Recognition of Local Anesthetics by αβ+ T Cells
1999; Elsevier BV; Volume: 112; Issue: 2 Linguagem: Inglês
10.1046/j.1523-1747.1999.00484.x
ISSN1523-1747
AutoresMartin P. Zanni, Salomé von Greyerz, Yvonne Hari, Benno Schnyder, Werner J. Pichler,
Tópico(s)Bioactive Compounds and Antitumor Agents
ResumoPatients with drug allergy show a specific immune response to drugs. Chemically nonreactive drugs like, for example, local anesthetics are directly recognized by aβ+ T cells in an HLA-DR restricted way, as neither drug metabolism nor protein processing is required for T cell stimulation. In this study we identified some of the structural requirements that determine cross-reactivity of T cells to local anesthetics, with the aim to improve the molecular basis for the selection of alternatives in individuals sensitized to a certain local anesthetic and to better understand presentation and T cell recognition of these drugs. Fifty-five clones (52 lidocaine specific, three mepivacaine specific from two allergic donors) were analyzed. Stimulatory compounds induced a downregulation of the T cell receptor, demonstrating that these non-peptide antigens are recognized by the T cell receptor itself. A consistent cross-reactivity between lidocaine and mepivacaine was found, as all except one lidocaine specific clone proliferated to both drugs tested. Sixteen chemically related local anesthetics (including ester local anesthetics, OH- and desalkylated metabolites) were used to identify structural requirements for T cell recognition. Each of the four clones examined in detail was uniquely sensitive to changes in the structures of the local anesthetic: clone SFT24, i.e., did not recognize any of the tested OH- or desalkylated metabolites, while the clone OFB2 proliferated to all OH-metabolites and other differently modified molecules. The broadly reactive clone OFB2 allowed us to propose a model, suggesting that the structure of the amine side chain of local anesthetics is essential for recognition by the T cell receptor. Patients with drug allergy show a specific immune response to drugs. Chemically nonreactive drugs like, for example, local anesthetics are directly recognized by aβ+ T cells in an HLA-DR restricted way, as neither drug metabolism nor protein processing is required for T cell stimulation. In this study we identified some of the structural requirements that determine cross-reactivity of T cells to local anesthetics, with the aim to improve the molecular basis for the selection of alternatives in individuals sensitized to a certain local anesthetic and to better understand presentation and T cell recognition of these drugs. Fifty-five clones (52 lidocaine specific, three mepivacaine specific from two allergic donors) were analyzed. Stimulatory compounds induced a downregulation of the T cell receptor, demonstrating that these non-peptide antigens are recognized by the T cell receptor itself. A consistent cross-reactivity between lidocaine and mepivacaine was found, as all except one lidocaine specific clone proliferated to both drugs tested. Sixteen chemically related local anesthetics (including ester local anesthetics, OH- and desalkylated metabolites) were used to identify structural requirements for T cell recognition. Each of the four clones examined in detail was uniquely sensitive to changes in the structures of the local anesthetic: clone SFT24, i.e., did not recognize any of the tested OH- or desalkylated metabolites, while the clone OFB2 proliferated to all OH-metabolites and other differently modified molecules. The broadly reactive clone OFB2 allowed us to propose a model, suggesting that the structure of the amine side chain of local anesthetics is essential for recognition by the T cell receptor. B lymphoblastoid cell line culture media local anesthetics lymphocyte transformation test peripheral blood mononuclear cells T cell clone T cell line Drugs are examples of non-peptide antigens. The immune response to drugs has been mainly investigated with penicillin G, where it has been shown that penicillin G binds covalently to proteins resulting in altered self structures, which are recognized by B and T cells (hapten model). Recognition of penicillins and other drugs by specific T cells is HLA restricted and antigen-presenting cell (APC) dependent (Brander et al., 1995Brander C. Mauri-Hellweg D. Bettens F. Rolli H. Goldman M. Pichler W.J. Heterogeneous T cell responses to β-lactam-modified self structures are observed in penicillin allergic individuals.J Immunol. 1995; 155: 2670-2678PubMed Google Scholar;Padovan et al., 1997Padovan E. Bauer T. Tongio M.M. Karlbacher H. Weltzien H.U. Penicilloyl peptides are recognized as T cell antigenic determinants in penicillin allergy.Eur J Immunol. 1997; 27: 1303-1307Crossref PubMed Scopus (135) Google Scholar;Pichler et al., 1998Pichler W.J. Schnyder B. Zanni M.P. Hari Y. von Greyerz S. Role of T-cells in drug allergies.Allergy. 1998; 53: 225-232Crossref PubMed Scopus (85) Google Scholar). The fine specificity of drug recognition by the immune system has been mainly investigated with the β-lactam model comparing different penicillins: A number of patients have been described, who form IgE antibodies (MoAb) that can discriminate between penicillin G, ampicillin or other semisynthetic penicillins (Blanca et al., 1989Blanca M. Fernandez J. Miranda A. Cross-reactivity between penicillins and cephalosporins: Clinical and immunogenic studies.J Allergy Clin Immunol. 1989; 83: 381-385Abstract Full Text PDF PubMed Scopus (116) Google Scholar;Miranda et al., 1996Miranda A. Blanca M. Vega J.M. Cross-reactivity between a penicillin and a cephalosporin with the same side chain.J Allergy Clin Immunol. 1996; 98: 671-677Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar; Silviu-Dan et al., 1992Silviu-Dan F. McPhillips S. Warrington R.J. The frequency of skin test reactions to side-chain penicillin determinants.J Allergy Clin Immunol. 1992; 91: 694-701Abstract Full Text PDF Scopus (108) Google Scholar). By using β-lactam specific T cell clones (TCC) it was found, that the antigenic epitope consisted of both the amide-linked side chain, which is different in every member of the penicillin family, as well as the thiazolidine ring common to all penicillin derivatives (Mauri-Hellweg et al., 1996Mauri-Hellweg D. Zanni M.P. Frei E. Bettens F. Brander C. Pichler W.J. et al.Crossreactivity of T cell lines and clones to β- lactam antibiotics.J Immunol. 1996; 157: 1071-1079PubMed Google Scholar;Padovan et al., 1996Padovan E. Mauri-Hellweg D. Pichler W.J. Weltzien H.U. T cell recognition of penicillinG: structural features determining antigenic specificity.Eur J Immunol. 1996; 26: 42-48Crossref PubMed Scopus (107) Google Scholar). Earlier studies in the mouse model with specific T cells for arsenylated ovalbumin showed that alterations in the aromatic side chain are not recognized by TCC specific for arsonate. Some of these analogs functioned even as inhibitors for arsonate specific T cells (Rao et al., 1984Rao A. Faas S.J. Cantor H. Analogs that compete for antigen binding to an arsonate-reactive T-cell response to arsonate.Cell. 1984; 36: 889-895Abstract Full Text PDF PubMed Scopus (29) Google Scholar). The hapten model was, however, recently questioned as the only model for drug recognition by our finding that glutaraldehyde fixed cells can present sulfamethoxazole as well as lidocaine to specific CD4+ and CD8+aβ+ T cells (Schnyder et al., 1997Schnyder B. Mauri-Hellweg D. Zanni M.P. Bettens F. Pichler W.J. Direct, MHC dependent presentation of the drug sulfamethoxazole to aβ+ T cell clones.J?Clin Invest. 1997; 100: 136-141Crossref PubMed Scopus (233) Google Scholar;Pichler et al., 1998Pichler W.J. Schnyder B. Zanni M.P. Hari Y. von Greyerz S. Role of T-cells in drug allergies.Allergy. 1998; 53: 225-232Crossref PubMed Scopus (85) Google Scholar;Zanni et al., 1998aZanni M.P. von Greyerz S. Schnyder B. Wendland T. Pichler W.J. Allele-unrestricted presentation of lidocaine by HLA-DR molecules to specific aβ+ T cell clones.Int Immunol. 1998; 10: 507-515Crossref PubMed Scopus (60) Google Scholar) and that pulsing of APC was not possible. Neither sulfamethoxazole nor lidocaine are chemically reactive, but are nevertheless presented and recognized by specific TCC: this indicates that some drugs like local anesthetics (LA) do not need drug metabolism or protein processing to become immunogenic and that not all drugs behave like classical haptens, which gain their immunogenicity only after covalent binding to endogenous proteins. The type of drug binding to major histocompatibility complex (MHC)/peptide and T cell receptor (TCR) is at present unclear, but might be similar to other pharmacologic drug–receptor interactions or the binding and recognition of metals like Ni-salts (Romagnoli et al., 1991Romagnoli P. Labhardt A.M. Sinigaglia F. Selective interaction of Ni with an MHC-bound peptide.Embo J. 1991; 10: 1303-1307Crossref PubMed Scopus (132) Google Scholar). In this study we investigated the potential of LA specific TCC to recognize chemically similar LA. We had three aims by analyzing the fine specificity to different LA: (i) obtaining a better understanding of the molecular basis for cross-reactivity, which has clinical implications for LA-sensitized individuals, namely an improved choice of an alternative drug; (ii) determining the fine specificity of TCC to a drug, which is presented in a noncovalent way; (iii)?obtaining some information about the localization or orientation of drug binding to the MHC/peptide complex or to the TCR by detecting structural requirements for TCR recognition, as the lability of drug binding makes usual binding studies unfeasible. To address these questions we tested the in?vivo response of patients by patch tests to six widely used LA and analyzed the cross-reactivity to different LA-specific T cell lines (TCL) and TCC. Our data underline the lack of cross-reactivity between ester/amide LA, but demonstrate a very high degree of cross-reactivity between lidocaine and mepivacaine. The combined analysis of the reactivity of the four TCC analyzed in detail revealed a possible core structure for T cell recognition. Moreover, based on the broad reactivity of one clone we propose a model, suggesting that the structure of the amine side chain of local anesthetics is essential for recognition by the TCR. All patients tested developed contact dermatitis-like symptoms upon topical application or subcutaneous injection of lidocaine (patients SF and OF) or mepivacaine (patient PG) (Zanni et al., 1997Zanni M.P. Mauri-Hellweg D. Brander C. Characterization of lidocaine specific T cells.J Immunol. 1997; 158: 1139-1148PubMed Google Scholar). Blood was taken, when patients had recovered from the allergic reaction and informed consent was obtained from all subjects before performing these studies. The HLA type of donor OF was: HLA-A2, A26(10), B7, BX, Bw6, DRB1*1501 or 1503/X, DQA1*0102/–, DQB1*0602/–, and DPB1*0401/–; and of patient SF: HLA-A3, A29(19), B7, BX, Bw6 and DRB1*1501 or 1503/X (Zanni et al., 1997Zanni M.P. Mauri-Hellweg D. Brander C. Characterization of lidocaine specific T cells.J Immunol. 1997; 158: 1139-1148PubMed Google Scholar). Culture media (CM) consisted of RPMI 1640 supplemented with 10% pooled heat-inactivated human AB serum (Swiss Red Cross, Bern, Switzerland), 25?mM HEPES buffer, 2?mM L-glutamine, 10?mg per ml streptomycin and 100?U per ml penicillin. CM+ used to culture TCL and TCC was enriched with 20?U per ml natural interleukin-2 (a kind gift from Dr. U. Schwulèra, Biotest, Frankfurt/Main, Germany) and 20?U per ml recombinant interleukin-2 (Dr. A. Cerny, Inselspital, Switzerland). The medium for culture of Epstein–Barr virus (EBV) transformed B lymphoblastoid cell lines (B-LCL) was RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Paisley, Scotland), 25?mM HEPES buffer, but no L-glutamine and no antibiotics. The EBV-B cell lines (B-LCL) were generated as described (Brander et al., 1995Brander C. Mauri-Hellweg D. Bettens F. Rolli H. Goldman M. Pichler W.J. Heterogeneous T cell responses to β-lactam-modified self structures are observed in penicillin allergic individuals.J Immunol. 1995; 155: 2670-2678PubMed Google Scholar). LA have an aromatic lipophilic constituent, which is coupled via a carbonyl to a hydrophilic tertiary amine. According to their benzoic substitution in position 1, LA are divided into ester LA and amide LA (for structure see Table 1). Ester LA have an additional amine substitution in position 4 of the benzene ring. While amine tails in ester LA are linear, several amide LA compounds show cyclic amine formation (mepivacaine, bupivacaine).Table 1Chemical structures of LA and in vivo (patch test) and in vitro (LTT) testsPatient:aResults from patients tested by lymphocyte transformation test (LTT) and patch test to the relevant LA (lidocaine and mepivacaine) and to a panel of related LA are summarized and correlated to the chemical structure of LA (13). Sensitized to: sex/year:OFlidocainef/1994SFlidocainef/1992PGmepivacainef/1995Drug testedSkin testsbPatch tests were evaluated with a 5% drug solution in PBS 24, 48, and 72 h after exposure to the allergen. Nonallergic control individuals did not show positive reactions in the patch test or LTT (n = 5).LTTcResults from LTT are given as stimulation indices (cpm in culture with antigen divided by cpm in culture without antigen). All compounds were tested with different concentrations (1-500 ?g per ml); only the concentration with maximal stimulation (100 ?g) is shown. Bold numbers show elevated specific stimulation indices (stimulation index > 3).Skin testsLTTSkin testsLTTlidocainepos15pos18.2pos24.9mepivacainepos9.2pos6pos26.3bupivacaineneg0.2posdOnly a weak reaction after 72 h visible.0.2neg5.5View Large Image Figure ViewerDownload (PPT)procaineneg0.7neg1,4neg1.1oxybuprocaineneg0.7neg3.5neg0.7tetracaineneg1.1neg1.4neg2.4a Results from patients tested by lymphocyte transformation test (LTT) and patch test to the relevant LA (lidocaine and mepivacaine) and to a panel of related LA are summarized and correlated to the chemical structure of LA (13).b Patch tests were evaluated with a 5% drug solution in PBS 24, 48, and 72 h after exposure to the allergen. Nonallergic control individuals did not show positive reactions in the patch test or LTT (n = 5).c Results from LTT are given as stimulation indices (cpm in culture with antigen divided by cpm in culture without antigen). All compounds were tested with different concentrations (1-500 ?g per ml); only the concentration with maximal stimulation (100 ?g) is shown. Bold numbers show elevated specific stimulation indices (stimulation index > 3).d Only a weak reaction after 72 h visible. Open table in a new tab All drugs used in these experiments have been previously tested for their inhibitory activity on the proliferative response to mitogens in nonallergic individuals and only nontoxic concentrations of the drugs have been used for in?vitro stimulations (data not shown). Preparation of drug solutions in CM was always done freshly just before use.Table 1 Following drugs were used: lidocaine, mepivacaine, bupivacaine, etidocaine, prilocaine (Astra, Uppsala, Sweden), encainide (Bristol-Myers Squibb,?Princeton, NJ), butanilicaine (Hoechst-Pharma, Unterschleissheim, Germany), procaine (Siegfried AG, Zofingen, Switzerland), oxybuprocaine (Dolder AG, Zürich, Switzerland), tetracaine (Hänseler AG, Herisau, Switzerland), and tocainide (kindly provided by U. Meyer, Biozentrum, Basel, Switzerland). Metabolites were provided by Astra, Uppsala, Sweden: 3-OH-lidocaine, Glycinexylidide HCl (GX), Monoethylglycinexylidide HCl (MEGX), 3-OH-bupivacaine, 4-OH-bupivacaine, 4-OH-mepivacaine, 2-amino-2'6'-butyroxylidide, 4-OH-6'methylalanine, and 4-OH-2'6'-dimethylalanine. Patients with presumable allergy to LA were tested routinely in a LTT for their reactivity to six LA (see Table 1). Peripheral blood mononuclear cells (PBMC) were obtained after the drug allergy symptoms have subsided. Briefly, freshly isolated PBMC (1–2×106 cells in 1?ml CM) from these patients were cultured in upright 12?ml tubes (Falcon, Franklin Lakes, NJ) in the presence of different drug concentrations. After 6?d, [3H]thymidine (0.5?mCi) was added for 14?h, cultures were harvested and cpm were measured in a β-counter (Packard, Canberra, Meriden, CT). Stimulation indices were calculated as "cpm in culture with antigen per cpm in culture without antigen". Freshly isolated PBMC of drug allergic patients were stimulated with lidocaine (100?µg per ml) or mepivacaine (100?µg per ml) in CM at a cell density of 2×106 per well in a 24 well plate (Falcon). To generate TCL, CM+ was added after 7?d of culture. After 11?d, 0.8×106 bulk cells were restimulated with 0.5×106 autologous irradiated (3000?rad) PBMC, 0.3×106 autologous irradiated (6000?rad) B-LCL and the same concentration of antigen used in primary stimulations. TCL were expanded in CM+, and restimulated every 14?d by the above described APCs and antigen. Drug-specific T cells were cloned by limiting dilution as described earlier (Zanni et al., 1997Zanni M.P. Mauri-Hellweg D. Brander C. Characterization of lidocaine specific T cells.J Immunol. 1997; 158: 1139-1148PubMed Google Scholar). Briefly, blast cells from specific TCL were seeded at 0.3, 1, or 3 cells per well with 25,000 allogeneic irradiated PBMC into a 96 well round bottom plate (Falcon) plus phytohemogluttinin (0.5?µg per ml) (Bacto, Difco Laboratories, Detroit, MI) in 150?µl per well of CM. Growing TCC were expanded in CM+ and restimulated every 14?d with allogeneic irradiated PBMC plus phytohemogluttinin (0.5?µg per ml). The phenotype of TCC was identified by immunofluorescence analysis with anti-CD4, anti-CD8, anti-CD3 and anti-TCR ?d MoAb (Becton Dickinson, Rutherford, NJ). To critically evaluate cross-reactivity of specific TCC, proof of clonality is essential. The four clones were analyzed and stained with a panel of 17 defined Vβ-antibodies obtained from Immunotech, SA (Marseille, France) (Vβ2, Vβ3, Vβ5.1, Vβ5.2, Vβ5.3, Vβ6, Vβ8, Vβ11, Vβ12, Vβ13.1, Vβ13.3, Vβ14, Vβ16, Vβ17, Vβ20, Vβ21, Vβ22). Three clones gave only a positive staining for one Vβ, namely Vβ14 (SFT24), Vβ5.1 (OFZ45), and Vβ17 (OFB2), but negative staining with all other Vβ-specific MoAb. Clone SFM8 was negative for all tested MoAb, indicating a TCR with a Vβ not stainable by MoAb. To measure the antigen specific proliferation of TCL and TCC, 3×104 cells were incubated with the same number of autologous irradiated (3000?rad) PBMC or 5×103 B-LCL (6000?rad) in the presence or absence of drugs at indicated concentrations in 200?µl CM in a 96-well round bottom plate (Falcon). After 48?h, [3H]thymidine (0.5?µCi) was added for 14?h, cells were harvested on glass fiber disks and counted in a microplate β-counter (Inotech Filter Counting System INB 384; Inotech, Dottikon, Switzerland). Stimulation indices were calculated as described above. Three times 104 clone cells were incubated with 5×104 autologous B-LCL in 200?µl RPMI 10% fetal bovine serum medium in U bottom plates in the presence of no antigen or drugs (100?µg per ml). The plates were centrifuged for 2?min to allow conjugate formation and incubated for 4 h at 37°C. Cells were washed with PBS containing 1% fetal bovine serum and 0.02% NaN3 and stained with a phycoerythrin-labeled anti-CD3 and a fluoroscein-isothiocyanate-labeled anti-CD19 MoAb to exclude B-LCL. The CD3 fluorescence was analyzed on an EPICS profile II flow cytometer (Coulter, Hialeah, FL). APC were gated out using both forward and side scatter (FSC/SSC) and green fluorescence. CD3, CD19 MoAb were obtained from Dako Diagnostics (Zug, Switzerland). In a previous study we characterized the T cell reactivity to LA regarding symptoms, induction of activation parameter, TCC phenotype, cytokine release, TCRVβ pattern and HLA restriction (Zanni et al., 1997Zanni M.P. Mauri-Hellweg D. Brander C. Characterization of lidocaine specific T cells.J Immunol. 1997; 158: 1139-1148PubMed Google Scholar). Table 1 shows the structures of LA tested and gives a summary of results obtained with in?vivo (patch tests) and in?vitro data (LTT). All three patients showed a positive patch test and LTT to the sensitizing drug. In addition, the two patients (SF and OF) sensitized to lidocaine reacted to mepivacaine and patient PG, originally showing an adverse reaction to mepivacaine cross-reacted to lidocaine. Interestingly, patients SF and OF revealed a positive skin test and LTT to mepivacaine, although they never have received mepivacaine treatment before. Except for patient SF, which showed a weakly positive skin test, the patients did not react to the third tested amide LA (bupivacaine). In general, we found a good correlation of patch test results with the LTT. The only exception was a negative skin test with oxybuprocaine but a weakly positive LTT to this compound for patient SF. Only a narrow range of concentrations resulted in a positive LTT. Concentrations between 10 and 100?µg per ml were found to be optimal for T cell stimulation. Higher concentrations (500?µg per ml) were already toxic and those lower (1?µg per ml) were nonstimulatory. All results were reproducible over the tested time period of 2?y. Local anesthetics can induce a specific TCR downregulation There is only indirect evidence that drug recognition is TCR mediated, as up to now no drug-specific hybridoma or TCR-transfected cell line exists As shown in Figure 1 the lidocaine-specific TCC OFB2 exhibits a TCR downregulation when incubated with stimulatory drugs such as lidocaine or mepivacaine, but not when stimulated with nonproliferation-inducing compounds, such as MEGX or butanilicaine The response is clearly APC dependent, as absence of APC failed to induce a TCR downregulation Figure 1b These results demonstrate that drug recognition by T cells is associated with a downregulation of the TCR itself. Cross-reactivity of lidocaine and mepivacaine specific TCL It is generally assumed, that there is no immunologic cross-reactivity between amide and ester type LA, but a detailed analysis of the in?vivo and in?vitro T cell cross-reactivity has not yet been described (Curley et al., 1986Curley R. Macfarlane K. King C.M. Contact sensitivity to the amide anesthetics lidocaine, prilocaine and mepivacaine.Arch Dermatol. 1986; 122: 924-926Crossref PubMed Scopus (73) Google Scholar To analyze further the in?vitro cross-reactivity, we established LA-specific TCL In spite of several in?vitro restimulations, we never succeeded to establish TCL specific to ester LA from the originally amide LA-sensitized donors Additionally, it was not possible to generate bupivacaine-specific TCL albeit this drug differs from mepivacaine only by showing a N-butyl-piperidine instead of N-methyl-piperidine substituent With the help of lidocaine and mepivacaine specific TCL from donors SF, OF and PG, we found that these lines are cross-reactive, but do not recognize the third tested amide LA, bupivacaine (see Table 2, for chemical structures Table 1). As expected from skin test results and LTT, none of the six established lines reacted to any of the analyzed LA of the ester type Table 2Table 2Cross-reactivity of lidocaine- and mepivacaine-specific TCLaTCL were analyzed for cross-reactivity with three amide LA and three ester LA (chemical structure see Table 1 ). Only lidocaine and mepivacaine revealed a positive proliferation with a maximal response when 100 µg drug per ml where used. Results are shown for drug concentrations which yielded maximum proliferation. Results of one of three experiments are shown.Restimulation (antigen)PatientNo antigen(cpm)T cell lines generated byLidocaine(SI)bSI, stimulation index.Mepivacaine(SI)Bupivacaine(SI)Procaine(SI)Oxybuprocaine(SI)Tetracaine(SI)OF42lidocaine156165.40.91.20.9222mepivacaine90.1157.21.81.71.53SF676lidocaine4.84.80.710.90.8207mepivacaine51.154.14.50.92.51.5PG2051lidocaine3.92.80.51.10.60.6560mepivacaine4.24.40.61.50.80.9a TCL were analyzed for cross-reactivity with three amide LA and three ester LA (chemical structure see Table 1 ). Only lidocaine and mepivacaine revealed a positive proliferation with a maximal response when 100 µg drug per ml where used. Results are shown for drug concentrations which yielded maximum proliferation. Results of one of three experiments are shown.b SI, stimulation index. Open table in a new tab To see whether patients with positive patch tests to lidocaine and mepivacaine do also show a cross-reactivity on a clonal level, lidocaine-specific and mepivacaine-specific TCC of two patients were generated. As shown in Table 3 for patient SF, all clones specific for lidocaine are cross-reactive to mepivacaine and, vice versa, the three tested mepivacaine-specific clones recognized lidocaine. The possibility of analyzing sister clones, could be ruled out as flow cytometry and polymerase chain reaction analysis performed with seven selected clones revealed different TCRVβ expression (data not shown). Additionally, the cross-reactive clones showed a distinct CD4+ or CD8+ phenotype. The two clones from patient OF were obtained at different time points. They demonstrate that lidocaine-specific cells exist, which do not recognize mepivacaine, as one clone reacted exclusively to lidocaine.Table 3Lidocaine- and mepivacaine-specific TCC cross-react to each otheraLidocaine-specific TCC of donor OF and SF were tested for recognition of mepivacaine and mepivacaine-specific clones obtained from donor SF were analyzed for lidocaine specificity. A positive reaction was determined as an stimulation index > 3 for both drugs.DrugbClone generated with drug indicated.PatientPhenotypeLidocaine specificcNumber of clones with a stimulation index >3.Mepivacaine specificlidocaineSFCD44343CD855γδ22OFCD421mepivacaineSFCD433a Lidocaine-specific TCC of donor OF and SF were tested for recognition of mepivacaine and mepivacaine-specific clones obtained from donor SF were analyzed for lidocaine specificity. A positive reaction was determined as an stimulation index > 3 for both drugs.b Clone generated with drug indicated.c Number of clones with a stimulation index >3. Open table in a new tab Fine specificity of lidocaine- and mepivacaine-specific TCC To better characterize the core structure for T cell recognition, LA specific clones were analyzed with different, structurally slightly modified LA and their metabolites (Table 4, 5, 6 and 7). All compounds are in clinical use, or are in?vivo formed metabolites. Three clones generated with lidocaine (SFT24 from donor SF, and OFB2, OFZ45 from donor OF) and one clone generated with mepivacaine (SFM8 from donor SF) were analyzed in detail. Clone OFB2 and OFZ45 as well as SFM8 were HLA-DRB1*15 restricted as evaluated by blocking experiments with anti-DR-antibodies and use of HLA-DR matched allogeneic APC (Zanni et al., 1997Zanni M.P. Mauri-Hellweg D. Brander C. Characterization of lidocaine specific T cells.J Immunol. 1997; 158: 1139-1148PubMed Google Scholar); clone SFT24 was HLA-DR restricted, but able to recognize lidocaine with certain allogeneic APC as well (Zanni et al., 1998aZanni M.P. von Greyerz S. Schnyder B. Wendland T. Pichler W.J. Allele-unrestricted presentation of lidocaine by HLA-DR molecules to specific aβ+ T cell clones.Int Immunol. 1998; 10: 507-515Crossref PubMed Scopus (60) Google Scholar).Table 3Table 4Cross-reactivity of LA-specific TCC to LA bearing cyclic amines and their OH-metabolitesaLidocaine- and mepivacaine-specific TCC do cross-react to compounds containing N-methyl-piperidine (mepivacaine) (clone SFT24 and SFM8), to N-butyl-piperidine (bupivacaine) (clone OFZ45) exclusively or tolerate both amine modifications (clone OFB12). OH-metabolites are not recognized by clone SFT24. Clone OFZ45 and SFM8 reacted with metabolites containing a hydroxylation in position 3, while clone OFB2 proliferates to both 3-OH and 4-OH metabolites. All compounds were tested with different concentrations (1–500 µg per ml). Only the highest values of proliferation are shown (100 µg per ml). One of three representative experiments is shown.CloneNo antigen(cpm)Lidocaine(SI)3-OH-lidocaine(SI)Mepivacaine(SI)4-OH-mepivacaine(SI)Bupivacaine(SI)3-OH-bupivacaine(SI)4-OH-bupivacaine(SI)SFT2463113.41.016.40.81.80.70.6OFZ4561217.314.41.61.16.96.91.2SFM816091.420.851.66.72.711.02.1OFB26239.810.020.011.615.911.88.7a Lidocaine- and mepivacaine-specific TCC do cross-react to compounds containing N-methyl-piperidine (mepivacaine) (clone SFT24 and SFM8), to N-butyl-piperidine (bupivacaine) (clone OFZ45) exclusively or tolerate both amine modifications (clone OFB12). OH-metabolites are not recognized by clone SFT24. Clone OFZ45 and SFM8 reacted with metabolites containing a hydroxylation in position 3, while clone OFB2 proliferates to both 3-OH and 4-OH metabolites. All compounds were tested with different concentrations (1–500 µg per ml). Only the highest values of proliferation are shown (100 µg per ml). One of three representative experiments is shown. Open table in a new tab Table 5Cross-reactivity of LA-specific TCC to LA bearing primary amine side chainsaLA-specific TCC react to tertiary amines, but not to primary amines. The major in vivo-metabolites MEGX and GX are not recognized. All compounds were tested with different concentrations (1–500 µg per ml). Only the highest values of proliferation are shown (100 µg per ml). One of three representative experiments is shown.CloneNo antigen(cpm)Lidocaine(SI)ButanilicainebButanilicaine bears a chloride instead of a methyl in position 2 of the benzene ring.(SI)MEGX(SI)GX(SI)SFT2463113.80.80.90.9OFZ4539031.01.21.51.6SFM85632.60.52.22.4OFB214237.00.70.20.3a LA-specific TCC react to tertiary amines, but not to primary amines. The majo
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