Degeneracy and additional alloreactivity of drug-specific human {alpha}ß+ T cell clones

Salome von Greyerz, Gregor Bültemann, Karin Schnyder, Christoph Burkhart, Barbara Lotti, Yvonne Hari and Werner J. Pichler Allergology, Clinic for Rheumatology and Clinical Immunology/Allergology, Inselspital, 3010 Bern, Switzerland

Correspondence to: W. J. Pichler


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
It has been well established that T cells can recognize small mol. wt compounds such as drugs. Results from previous studies revealing a high heterogeneity and cross-reactivity of drug-specific T cell clones (TCC) in individual patients prompted us to analyze the degeneracy of drug-reactive TCR in detail. Hence, we analyzed the MHC restriction pattern of a panel of 100 drug-specific TCC isolated from different drug-allergic donors. We found that 28 of the tested clones showed an MHC allele-unrestricted drug recognition. Most of these clones were at the same time highly drug specific, i.e. they could only be stimulated by the original drug and not by any drug derivatives. In contrast, TCC with the ability to interact with different drug derivatives displayed a clearly MHC allele-restricted drug recognition. Therefore, we concluded that the TCR of these clones is mainly interacting with side chains of the appropriate drug molecules and hence able to tolerate alterations in the MHC molecule. Moreover, we tested all clones for additional alloreactivity and found that 27 clones could be stimulated by a self-MHC–peptide–drug complex as well as by a non-self-MHC–peptide complex. This cross-reactivity with allogeneic MHC molecules was substantially higher in drug-specific TCC compared to tetanus toxoid-specific clones from the same donors. This suggests that from the point of view of drug-specific TCR, non-self-MHC–peptide complexes have a higher incidence to mimic the `original' self-MHC–peptide–drug complex and this may occur for TCR recognizing self-MHC–pathogen-derived peptide complexes. Finally, the biological functions of bispecific TCC were not influenced by the nature of the stimulating ligand. Both drug as well as allogeneic stimulation led to similar reaction patterns in the analyzed TCC.

Keywords: allergy, allorecognition, drug, non-peptide antigens, T cells


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In addition to the recognition of pathogen-derived peptides embedded in the groove of self-MHC molecules, T cells can also recognize non-peptide antigens (1). In addition to lipids, glycolipids (2) and carbohydrates (3), T cells can also be triggered by small mol. wt compounds such as drugs (4) or contact sensitizers like TNP or metal salts (5).

Drugs like ß-lactam antibiotics, sulfonamides or local anesthetics are often responsible for allergic reactions in humans. The T cell involvement in such drug allergic reactions is well established (for review, see 6). However, several molecular aspects of the recognition of drugs by the TCR of drug-reactive T cell clones (TCC) remain still unclear. In principle two groups of drugs can be distinguished: (i) chemically reactive compounds with the ability to covalently modify side chains of amino acids such as ß-lactam antibiotics or reactive drug metabolites and (ii) drugs like lidocaine or sulfamethoxazole (SMX) which are not per se chemically reactive. Covalent modification of serum or cellular proteins can lead to presentation of modified self structures (haptenated peptides) by MHC class I or class II molecules to reactive T cells (79). However, recent work has elucidated that covalent binding is not a necessary prerequisite for drug presentation: preincubation and subsequent washing of antigen-presenting cells (APC) with chemically inert drugs like lidocaine or SMX did not lead to any detectable T cell responses (10,11). We postulated, therefore, that these compounds can associate with MHC and/or embedded peptides in a non-covalent way, forming rather unstable trimolecular MHC–peptide–drug complexes.

Although self-MHC-restricted T cell recognition has sufficient specificity to discriminate between an enormous variety of antigenic peptides, recent studies on TCR recognition have begun to emphasize the importance of a significant level of cross-reactivity on different peptides, which is necessary to produce an adequate TCR repertoire capable of responding to the universe of pathogens (reviewed in 12). Several studies of the cross-reactivity and fine specificity pattern of drug-specific TCC revealed highly heterogeneous T cell responses in the analyzed donors (1316).

It is generally agreed that alloreactivity is the result of cross-reactive recognition by self-MHC-restricted T cells and is not a function of a particular population of T cells that has escaped the constraints of the thymic selection (17). A recent study form Daniel et al. proposes that the majority of the alloreactivity represents a variation of self-restricted responses in which there is a shift in the relative contribution of the MHC and peptide residues toward the overall strength of binding between the TCR and the MHC–peptide ligand. More energy is provided by the TCR–MHC interactions and less from the TCR–peptide interactions; however, the latter are still necessary to reach the threshold required for T cell activation (18).

The aim of this study was to study the degeneracy of the TCR of drug-specific TCC isolated from patients with hypersensitivity reactions to different drugs. We analyzed the MHC restriction pattern of the different clones as well as a possible cross-recognition of allogeneic MHC alleles. In addition we compared the activation mechanisms in bispecific clones.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Culture media (CM)
CM consisted of RPMI 1640 supplemented with 10% pooled heat-inactivated human antibody 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), 100 µg/ml streptomycin and 100 U/ml penicillin. The culture medium CM+, used to culture TCC, was additionally enriched with 50 U/ml recombinant IL-2 (obtained from Dr D. Wrann, Novartis Research Institute, Vienna, Austria).

Drugs used for stimulation
Amoxicillin, ceftriaxone, penicillin G (Pen G) and sulfonamide derivatives were purchased from Sigma (St Louis, MO), and lidocaine from Grogg Chemie (Stetteln, Switzerland). All drugs used have been tested previously for their inhibitory activity on the proliferative response to mitogens in non-allergic individuals. Stock solutions of each drug were always freshly prepared just before use. Sulfonamide derivatives were dissolved in RPMI/0.05 M NaOH, all other drugs in RPMI. Tetanus toxoid (TT) was kindly provided by Dr J. Cryz (Serum und Impfinstitut, Bern, Switzerland).

Generation of drug- or TT-specific TCC
TCC from peripheral blood mononuclear cells (PBMC).
Freshly isolated PBMC from donors allergic to lidocaine (19), ß-lactam antibiotics (13) and SMX (10) were stimulated with the corresponding drug in CM at a cell density of 2x106 cells/well in 24-well plates. The following drug concentrations were used: amoxicillin (500 µg/ml), Pen G (500 µg/ml), lidocaine (100 µg/ml) and SMX (200 µg/ml). CM+ was added after 7 days of culture. After 14 days, bulk cultures were re-stimulated with autologous irradiated (4000 rad) PBMC and the respective antigen. Specific T cell lines (TCL) were cloned by limiting dilution as described previously (20). Growing TCC were expanded in CM+ and re-stimulated every 14 days with allogeneic irradiated (6000 rad) PBMC plus phytohemagglutinin (PHA, 1 µg/ml, Bacto; Difco, Detroit, MI). From three donors (KB, SE and SF; see Table 5Go), freshly isolated PBMC were additionally simulated with TT (5 µg/ml) and TT-specific TCC were generated after cloning by limiting dilution as described above.


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Table 5. Comparison of drug- and TT-specific TCC
 
TCC from skin biopsies.
Skin punch biopsies were taken from dermal reaction centers from a patient allergic to amoxicillin and a patient allergic to ceftriaxone (21). T cells were isolated as described (21). Briefly dermal T cells were cultured with 25,000 irradiated allogeneic PBMC plus PHA (2 µg/ml) in 100 µl CM in 96-well round-bottomed plates. Growing T cell lines were expanded in CM+ and subsequently cloned by limiting dilution as described (20).

Immunofluorescence and PCR-based TCR Vß analysis
Monoclonality of the TCC used was proven either by staining with a panel of 22 different mAb recognizing different Vß gene products (Immunotech, Marseilles, France) or by RT-PCR-based TCR oligotyping as described previously (8). Additionally, cross-reactive clones were tested for TCR V{alpha} expression by RT-PCR-based TCR oligotyping as described (8).

Proliferation assay
In order to reveal the MHC restriction pattern as well as to identify the alloreactive potential of the drug- or TT-specific TCC we used a panel of allogeneic HLA-typed PBMC from healthy donors (see Tables 2 and 3GoGo). HLA class II phenotyping was performed by PCR amplification using sequence-specific primers (Protrans Endotell, Allschwil, Switzerland). To determine the proliferation of the TCC to the different allogeneic PBMC, 5x104 TCC were incubated either with 5x104 of the respective irradiated PBMC or with 5x103 autologous B-LCL in the presence or absence of the indicated drug or TT (5 µg/ml) in 200 µl CM in round-bottomed microplates (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 ß-counter (Inotech filter counting system INB 384; Dottikon, Switzerland).


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Table 2. Origin of drug-specific TCC
 

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Table 3. MHC restriction pattern of selected drug-specific TCCa
 
Proliferation assay with TCC of multiple specificity
TCC cells (5x104) were incubated in round-bottomed microplates (Falcon no. 3077) with different concentrations of drugs or allogeneic B-LCL. Irradiated (6000 rad) HLA-matched B-LCL (2x104) were added to drug-stimulated cultures. Cultures were pulsed with [3H]thymidine (0.5 µCi) for the last 8 h to determine proliferation as described above.

FACS analysis of CD25, CD69 expression
For immunofluorescence analysis of CD25, CD69 expression, 5x104 TCC cells were incubated with different concentrations of the antigens; again, 2x104 irradiated (6000 rad) HLA-matched B-LCL were added to drug-stimulated cultures. After 12 h, the supernatant was harvested for cytokine measurement and cells were washed in PBS containing 0.5 mM EDTA to break the conjugates. Cells were then stained either with anti-CD25–FITC (Dako Research, Besancon, France) or anti-CD69–FITC (PharMingen, San Diego, CA). Analysis was performed on a Coulter Epics XL-MCL flow cytometer (Beckman Coulter, Nyon, Switzerland).

Cytokine measurement
To detect cytokines produced after stimulation with the different antigens, supernatants of cells stimulated as described above were collected after 12 or 48 h and cytokines were quantified by a sandwich ELISA according to standard protocols (PharMingen) (22). Detection limits were 10 pg/ml.

Measurement of TCR down-regulation
For immunofluorescence analysis of CD25, CD69 expression, TCR down-regulation induced by different compounds was determined as described previously (10,16). TCC cells (5x104) were incubated with different concentrations of the antigens in 200 µl CM in U-bottom microtiter plates; again, 2x104 irradiated (6000 rad) HLA-matched B-LCL were added to drug-stimulated cultures. The plates were centrifuged at 1200 r.p.m. for 2 min to allow conjugate formation and incubated at 37°C. After 12 h, cells were resuspended, washed in PBS containing 0.5 mM EDTA to break the conjugates and stained with anti-CD3 (UCHT-1; Dako Diagnostics, Zug, Switzerland) followed by an FITC-labeled goat anti-mouse Ig (Dako Diagnostics). Analysis was performed on a Coulter Epics XL-MCL flow cytometer (Beckman Coulter). The absolute number of CD3 molecules per cell was estimated by reference to a standard curve of beads coated with known amounts of mouse Ig (Qifikit; Dako Diagnostics). Cytokine production was measured in the culture supernatant using an ELISA as described above.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Heterogeneous MHC restriction pattern of drug-specific TCC
In previous studies we showed that drug-specific TCC have a high heterogeneity in their ability to respond to different drugs with the same core structure (13,15,16). Table 1Go gives a summary of the observed cross-reactivity pattern of SMX-specific TCC to a panel of different sulfonamide derivatives. On one hand, nine TCC were isolated, which were highly specific as they could be stimulated by the parent drug SMX only and did non-tolerate even slight modifications of the drug molecule. On the other hand, several clones, such as H9, showed a broad cross-reactivity pattern in as much as they could recognize up to nine different compounds sharing only minor structural similarities (16). Interestingly, for several cross-reactive clones, such as 8.15, the different compounds stimulating these clones differed in their capacity to induce proliferation (data not shown and 16). Few compounds had the same full agonistic properties as the parent drug SMX, whereas other compounds needed a 10- to 100-fold higher concentration to achieve half-maximal proliferation and could therefore be classified as weak agonists.


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Table 1. Cross-reactivity-pattern of 21 different SMX-specific TCCa
 
We concluded that the drug-specific T cell responses in most of the analyzed patients were polyclonal and heterogeneous. In addition, another preceding analysis revealed that certain drug-specific TCC displayed a MHC allele unrestricted recognition pattern, implicating that they recognize non-polymorphic residues of the MHC molecules in addition to certain side chains of the drug molecule (23). The aim of this study was to characterize the degeneracy of drug-specific TCC in detail. In order to do that we analyzed a total number of 100 drug-specific TCC isolated either from PBMC or from skin biopsies of seven different drug-allergic donors (see Table 2Go). To compare drug-specific TCC with clones specific for pathogen-derived peptides we tested in addition a panel of 58 TT-specific TCC obtained from three different TT-immunized and drug-allergic patients (donors KB, SE and SF). In a first set of experiments, we analyzed the MHC restriction pattern of both groups of clones. For that purpose we used a panel of 10 different allogeneic PBMC expressing a wide variety of different HLA-DRB1 molecules. Most of the drug-specific clones (72 out of 100 tested clones) recognized the respective antigen only in the presence of HLA-matched allogeneic APC as shown for clone DB3 in Table 3Go. For this clone the ceftriaxone recognition is clearly HLA-DRB1*1101-1104 restricted. However, 28 of the tested clones showed a drug-specific proliferative response irrespective of the HLA phenotype of the APC used. As shown in Table 3Go, clone ABH47 could be stimulated in the presence of amoxicillin by any of the used allogeneic feeder cells. This phenomenon could also be observed in TCC specific for non-covalently binding drugs such as lidocaine and SMX (see Table 5Go and data not shown). The drug-specific response of all these HLA allele-non-restricted clones could easily be blocked by addition of anti-DR mAb (data not shown). Thereby we could exclude that in these clones the drug molecules were presented by HLA-DP or HLA-DQ, or by non-classical antigen-presenting molecules such as CD1. None of the 58 tested TT-specific TCC showed an MHC allele-unrestricted antigen recognition.

Furthermore, a third group of clones could be detected where the need of professional APC was not required: clone H4 had the ability to recognize the drug SMX presented on MHC molecules expressed on the surface of the T cells themselves (see Table 3Go). Twelve other clones from three different donors showed the same `T cell responder' features (data not shown and 20).

Alloreactivity of drug-specific TCC
In addition to the above described clones, we could detect in total 27 clones which reacted in the absence of stimulatory drug molecules in an alloreactive manner to a certain non-self-MHC allele. By RT-PCR oligotyping of the TCR V{alpha} chains we could exclude that the additional alloreactivity of the drug-specific clones was due to expression of two different V{alpha} chains. Table 4Go shows the representative reactivity pattern of three alloreactive drug-specific TCC from three different donors. Clone OFB12 showed lidocaine recognition in the context of HLA-DRB1*1501-03 (donors WP and autologous B-LCL) and an additional alloreactivity to the HLA-DRB1*0801 allele (donors SK and CB). A similar pattern could be observed for clone H13 which recognized the stimulating drug SMX in the context of HLA-DRB1*1001 (donor KV and autologous B-LCL) and revealed alloreactivity to the HLA-DRB1*1303-04 allele (donors MA, YH and SK). The same cross-reactivity could also be observed in TCC reactive to covalently binding drugs such as Pen G and other ß-lactam antibiotics: clone KP26 could be stimulated by Pen G presented in the context of HLA-DRB1*1501-03 (donor WP and autologous B-LCL) and showed an additional allospecific response to the HLA-DRB1*1101-04 allele (donors SK and BL). In all allospecific TCC the alloreactive MHC allele could be clearly evaluated (data not shown). Additionally, in only one female donor (with two children) five TT-specific TCC with an additional alloreactivity could be detected. Therefore, we concluded that the ability to cross-recognize non-self-MHC–peptide complexes must be substantially higher for drug-specific TCC than for T cells initially primed by pathogen-derived peptides (unpaired Student's t-test: P = 0.05).


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Table 4. Representative drug-specific TCC with additional alloreactivitya
 
Comparison of drug and allo-stimulation
It has been shown for peptide antigens, that the recognition of slightly altered ligands by {alpha}ß+ TCR can have dramatic functional consequences for the T cells (24). The responses to such altered peptide ligands (APL) include cytokine production in the absence of proliferation, differential cytokine production, anergy and antagonism of the response to the wild-type antigen (2528). We therefore asked if the different antigenic structures (self-MHC–peptide–drug complexes versus non-self-MHC–peptide complexes) may elicit a similar reaction pattern in bispecific TCC. Two TCC, OFB12 and H13 (see Table 4Go), were analyzed in detail. As previously shown, the kinetics of T cell triggering after drug stimulation resembled the ones observed after activation by allogeneic B-LCL (11). In addition, the comparison of the dose–response curves in different readout systems, i.e. up-regulation of activation markers, proliferation and cytokine production, after drug and allogeneic stimulation did not show any significant differences (Fig. 1Go). As the ligand density of APC presenting unstably associated drug molecules and APC bearing non-self-MHC–peptide complexes cannot be measured up directly, we compared the extent of different biological responses, measurable after TCR triggering by different ligands. Both stimuli elicited production of a similar cytokine pattern and showed similar extents of TCR down-regulation required for the induction of half-maximal effector functions (see Fig. 2Go). Therefore, we concluded that in the case of the analyzed TCC the activation by different antigens such as self-MHC–peptide–drug complexes and non-self-MHC–peptide complexes did not influence the biochemical events following TCR–ligand interaction.



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Fig. 1. Dose-dependent reactions of a drug-specific TCC with additional alloreactivity. A representative comparison of the dose-dependent CD25 and CD69 expression (A and D), the IL-4 and IL-5 production (B and E) as well as the proliferative response (C and F) of TCC OFB12 after stimulation with SMX (A–C) or allogeneic APC (D–F) is shown. For experimental details, see Methods. Similar results were obtained with other drug-specific TCC with additional alloreactivity.

 


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Fig. 2. Comparison of drug and allo-stimulations. A comparison of TCR down-regulation and effector functions (obtained in the same experiment) for TCC OFB12 (A) and TCC H13 (B) was performed. The level of TCR expression required to induce 50% activation of the different effector functions is shown for the two different ligands. The CD3 MFI of unstimulated T cells was 90.0 for OFB12 and 134.7 for H13.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we analyzed the influence of different HLA-DR alleles on the interaction of a drug-specific TCR with MHC–peptide–drug complexes. We found that 28 out of 100 tested drug-specific clones from different donors showed a proliferative response after drug stimulation irrespective of the presenting HLA-DR allele. Interestingly, this HLA allele-unrestricted drug presentation was found for clones that react to covalently binding drugs such as Pen G or amoxicillin as well as for clones specific for the non-covalently binding drugs lidocaine and SMX. As the responses could easily be blocked by addition of anti-DR mAb, we could exclude that these clones recognize the drug molecules in the context of other presenting molecules such as CD1. Similar observation have been made recently for nickel-specific T cells (29), as well as for peptide-specific clones (30). In addition, murine T cell hybridomas specific for the contact sensitizer TNP showed a similar freedom in their ability to tolerate different presenting MHC alleles (31).

It has been shown by crystallographic analysis of TCR–ligand interactions that the MHC residues that are engaged by the TCR are mostly conserved amino acids, which do not vary between different DR alleles (32,33). The TCR–MHC–peptide structures determined to date (34,35) indicate that the required degree of peptide cross-reactivity can be produced in at least two ways: (i) the TCR `focus' on only few amino acid side chains of the peptide and can accommodate peptides with other side chains, depending on the size and surface chemistry of the TCR contact surfaces, and (ii) the structural flexibility of the CDR3 loops that contact the peptide allow a degree of accommodation of binding to multiple peptide ligands (33). These findings, together with the data summarized in Table 5Go, prompted us to establish a model of the molecular basis of the recognition of drug molecules by the TCR of specific T cells (see Fig. 3Go): allele-restricted and allele-unrestricted clones vary in the degree by which MHC residues and peptide–drug side chains contribute to the overall strength of the TCR–ligand interactions. This would imply that in the case of allele-unrestricted clones the main part of the energy required for a productive TCR engagement is provided by the TCR–peptide–drug interaction. Therefore, it is feasible that alteration in the involved MHC residues may be allowed. In addition, such clones are highly sensitive to alteration of the presented drug molecule, as they recognize only the parent drug and do not tolerate even slight modifications of the drug molecules. In contrast, the TCR of allele-restricted TCC interacts predominantly with amino acid residues of the MHC molecule and thus does not allow larger modifications of the presenting MHC molecule (18,36). In consequence, they allow broader alterations of the embedded drug molecule. However, for the activation of these clones the presence of drug molecules in the stimulating MHC–peptide–drug complex is still necessary as they do not proliferate in the absence of the antigenic drug.



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Fig. 3. Models of degenerate TCR–ligand interactions. The scheme delineates a possible explanation for the degeneracy of drug recognition by TCC (see also Table 1Go). The depicted MHC–peptide–drug complex shows a schematic view of the unstable, non-covalent association of a non-reactive drug (SMX) with a MHC–peptide complex. Notice that the thickness of the arrows corresponds to the energy provided for the overall strength of the TCR–ligand interaction. (A) In the case of highly drug-specific clones the TCR is mainly triggered by interactions with side chains of the drug molecule and therefore even slight alterations of the three-dimensional structure of the ligand surface (i.e. the drug molecule) are not tolerated. If the main part of the energy required for a productive TCR engagement is provided by the interaction of the TCR with the peptide–drug complex, then it seems feasible that alteration in the involved MHC residues may also be allowed. Therefore, highly specific TCC show mainly a MHC allele-unrestricted recognition. (B) Broadly drug-reactive TCC interact predominantly with amino acid residues of the MHC molecule and thus allow larger modifications of the embedded drug molecule. As a consequence, these clones show a clearly MHC allele-restricted recognition, as modifications of the relevant MHC residues abrogate the interaction.

 
The data obtained from the analysis of the MHC restriction pattern of drug-specific TCC revealed an other interesting phenomenon: 12 clones, isolated from three different donors, did not need the presence of professional APC for the induction of a productive drug-specific response. These clones had the ability to recognize the drug molecules presented on MHC molecules expressed on the surface of the T cells themselves. It is known from studies with peptide-specific TCC that the ability to react to T cells as APC is a feature of the responding T cells and not of the presenting T cells. In analogy, these drug-specific clones can also be classified as `T cell responder clones' (20). The functional differences between `responder' and `non-responder' T cells have not yet been fully elucidated; however, the data obtained with peptide-specific clones indicate that these two groups of clones differ in their expression of co-stimulatory ligands, mainly CD28 (20).

Under normal conditions, T cells will not encounter allo-MHC molecules during their maturation. This fact has led to the hypothesis that alloreactivity is the result of cross-reactivity of T cells selected for self-restricted responses (37). This also implies that alloreactive T cells are not a particular population of the mature T cell repertoire that has escaped the constraints of the positive selection process in the thymus (17). This directs us to the interesting phenomenon that, although TCR-mediated recognition appears to be sensitive to minute changes in the peptide or the MHC molecule, interaction of the TCR with MHC–peptide complexes is permissive to various combinations of peptides and MHC molecules (37). From the point of view of bispecific TCR, self-MHC–peptide–drug complexes must show a similar three-dimensional structure as non-self-MHC–peptide complexes. The data shown in Table 5Go indicate that the degeneracy of drug-specific TCR might be higher compared to peptide-specific TCR. As a consequence, a much higher proportion of drug-specific clones (27% compared to 8.6% in peptide-specific clones) has the ability to recognize a non-self-MHC molecule in addition to the original self-MHC–peptide drug complex. The data could also be interpreted in the way that self-MHC–peptide–drug complexes can more easily mimic non-self-MHC–peptide complexes than self-MHC–pathogenic peptide complexes. Interestingly the two different antigenic stimuli lead to similar T cell activation pattern as no altered functions of drug-specific TCC after simulation with allogeneic B-LCL could be observed.

In summary, the obtained data indicate that drug-specific T cells isolated from allergic donors comprise a heterogeneous population of highly degenerate T cells. This finding leads to the assumption that drugs can be presented to specific T cells in a multitude of ways. Therefore, a high predominance of MHC allele-unrestricted drug recognition as well as additional alloreactivity in drug-specific TCC might be a peculiar feature of the activation of the specific immune system by drug antigens.


    Acknowledgments
 
We thank all blood donors for their friendly collaboration, E. Frei for TCR oligotyping, I. Strasser and P. Stünzi for HLA phenotyping, and M. Zanni and M. Britschgi for helpful discussions. This work was supported by grant no. 97.0431 of the Federal Office of Education and Science (EU Research Program BIOMED) (to W. J. P.).


    Abbreviations
 
APC antigen-presenting cell
APL altered peptide ligand
B-LCL B lymphoblastoid cell line
CM culture media
PBMC peripheral blood mononuclear cell
Pen G penicillin G
PHA phytohemagglutinin
SMX sulfamethoxazole
TCC T cell clone
TCL T cell line
TT tetanus toxoid

    Notes
 
Transmitting editor: A. McMichael

Received 5 April 2000, accepted 2 April 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Porcelli, S. A., Morita, C. T. and Modlin, R. L. 1996. T-cell recognition of non-peptide antigens. Curr. Opin. Immunol. 8:510.[ISI][Medline]
  2. Grant, E. P., Degano, M., Rosat, J. P., Stenger, S., Modlin, R. L., Wilson, I. A., Porcelli, S. A. and Brenner, M. B. 1999. Molecular recognition of lipid antigens by T cell receptors. J. Exp. Med. 189:195.[Abstract/Free Full Text]
  3. Corinti, S., De Palma, R., Fontana, A., Gagliardi, C., Pini, C. and Sallusto, F. 1997. Major histocompatibility complex-independent recognition of a distinctive pollen antigen, most likely a carbohydrate, by human CD8+ {alpha}/ß T cells. J. Exp. Med. 186:899.[Abstract/Free Full Text]
  4. Zanni, M. P., Schnyder, B., von Greyerz, S. and Pichler, W. J. 1998. Involvement of T cells in drug-induced allergies. Trends Pharm. Sci. 19:308.[ISI][Medline]
  5. Weltzien, H. U., Moulin, C., Martin, S., Padovan, E., Hartmann, U. and Kohler, J. 1996. T cell immune response to haptens: structural models for allergic and autoimmune reactions. Toxicology 107:141.[ISI][Medline]
  6. Pichler, W. J., Schnyder, B., Zanni, M. P., Hari, Y. and von Greyerz, S. 1998. Role of T cells in drug allergies. Allergy 53:225.[ISI][Medline]
  7. Brander, C., Mauri-Hellweg, D., Bettens, F., Rolli, H., Goldman, M. and Pichler, W. J. 1995. Heterogeneous T cell responses to ß-lactam-modified self-structures are observed in penicillin-allergic individuals. J. Immunol. 155:2670.[Abstract]
  8. Mauri-Hellweg, D., Bettens, F., Mauri, D., Brander, C., Hunziker, T. and Pichler, W. J. 1995. Activation of drug-specific CD4+ and CD8+ T cells in individuals allergic to sulfonamides, phenytoine and carbamazepine. J. Immunol. 155:462.[Abstract]
  9. Weltzien, H. U. and Padovan, E. 1998. Molecular features of penicillin allergy. J. Invest. Dermatol. 110:203.[Abstract]
  10. Schnyder, B., Mauri-Hellweg, D., Zanni, M., Bettens, F. and Pichler, W. J. 1997. Direct, MHC-dependent presentation of the drug sulfamethoxazole to human {alpha}ß T cell clones. J. Clin. Invest. 100:136.[Abstract/Free Full Text]
  11. Zanni, M. P., von Greyerz, S., Schnyder, B., Brander, K. A., Frutig, K., Hari, Y., Valitutti, S. and Pichler, W. J. 1998. HLA-restricted, processing- and metabolism-independent pathway of drug recognition by human {alpha}ß T lymphocytes. J. Clin. Invest. 102:1591.[Abstract/Free Full Text]
  12. Mason, D. 1998. A very high level of crossreactivity is an essential feature of the T-cell receptor. Immunol. Today 19:395.[ISI][Medline]
  13. Mauri-Hellweg, D., Zanni, M. P., Frei, E., Bettens, F., Brander, C. and Pichler, W. J. 1996. Crossreactivity of T cell lines and clones to ß-lactam antibiotics. J. Immunol. 157:1071.[Abstract]
  14. Padovan, E., Mauri-Hellweg, D., Pichler, W. J. and Weltzien, H. U. 1996. T cell recognition of penicillin G: structural features determining antigenic specificity. Eur. J. Immunol. 26:41.
  15. Zanni, M. P., von Greyerz, S., Hari, Y., Schnyder, B. and Pichler, W. J. 1999. Recognition of local anesthetics by {alpha}ß+ T cells. J. Invest. Dermatol. 112:197.[Abstract/Free Full Text]
  16. von Greyerz, S., Zanni, M. P., Frutig, K., Schnyder, B., Burkhart, C. and Pichler, W. J. 1999. Interaction of sulfonamide derivatives with the TCR of sulfamethoxazole-specific human {alpha}ß+ T cell clones. J. Immunol. 162:595.[Abstract/Free Full Text]
  17. Lechler, R., Batchelor, R. and Lombardi, G. 1991. The relationship between MHC restricted and allospecific T cell recognition. Immunol. Lett. 29:41.[ISI][Medline]
  18. Daniel, C., Horvath, S. and Allen, P. M. 1998. A basis for alloreactivity: MHC helical residues broaden peptide recognition by the TCR. Immunity 8:543.[ISI][Medline]
  19. Zanni, M. P., Mauri-Hellweg, D., Brander, C., Wendland, T., Schnyder, B., Frei, E., von Greyerz, S., Bircher, A. and Pichler, W. J. 1997. Characterization of lidocaine specific T cells. J. Immunol. 158:1139.[Abstract]
  20. Wyss-Coray, T., Brander, C., Frutig, K. and Pichler, W. J. 1992. Discrimination of human CD4 T cell clones based on their reactivity with antigen-presenting T cells. Eur. J. Immunol. 22:2295.[ISI][Medline]
  21. Hari, Y., FruStig-Schnyder, K., HuSrni, K., Yawalkar, N., Zanni, M. P., Schyder, B., Kappeler, A., von Greyerz, S., Braathen, L. and Pichler, W. J. 2001. T cell involvement in cutaneous drug eruption. Clin. Exp. Allergy in press.
  22. Elsasser-Beile, U., von Kleist, S. and Gallati, H. 1991. Evaluation of a test for measuring cytokine production in human whole blood cell cultures. J. Immunol. Methods 139:191.[ISI][Medline]
  23. Zanni, M. P., von Greyerz, S., Schnyder, B., Wendland, T. and Pichler, W. J. 1998. Allele-unrestricted presentation of lidocaine by HLA-DR molecules to specific {alpha}ß+ T cell clones. Int. Immunol. 10:507.[Abstract]
  24. Kersh, G. J. and Allen, P. M. 1996. Structural basis from T cell recognition of altered peptide ligands: a single T cell receptor can productively recognize a large continuum of related ligands. J. Exp. Med. 184:1259.[Abstract]
  25. Evavold, B. D. and Allen, P. M. 1991. Separation of IL-4 production from Th cell proliferation by an altered T cell receptor ligand. Science 252:1308.[ISI][Medline]
  26. De Magistris, M. T., Alexander, J., Coggeshalll, M., Altman, A., Gaeta, F. C. A., Grey, H. M. and Sette, A. 1992. Antigen analog–major histocompatibility complexes act as antagonists of the T cell receptor. Cell 68:625.[ISI][Medline]
  27. Sloan-Lancaster J., Evavold, B. D. and Allen, P. M. 1993. Induction of T cell anergy by altered T-cell-receptor ligand on live antigen-presenting cells. Nature 363:156.[ISI][Medline]
  28. Sloan-Lancaster, J. and Allen, P. M. 1996. Altered peptide ligand-induced partial T cell activation: molecular mechanisms and role in T cell biology. Annu. Rev. Immunol. 14:1.[ISI][Medline]
  29. Moulon, C., Wild, D., Dormoy, A. and Weltzien, H. U. 1998. MHC-dependent and -independent activation of human nickel-specific CD8+ cytotoxic cells from allergic donors. J. Invest. Dermatol. 111:360.[Abstract]
  30. Panina-Bordignon, P., Tan, A., Termijtelen, A., Demotz, S., Corradin, G. and Lanzavecchia, A. 1989. Universally immunogenic T cell epitopes: promiscuous binding to human class II and promiscuous recognition by T cells. Eur. J. Immunol. 19:2237.[ISI][Medline]
  31. Kohler, J., Hartmann, U., Grimm, R., Pflugefelder, U. and Weltzien, H. U. 1997. Carrier-independent hapten recognition and promiscuous MHC restriction by CD4 T cells induced by trinitrophenylated peptides. J. Immunol. 158:591.[Abstract]
  32. Ding, Y. H., Smith, K. J., Garboczi, D. N., Utz, U., Biddison, W. E. and Wiley, D. C. 1998. Two human T cell receptors bind in a similar diagonal mode to the HLA-A2/tax peptide complex using different TCR amino acids. Immunity 8:403.[ISI][Medline]
  33. Garboczi, D. N. and Biddison, W. E. 1999. Shapes of MHC restriction. Immunity 10:1.[ISI][Medline]
  34. Garcia, K. C., Degano, M., Stanfield, R. L., Brunmark, A., Jackson, M. R., Peterson, P. A., Teyton, L. and Wilson, I. A. 1996. An {alpha}ß T cell receptor structure at 2. 5 A and its orientation in the TCR–MHC complex. Science 274:209.[Abstract/Free Full Text]
  35. Garboczi, D. N., Ghosh, P., Utz, U., Fan, Q. R., Biddison, W. E. and Wiley, D. C. 1996. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2. Nature 384:134.[ISI][Medline]
  36. Hemmer, B., Vergelli, M., Pinilla, C., Houghten, R. and Martin, R. 1998. Probing degeneracy in T cell recognition using peptide combinatorial libraries. Immunol. Today 19:163.[ISI][Medline]
  37. Udaka, K., Wiemüller, K. H., Kienle, S., Jung, G. and Walden, P. 1996. Self-MHC-restricted peptides recognized by an alloreactive T lymphocyte clone. J. Immunol. 157:670.[Abstract]