specific and recognized only SMX. They did not react to any other compound even when concentrations up to 8 mM were used (data not shown). The three TCC in group II proliferated in the presence of the two most similar compounds showing only moderate differences in terms of electronic volume and density: SMX and sulfamethizole (SMT). All 5 remaining clones recognized at least three different compounds: SMX, SMT, and sulfathiazole (STH), although STH triggered all reactive TCC with much lower efficiency than the two other compounds. Group IV includes two clones, 9.3 and 8.21, which could be stimulated by an additional compound: sulfamethoxipyridazine (SMP). TCC 9.5 (group V) was the only clone tested that reacted with high efficiency to the pyridine-derivative sulfapyridine (SPD). Finally, group VI contains the broadly reactive TCC 8.15, which could be stimulated by the continuous presence of six different compounds displaying a quite high heterogeneity in their molecular structure. Others drugs, such as penicillin G or lidocaine, did not stimulate the SMX-specific clones.

Five of the 13 different sulfonamide derivatives, namely sulfamoxole (SMO) and the four pyrimidine derivatives containing a guanidine partial structure [sulfadiazine (SDZ), sulfamerazine (SMR), sulfamethazine (SMZ), and sulfadimethoxidiazine (SMD)], were not recognized by any of the 13 clones tested.

As it has been shown that APLs can induce cytokine production in the absence of a measurable proliferation (9), we analyzed the cytokine production of all TCC in response to different sulfonamide derivatives. None of the nonstimulating compounds induced a detectable amount of IL-4, IL-5, or IFN- in any of the clones tested (data not shown). Furthermore, none of the stimulating compounds induced a cytokine pattern in the reactive TCC differing from the one obtained by stimulation with the parent drug SMX (data not shown).

Compounds stimulating TCC 8.15 have stronger or weaker agonistic properties

Although we could not detect "altered drug ligands," it was already clear from the cross-reactivity experiments shown in Table I that the six compounds stimulating the broadly reactive TCC 8.15 were differing in their capacity to induce proliferation. To further analyze the distinct abilities of these compounds to activate TCC 8.15, we incubated the clone cells with titrated amounts of the stimulating compounds in the presence of autologous B-LCL as APCs. By comparing the efficiency of the induction of a significant proliferative response by the different sulfonamide derivatives, two groups of compounds could be distinguished (Fig. 2A). Sulfisomidine (SID) had the same full agonistic properties as the parent drug SMX, whereas all other compounds needed a 10- to 100-fold higher concentration to achieve half-maximal proliferation and could therefore be classified as weak agonists. An identical pattern could be observed when cytokine production after 24 h was analyzed (Fig. 2, B–D). To our knowledge, these are the first data demonstrating that unstable-presented nonpeptide Ags can have different agonistic properties.

An early phenotypic event that occurs subsequent to TCR engagement by an antigenic ligand is the down-regulation of TCR expression on the surface of T cells. This process starts within minutes following the initial interaction and reaches a peak after a few hours. In our system, the plateau of TCR down-regulation was reached after 5–6 h. To quantify the distinct interactions of strong and weak agonists with the 8.15 TCR, we determined the extent of TCR down-regulation induced by the different compounds after 6 h. The sulfonamide derivatives with weak agonistic properties again needed up to 100 fold higher concentrations to remove a similar percentage of TCRs from the cell surface (Fig. 3A). Nonstimulating compounds did not lead to any detectable TCR down-regulation (data not shown). In consequence, the question arose whether the lower response of the TCC after stimulation with the weak agonistic sulfonamide derivatives reflected a lower extent of TCR triggering or rather an altered signal leading to qualitatively different responses. Therefore, we correlated TCR down-regulation and cytokine production after stimulation with the strong or the weak agonists. As shown in Fig. 3, B–D, in spite of a decreased efficiency of TCR triggering, the threshold of T cell activation and the type of cytokines produced were not affected by the agonistic capacities of the different compounds.

View larger version (35K): [in this window] [in a new window]   FIGURE 3. Dose-dependent TCR down-regulation in TCC 8.15 induced by different compounds. Clone 8.15 was incubated with various concentrations of the stimulating compounds in the presence of autologous B-LCL. TCR down-regulation was determined after 6 h (A) and is indicated as the percentage of CD3 mean fluorescence calculated from values without drugs. B–D, Levels of IL-4 (B), IL-5 (C), and IFN- (D) produced after 6 h as a function of the number of TCRs down-regulated. No cytokine production was observed in cultures without drug stimulation.

From the data obtained with clone 8.15, the question arose whether the distinct agonistic properties of the compounds to stimulate this clone were an intrinsic feature of the respective compound or if they reflected differences in the ability of the TCR 8.15 to interact with distinct MHC-peptide-drug complexes. The comparison of the dose-response curves of sulfonamide derivatives stimulating TCC 9.3 and 9.5 gave further insights in the interaction of different clones with compounds with distinct triggering capacities. Whereas for TCC 9.5 <20 µM SMT (see Fig. 4, A and C) were sufficient to induce a significant TCR down-regulation and were additionally able to trigger a proliferative response, TCC 9.3 needed up to 2000 µM SMT for comparable responses (Fig. 4, B and D). In contrast, both clones required similar amounts of SMX (10 µM) for the induction of significant responses. The obtained data demonstrate that different TCRs can recognize the same drug with different efficiencies.

View larger version (34K): [in this window] [in a new window]   FIGURE 4. Different SMT dose-curves observed in TCC 9.3 and 9.5. T cell clones 9.5 (A and C) and 9.3 (B and D) were incubated with different concentrations of SMX, sulfamethizole (SMT), and sulfathiazole (STH) by using autologous B-LCL cells as APCs. A and B, TCR down-regulation determined after 6 h. C and D, Proliferative response measured after 48 h by [3H]thymidine incorporation. The experiments were repeated at least three times.

The concept of serial TCR engagement suggests that the rate of TCR-ligand complex dissociation has a main influence on the resulting T cell activation, because the magnitude of the response depends on the frequency of TCR engagement (18, 19). The differences in the agonistic properties of the compounds stimulating clone 8.15 and the different abilities of clones 9.3 and 9.5 to react to SMT could easily be explained by distinct strengths of the association of the TCR with the respective MHC-peptide-drug complex. Therefore, we had to prove that unstable trimolecular MHC-peptide-drug complexes activate specific T cells by serial TCR engagement. To do that, we analyzed the relationship between the number of TCR ligands expressed as the drug concentration in the cultures and the number of TCRs triggered after 6 h. As depicted in Fig. 5A for both clones, a logarithmic function of the number of TCRs down-regulated compared with the ligand concentration offered could be revealed, a correlation that is only compatible with the so-called "serial triggering" model, where low numbers of ligands can successfully trigger high numbers of TCRs. In the case of the two clones shown, the differences in their efficiency to react to SMX can be explained by different levels of TCR expression on both clones (TCC 8.21, 90,000 TCRs/cell; TCC 9.3, 140,000 TCRs/cell). By measuring proliferation and cytokine production, a similar pattern could be observed (data not shown). However, despite the different prerequisites for ligand concentration both TCC had comparable activation thresholds of approximately 15,000 TCRs (Fig. 5B).

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Number of TCRs triggered is a logarithmic function of the ligand concentration. A, Clones 8.21 and 9.3 were incubated with different concentrations of SMX in the presence of autologous B-LCL cells, and the number of triggered TCRs was determined after 6 h. B, Levels of IFN- produced after 6 h as a function of the number of TCRs down-regulated.

	Discussion

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The 13 SMX-specific TCC used in this study showed a quite high diversity in their ability to respond to different sulfonamide derivatives. On one hand, the five clones comprised in group I (see Table I) were highly specific and could be stimulated by SMX only. Quite surprisingly, even a chemically very similar compound like SMT was not recognized by these five TCC. On the other hand, TCC 8.15 showed a broad cross-reactivity as it was responding to six different compounds sharing only a minor structural similarity.

A detailed analysis of the different cross-reactivity pattern by using images of the electronic structure, obtained by molecular modeling of the different compounds, did not lead to a clear relationship between chemical structure and the ability to activate a given TCC. However, some cross-reactivity pattern could be explained. In the case of the clones 8.21 and 9.3, the recognition of SMX, SMT, and SMP might be elucidated by the fact that all three compounds bear in the -position of the first electron-negative N atom of the heterocycle a second nucleophilic N or O atom, both of them able to act as electron donors for H bond interactions. As the interaction of the TCR with its ligands can involve H bond interactions with side chains of peptide residues (29, 30), it is feasible that also in the case of MHC-peptide-drug complexes the capacity to undergo H bond interactions can influence the recognition by a given TCR.

Five sulfonamide derivatives (SMO, SDZ, SMR, SMZ, and SMD) did not stimulate any of the clones tested. However, different SMX-specific polyclonal TCL from the same donor could be stimulated in the presence of autologous B-LCL cells with all compounds used (data not shown). Hence, we could exclude the possibility that these compounds, due to their chemical properties, could either not associate with MHC-peptide complexes or could not be recognized by SMX-specific T cells.

The high heterogeneity of the observed cross-reactivity pattern in addition to the different Vß gene usage of the tested clones indicates that although oligoclonal outgrowth of drug-specific T cells has been described (25, 31, 32), in this patient the T cell-mediated response to SMX was polyclonal and heterogeneous. This implies that each of the TCC generated to SMX recognizes the drug in a different context. Therefore, it seems likely that the per se nonreactive drug SMX has the possibility to interact with MHC-peptide complexes in several ways, generating distinct antigenic determinants. This would suggest that already during the induction phase of the drug allergy several quite distinct immunogenic structures did activate the SMX-reactive T cells (33). Therefore, the recognition of the noncovalently bound drug SMX by a given TCR is not a randomly occurring event as the observed cross-reactivity pattern as well as the different strength of the TCR engagement were highly reproducible.

None of the tested clones reacted to the used sulfonamide derivatives with an "altered drug ligand"-like response. However, the analysis of the dose-response relationships of broader reactive clones revealed that compounds with weaker or stronger agonistic properties could be distinguished. This phenomenon is widely known for single amino acid-substituted peptide Ags (34), but has not yet been described for nonpeptide Ags, especially for noncovalently binding drugs.

One of the factors influencing the efficiency of the TCR-ligand interaction is the stability of the antigenic MHC-peptide complex, governed by the affinity of the peptide to the restricting MHC molecule (35). The unstable association of the drugs with the MHC-peptide complex did not allow the determination of the affinity of the different compounds to the MHC-complex. However, the comparison of the dose relationships of SMX recognition by the two clones 9.3 and 9.5 gives further insights in the possible mechanism of interaction of a given TCR with distinct but chemically minutely different compounds. Both clones were expressing similar numbers of TCRs on their surface (see below) and could be activated by SMX with the same efficiency. In contrast, the SMT dose needed to induce a significant TCR down-regulation or proliferation differed in order of two magnitudes between the two clones. These data can only be explained by different affinities of the two TCRs to the MHC-peptide-SMT complexes. Hence, these findings indicate that not distinct affinities of the different sulfonamide derivatives to the MHC-peptide-complexes influence the efficiency of the TCR-ligand interaction. Moreover, they also reveal that weaker or stronger agonistic properties are not a characteristic of a given sulfonamide derivative but rather an intrinsic feature of the reacting TCR. In addition, these data clearly show that the distinct efficiencies of the six different compounds stimulating TCC 8.15 might not be explained by different affinities of the sulfonamide derivatives to MHC-peptide complexes and/or different ligand densities formed on the APC, but by distinct interactions of the 8.15 TCR with the different MHC-peptide-drug complexes.

It is known that T cell activation requires the assembly of multiple layers of proteins to the phosphorylated TCR (36). A certain amount of time is required to assemble the correct signaling complex, and, therefore, the formation of the complete complex will require stimuli that exceed a certain threshold of strength and duration (37, 38). This model would imply that the 8.15 TCR interacts with weak agonistic sulfonamide derivatives with suboptimal kinetics, so that only a certain percentage of interactions are stable enough to deliver a full signal. However, correlation of TCR down-regulation and cytokine production, as shown in Fig. 3, B–D, clearly demonstrates that despite of a decreased efficiency of TCR triggering, the threshold of T cell activation and the type of cytokines produced were not affected by the agonistic capacities of the different compounds. Therefore, the lower response of the TCC after stimulation with the weak agonistic sulfonamide derivatives reflects rather a lower extent of TCR triggering than an altered signal leading to a qualitatively different response.

Our results implicate a T cell activation by serial TCR engagement. In all clones used in this study, we could find a logarithmic correlation of the number of down-regulated TCRs compared with the offered ligand concentration. This relationship indicates that TCR triggering is most efficient when only few antigenic complexes were available on the APC surface and becomes less and less efficient with increasing numbers of ligands. According to the work of the group of Lanzavecchia (15, 19), this experimental finding is difficult to reconcile with a model of TCR cross-linking where the number of triggered TCRs would exponentially increase with an increase in the number of ligands offered. The data obtained clearly show that unstable MHC-peptide-drug complexes can activate specific TCC by serial TCR engagement.

These data give additional information for the understanding of the interaction of the trimolecular MHC-peptide-drug complex with a reactive TCR. Whereas the MHC-peptide complexes are stable and can be presented for a long time on the surface of the APC (39, 40), the association of the TCR with its ligand has a half-life of seconds (41). Therefore, even a very low number of such complexes may be sufficient to engage, in several rounds of ligation, a relatively high number of TCRs, thus triggering T cell responses (14, 15). In the case of sulfonamide derivatives, the binding of the drug to MHC-peptide complexes is much more unstable, as washing of drug-prepulsed APCs abrogates their ability to stimulate reactive T cells (6, 7, 8). However, the kinetics of the T cell triggering resemble very closely the serial triggering model, indicating that the half-life time of a MHC-peptide-drug complex must be considerably higher than the time needed to trigger a single TCR.

In summary, the reported data indicate that, despite the special way of Ag presentation, noncovalently binding drugs can interact with a specific TCR similar as nominal peptide Ags.

	Acknowledgments

 
We thank blood donor B.K. for his friendly collaboration, E. Frei for TCR oligotyping, D. Rognon for the molecular modeling of the electronic structure of the different sulfonamide derivatives, and T. Wendland for helpful discussion.

	Footnotes

 
¹ This work was supported by Grant NR.31-50482.97 of the Swiss National Research Foundation (to W.J.P.) and Grant NR97.0431 of the Federal Office for Education and Science (EU-Research Programme BIOMED).

² Address correspondence and reprint requests to Dr. Werner J. Pichler, Institute of Immunology and Allergology, Inselspital, Bern, Switzerland. E-mail address:

³ Abbreviations used in this paper: APL, altered peptide ligand; B-LCL, B lymphoblastoid cell line; CM, culture medium; SMX, Sulfamethoxazole; TCC, T cell clone; TCL, T cell line.

Received for publication May 29, 1998. Accepted for publication September 16, 1998.

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