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Molecular Features of Penicillin Allergy

1998; Elsevier BV; Volume: 110; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1747.1998.00122.x

1523-1747

Hans Ulrich Weltzien, Elisabetta Padovan,

Asthma and respiratory diseases

Haptens, such as drugs and other low molecular weight chemicals, become immunogenic only upon binding to proteins. Among antibiotics, penicillins are most commonly used for the treatment of bacterial infections and constitute a typical example of allergy inducing drugs in humans. Previous work on their immunologic properties focused mainly on the examination of IgE-mediated hypersensitivity reactions; however, drug-specific T cell reactions are also involved in causing a serious allergic inflammatory response. This review will focus on the interaction between antibiotic molecules and penicillin-specific T lymphocytes in humans. Experimental data accumulated so far on the reactivity of T cells with penicillin G point to penicilloyl-modified, major histocompatibility complex-associated peptides as T cell epitopes. The recognition specificity of the respective T cell receptors appears to be directed at both the backbone and the specific side chain of penicillin. In contrast, the sequence of the carrier peptides appears to contribute little to the antigenic specificity, mainly as a holder for the haptenic determinant. Finally, recent results demonstrating the capacity of penicillins to modulate, in vitro, the Th0/Th2 phenotype of established T cell clones will be presented and discussed in relation to possible therapeutic applications. Haptens, such as drugs and other low molecular weight chemicals, become immunogenic only upon binding to proteins. Among antibiotics, penicillins are most commonly used for the treatment of bacterial infections and constitute a typical example of allergy inducing drugs in humans. Previous work on their immunologic properties focused mainly on the examination of IgE-mediated hypersensitivity reactions; however, drug-specific T cell reactions are also involved in causing a serious allergic inflammatory response. This review will focus on the interaction between antibiotic molecules and penicillin-specific T lymphocytes in humans. Experimental data accumulated so far on the reactivity of T cells with penicillin G point to penicilloyl-modified, major histocompatibility complex-associated peptides as T cell epitopes. The recognition specificity of the respective T cell receptors appears to be directed at both the backbone and the specific side chain of penicillin. In contrast, the sequence of the carrier peptides appears to contribute little to the antigenic specificity, mainly as a holder for the haptenic determinant. Finally, recent results demonstrating the capacity of penicillins to modulate, in vitro, the Th0/Th2 phenotype of established T cell clones will be presented and discussed in relation to possible therapeutic applications. ampicillin penicillin G penicillin V Adverse reactions to antibiotics may cause serious problems in practical medicine. On the one hand, such reactions include side-effects due to the toxicity of the drug. On the other hand, the induction of drug-specific immune reactions may result in immediate and delayed types of hypersensitivity. In analogy to other chemical haptens, the ability of any drug to induce an immune response depends on its reactivity with proteins. Thus, drugs like β-lactam antibiotics, which easily form covalent bonds with free amino groups, are generally more immunogenic than, for example, sulfonamides, which are intrinsically unreactive and require cellular metabolism to produce reactive intermediates (Park et al., 1987Park B.K. Coleman J.W. Kitteringham N.R. Drug disposition and drug hypersensitivity.Biochem Pharmacol. 1987; 36: 581-590Crossref PubMed Scopus (175) Google Scholar). The molecular targets of such haptenation processes may be soluble or cell-bound proteins, including major histocompatibility complex (MHC) molecules and the associated peptides. In the mouse, T cell responses to model haptens are induced by haptenated peptides presented by MHC molecules to specific antigen receptors of T lymphocytes (Nalefski and Rao, 1993Nalefski E.A. Rao A. Nature of the ligand recognized by a hapten-specific and carrier- specific.MHC-restricted T cell receptor. J Immunol. 1993; 150: 3806-3816PubMed Google Scholar, Martin and Weltzien, 1994Martin S. Weltzien H.U. T cell recognition of haptens: a molecular view.Int Arch Allergy Immunol. 1994; 104: 10-16Crossref PubMed Scopus (102) Google Scholar, Luescher et al., 1995Luescher I.F. Anjuere F. Peitsch M.C. Jongeneel C.V. Cerottini J.C. Romero P. Structural- analysis of TCR-ligand interections studied on H-2Kd-restricted cloned CTL specific for a photoreactive peptide derivative.Immunity. 1995; 3: 51-63Abstract Full Text PDF PubMed Scopus (50) Google Scholar); however, the nature of hapten determinants, formed by drugs and other chemicals and relevant for human T cells, has only recently begun to be intensively investigated. Although the molecular interactions of chemical allergens with human T cells bear similarities to the recognition of nominal peptide antigens, they also exhibit particular characteristics and peculiarities. In this review we will concentrate on recent developments in the understanding of penicillin-specific human T cell responses. Penicillins constitute a large family of compounds whose common structural basis is a β-lactam ring condensed to a thiazolidine ring. Connected to this common backbone structure are characteristically different amide-bonded side chains in every member of this class of compounds. The β-lactam ring is a target for nucleophilic attack by free amino groups of proteins, leading to ring opening and covalent amide bonding of the penicilloyl group (Batchelor et al., 1965Batchelor F.R. Janet M.D. Gazzard D. Penicillin allergy: the formation of the penicilloyl determinant.Nature. 1965; 206: 362-364Crossref PubMed Scopus (123) Google Scholar). The penicilloyl configuration, where the hapten determinant is covalently linked to ε-amino groups of lysine residues of proteins, constitutes more than 90% of the reaction products between proteins (Fig.1), and is also referred to as the major antigenic determinant for antibodies. It plays a role in 75% of IgE mediated allergic reactions and is the target for most other types of hypersensitivity reactions, including delayed type hypersensitivity (Sullivan, 1993Sullivan T.J. Drug allergy.in: Middleton E. Reed C.E. Ellis E.F. Franklin Adkinson N. Yunginger J.W. Busse W.W. Allergy. Mosby-Year Book, St Louis1993: 1726-1746Google Scholar). Other conjugation products can be formed by degradation of penicillin in solution to penicillenic or penicilloic acid, which form disulfide bonds with sulfhydryl groups of cysteine producing, respectively, penicillenyl- and penicillamine-protein conjugates (Fig 1). These so-called minor antigenic determinants are present only at low abundance, but may also contribute to the induction of IgE mediated reactions (Levine and Redmond, 1969Levine B.B. Redmond A.P. Minor haptenic determinant-specific reagents of penicillin hypersensitivity in man.Int Arch Allergy Appl Immunol. 1969; 35: 445-449Crossref PubMed Scopus (133) Google Scholar). Several experimental data support the involvement ofT cell responses in penicillin allergy. It has been demonstrated that peripheral blood lymphocytes of penicillin allergic patients proliferate, in vitro, in response to the appropriate β-lactam antibiotic. Antigen-specific T cell clones isolated from such cultures were of the CD4+ or CD8+ phenotype and secreted heterogeneous patterns of cytokines upon antigenic stimulation (Brander et al., 1995Brander C. Mauri-Hellweg D. Bettens F. Rolli H.P. 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: 2670-2680PubMed Google Scholar). T lymphocytes in allergic skin lesions were identified as mainly CD8+ and when expanded in vitro they expressed cytolytic activity against epidermal keratinocytes and autologous B cells upon cross-linking of their T cell receptor (TCR) (Hertl et al., 1993Hertl M. Bohlen H. Jugert F. Merk H.F. Predominance of epidermal CD8+ T lymphocytes in bullous cutaneous reactions caused by β-lactam antibiotics.J Invest Dermatol. 1993; 101: 794-799Abstract Full Text PDF PubMed Google Scholar), pointing to the skin as the target organ of drug-induced CD8+ T cells (Hertl and Merk, 1995Hertl M. Merk H.F. Lymphocyte activation in coutaneous drug reactions.J Invest Dermatol. 1995; 105: 95s-98sAbstract Full Text PDF PubMed Scopus (114) Google Scholar); however, the majority of penicillin-specific T lymphocytes, particularly ifisolated from peripheral blood, appear to be of the CD4+ phenotype (Brander et al., 1995Brander C. Mauri-Hellweg D. Bettens F. Rolli H.P. 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: 2670-2680PubMed Google Scholar; Padovan et al., 1996Padovan E. Mauri-Hellweg D. Pichler W.J. Weltzien H.U. T cell recognition of penicillin G. structural features determining antigenic specificity.Eur J Immunol. 1996; 26: 42-48Crossref PubMed Scopus (107) Google Scholar). Despite several recent studies on drug-reactive T cells (Kalish et al., 1994Kalish R.S. La Porte A. Wood J.A. Johnson K.L. Sulfonamide-reactive lymphocytes detected at very low frequency in the peripheral blood of patients with drug-induced eruptions.J Allergy Clin Immunol. 1994; 94: 465-472Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, Mauri-Hellweg et al., 1995Mauri-Hellweg D. Bettens F. Mauri D. Brander C. Hunziker T. Pichler "WJ: Activation of drug-specific CD4+ and CD8+ T cells in individuals allergic to sulfonamides, phenytoin and carbamazepine.J Immunol. 1995; 155: 462-472PubMed Google Scholar, Zanni et al., 1997Zanni M.P. Mauri-Hellweg D. Brander C. et al.Characterization of lidocaine-specific T cells.J Immunol. 1997; 158: 1139-1148PubMed Google Scholar), questions on the identity of the molecular structure of the antigenic epitopes involved in these reactions are only beginning to be addressed. Concerning their function as haptens, β-lactam antibiotics represent a particularly convenient tool of investigation. The bicyclic thiazolidine/ β-lactam backbone is identical for all penicillins and distinguishes them from other β-lactam antibiotics, e.g., cyclosporines. On the other hand, many different side chains allow a detailed analysis of the fine specificity of recognition by penicillin-specific TCR. Considering this, numerous independent penicillin G (Pen G)-specific CD4+ T cell clones were analyzed for their capacity to proliferate in response to Pen G, penicillin V (Pen V), and ampicillin (Amp), differing exclusively in the region joining the phenyl group of the side chain with the β-lactam ring. Typical patterns of reactivity are summarized in Fig 2(A—D): the clones examined could be divided into four types, depending on their sensitivity to subtle chemical variations in the side chain of the antibiotic molecule. Among those, type I clones (Fig 2A), highly specific for Pen G, and clones of type III (Fig 2C), showing a broad cross-reaction, each with 35%, represented a majority (Padovan et al., 1996Padovan E. Mauri-Hellweg D. Pichler W.J. Weltzien H.U. T cell recognition of penicillin G. structural features determining antigenic specificity.Eur J Immunol. 1996; 26: 42-48Crossref PubMed Scopus (107) Google Scholar). The molecular epitope involved in this recognition was further deduced from the fact that not even type III clones reacted to either 6-aminopenicillenic acid or cefaclor (Fig 2E). 6-aminopenicillenic acid completely lacks a side chain whereas in cefaclor the thiazolidine ring is replaced by a six-membered ring. The most straightforward explanation of these data is that the antigenic determinant contacted by the specific TCR is formed by the contributions of both the side chain and the backbone structure of the penicillin molecule. Experimental evidence suggested that the molecular constraints in the presentation of Pen G are similar to those for nominal antigens. The penicillin-specific T cell response is MHC restricted, with a strong predominance of HLA-DR restriction for CD4+ T cell clones (Padovan et al., 1996Padovan E. Mauri-Hellweg D. Pichler W.J. Weltzien H.U. T cell recognition of penicillin G. structural features determining antigenic specificity.Eur J Immunol. 1996; 26: 42-48Crossref PubMed Scopus (107) Google Scholar). Moreover, penicillin can be presented to specific T cells by glutaraldehyde-fixed autologous B cells, implying the covalent formation of antigenic epitopes without requirement of intracellular processing (Brander et al., 1995Brander C. Mauri-Hellweg D. Bettens F. Rolli H.P. 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: 2670-2680PubMed Google Scholar; Padovan et al., 1996Padovan E. Mauri-Hellweg D. Pichler W.J. Weltzien H.U. T cell recognition of penicillin G. structural features determining antigenic specificity.Eur J Immunol. 1996; 26: 42-48Crossref PubMed Scopus (107) Google Scholar). Such information supported the idea of T cell activation as a consequence of TCR interaction with penicillin-modified peptides bound in the grove of surface MHC molecules. We indeed demonstrated the central role of hapten-modified peptides in eliciting these immune responses by using synthetic penicilloyl-peptides (Padovan et al., 1997Padovan E. Bauer T. Tongio M.M. Kalbacher 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). As shown in Table I, T cell clones reactive to Pen G presented by homozygous DRB1*0401 B cells, proliferated in response to designer peptides containing the appropriate HLA-DR anchor residues and carrying a lysine-bound penicilloyl group in specific TCR contact sites. In those experiments a precise positioning of the hapten molecule on the peptide backbone was required for optimal stimulation, and no stimulation was induced by the unmodified peptides. Moreover, the covalent modification by Pen G of peptide MCP 315-328, a natural DRB1*1101 binder (Verreck et al., 1996Verreck F.A.W. de Poel A. Drijfhout J.W. Amons R. Coligan J.E. Koning F. Natural peptides isolated from Gly86/Val86-containing variants of HLA-DR1, -DR11, DR13, and DR52.Immunogenetics. 1996; 43: 392-397Crossref PubMed Scopus (40) Google Scholar), did confer antigenic properties for Pen G reactive T cells to this sequence (Table I). The cross-reactive recognition of Pen G-modified self-peptides as well as of non-natural penicilloyl designer peptides or Pen G-modified whole presenter cells, clearly indicates a carrier independence of these reactivities.Table IEfficiency of Pen G-modified designer and natural peptides in triggering T cell proliferation of clones Rl12 and clone ES4.4PeptidesLD50 of prolifereationNameSequenceRl12ES4.4–+–+PEP1aThe 12mer polyalanine peptides PEP1, PEP2, and PEP3 contain Y in position P1 as general DR anchor (Hammer at al, 1993), and S in P6 as a DRB1*0401-specific anchor (McNicoll et al, 1995). E in position -2 was introduced to increased peptide solubility. Each peptide contains a K residue in positions corresponding to TCR contact sites (Stern et al, 1994). * indicates the position of the possible Pen G modification. +/- indicates whether or not peptide were modified with Pen G. nr, no reaction; nt, not testedE A Y A K*A A S A A Anrnrnr0.6dReaction of clone ES4.4 to PEP1-Pen G was restricted by DRB1*0401.PEP2aThe 12mer polyalanine peptides PEP1, PEP2, and PEP3 contain Y in position P1 as general DR anchor (Hammer at al, 1993), and S in P6 as a DRB1*0401-specific anchor (McNicoll et al, 1995). E in position -2 was introduced to increased peptide solubility. Each peptide contains a K residue in positions corresponding to TCR contact sites (Stern et al, 1994). * indicates the position of the possible Pen G modification. +/- indicates whether or not peptide were modified with Pen G. nr, no reaction; nt, not testedE A Y A A A K*S A A Anr10cReactions of clone Rl12 to PEP2-Pen G and PEP3-Pen G were restricted by DRB1*0401, whereas reactivity to MCP-Pen G was DRB1*1101 restricted. The efficiency of T cell stimulation is expressed as concentration of peptide (μM) inducing 50% of proliferation.nrnrPEP3aThe 12mer polyalanine peptides PEP1, PEP2, and PEP3 contain Y in position P1 as general DR anchor (Hammer at al, 1993), and S in P6 as a DRB1*0401-specific anchor (McNicoll et al, 1995). E in position -2 was introduced to increased peptide solubility. Each peptide contains a K residue in positions corresponding to TCR contact sites (Stern et al, 1994). * indicates the position of the possible Pen G modification. +/- indicates whether or not peptide were modified with Pen G. nr, no reaction; nt, not testedE A Y A A A A S A K*Anr0.1cReactions of clone Rl12 to PEP2-Pen G and PEP3-Pen G were restricted by DRB1*0401, whereas reactivity to MCP-Pen G was DRB1*1101 restricted. The efficiency of T cell stimulation is expressed as concentration of peptide (μM) inducing 50% of proliferation.nrnrMCPbThe sequence MCP 315-328, derived from the membrane cofactor protein, has been described among the natural peptide eluted from DRB1*1101 molecules (Verreck et al, 1996). For this peptide the modification occurred on all K residues present in the sequence (Kalbacher H, personal communication).V P Y R Y L Q R R K*K*K*G K*nr50cReactions of clone Rl12 to PEP2-Pen G and PEP3-Pen G were restricted by DRB1*0401, whereas reactivity to MCP-Pen G was DRB1*1101 restricted. The efficiency of T cell stimulation is expressed as concentration of peptide (μM) inducing 50% of proliferation.ntnta The 12mer polyalanine peptides PEP1, PEP2, and PEP3 contain Y in position P1 as general DR anchor (Hammer et al., 1993Hammer J. Valsasnini P. Tolba K. Bolin D. Higelin J. Takacs B. Sinigaglia F. Promiscuous and allele-specific anchors in HLA-DR-binding peptides.Cell. 1993; 74: 197-203Abstract Full Text PDF PubMed Scopus (375) Google Scholar), and S in P6 as a DRB1*0401-specific anchor (McNicoll et al., 1995McNicoll J.M. Withworth W.C. Oftung F. et al.Structural requirement of peptide and MHC for DR (α, β1*0401) -restricted T cell antigen recognition.J Immunol. 1995; 155: 1951-1963Google Scholar). E in position -2 was introduced to increased peptide solubility. Each peptide contains a K residue in positions corresponding to TCR contact sites (Stern et al., 1994Stern L.J. Brown J.H. Jardesky T.S. Gorga J.C. Urban R.G. Strominger J.L. Wiley D.C. Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide.Nature. 1994; 368: 215-221Crossref PubMed Scopus (1408) Google Scholar). * indicates the position of the possible Pen G modification. +/- indicates whether or not peptide were modified with Pen G. nr, no reaction; nt, not testedb The sequence MCP 315-328, derived from the membrane cofactor protein, has been described among the natural peptide eluted from DRB1*1101 molecules (Verreck et al., 1996Verreck F.A.W. de Poel A. Drijfhout J.W. Amons R. Coligan J.E. Koning F. Natural peptides isolated from Gly86/Val86-containing variants of HLA-DR1, -DR11, DR13, and DR52.Immunogenetics. 1996; 43: 392-397Crossref PubMed Scopus (40) Google Scholar). For this peptide the modification occurred on all K residues present in the sequence (Kalbacher H, personal communication).c Reactions of clone Rl12 to PEP2-Pen G and PEP3-Pen G were restricted by DRB1*0401, whereas reactivity to MCP-Pen G was DRB1*1101 restricted. The efficiency of T cell stimulation is expressed as concentration of peptide (μM) inducing 50% of proliferation.d Reaction of clone ES4.4 to PEP1-Pen G was restricted by DRB1*0401. Open table in a new tab As previously discussed for trinitrophenyl (TNP) responses in the mouse (Weltzien et al., 1995Weltzien H.U. Moulon C. Martin S. Padovan E. Hartmann U. Kohler J. T cell immune responses to haptens.Structural models for allergic and autoimmune reactions. Toxicol. 1995; 107: 141-151Google Scholar), and now formally proven for penicillin hypersensitivity in humans, antigenic determinants generated by haptens can derive from covalent modification of extra- or intracellular proteins as well as of MHC-associated peptides. Considering the selectivity of amino acid modifications by individual haptens, one might expect the availability of modification sites on a protein to be very limited. Three- dimensional structures and chemical factors are expected to further reduce the accessibility of target amino acids for modification. In this scenario, the direct haptenization of MHC-associated natural peptides might be more relevant than previously thought. In the case of carrier independent hapten recognition, specificity is defined mainly by the positioning of modifyable amino acids in the MHC groove, and the density of reactive epitopes for any given TCR on the surface of antigen-presenting cells might be relatively high, compared with those formed by nominal antigens. It is tempting to consider this as one of the decisive factors in defining the allergic potential of haptens. The role of minor antigenic determinants, like penicillenyl or penicillamine groups (Fig 1), in inducing penicillin-specific cellular responses, remains an open question. For a better understanding of the immunopathology of these allergic reactions it would be relevant to determine whether these structures can activate penicillin-specific T cells. From the data presented above, the analogies of the immune recognition of haptens in humans and mice are clearly evident. In both systems, the activation ofhapten-specific T cells is mediated by hapten-modified peptides. The fact that the responses of the human T cell clones studied in detail were carrier independent (Padovan et al., 1997Padovan E. Bauer T. Tongio M.M. Kalbacher 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) does not exclude the existence of TCR contacting haptenic and peptidic determinants at the same time; however, this latter way of recognition, well documented for TNP responses in the mouse (Martin and Weltzien, 1994Martin S. Weltzien H.U. T cell recognition of haptens: a molecular view.Int Arch Allergy Immunol. 1994; 104: 10-16Crossref PubMed Scopus (102) Google Scholar), could be detected only as a consequence of in vitro priming with TNP-modified peptides. A molecular modeling of penicillin recognition by specific TCR can be envisaged. It is likely that the thyazolidine ring and the phenyl group of the side chain of the molecule rotate and dispose in the space in order to be simultaneously recognized as such. The movement will be certainly influenced by the flexibility of the side chain of the penicillin derivative. The rotation may even result in the formation of a big hydrophobic group contacting particular TCR sites (Garcia et al., 1996Garcia K.C. Degano M. Stanfield R.L. et al.An αβ T cell receptor structure at 2.5A and its orientation in the TCR-MHC complex.Science. 1996; 274: 209-219Crossref PubMed Scopus (1033) Google Scholar, Garboczi et al., 1996Garboczi D.N. Ghosh P. Utz U. Fan Q.R. Biddison W.E. Wiley D.C. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2.Nature. 1996; 384: 134-140Crossref PubMed Scopus (1173) Google Scholar). As with any other lymphocytes, hapten-specific cells are also selected in the thymus in the absence of those chemical compounds. Cross- reactivity to completely irrelevant antigens, leading to selection events, might be a possible explanation; however, one could also imagine that for a TCR in that environment, the recognition of a molecular volume more than a precise chemical structure could be sufficient for the transmission of a survival signal. If this is the case a selection event might result from the interaction of hapten-specific TCR with particular amino acid sequences bound to MHC molecules forming threedimensional structures that mimic the hapten determinant (Lepoittevin and Leblond, 1997Lepoittevin J.P. Leblond I. Hapten-peptide-T cell receptor interactions: molecular basis for the recognition of haptens by T lymphocytes.Eur J Dermatol. 1997; 7: 151-154Google Scholar). The activation of CD4+ T cells by haptens plays a critical role in an allergic inflammatory immune response. The subsequent differentiation of T helper (Th) lymphocytes into Th1 or Th2 cells affects the recruitment of the many other cell types participating in the response. In both mice and men, immune activation events drive the differenti- ation of naive Th lymphocytes into two distinct subsets of effector cells, which are phenotypically and functionally different (Abbas et al., 1996Abbas A.K. Murphy K.M. Sher A. Functional diversity of helper T lymphocytes.Nature. 1996; 383: 787-793Crossref PubMed Scopus (3760) Google Scholar). Th1 cells produce cytokines associated with inflammation, such as interleukin (IL)-2 and interferon (IFN)-g, and induce cell-mediated immune responses. IL-4 and IL-5, in contrast, are characteristic for the Th2 subset, which is associated with humoral immune responses (Mossmann and Sad, 1996Mossmann T.R. Sad S. The expanding universe of T cell subsets: Th1, Th2 and more.Immunol Today. 1996; 17: 138-146Abstract Full Text PDF PubMed Scopus (3250) Google Scholar). The predominance of Th1 responses in organ-specific autoimmune diseases, like diabetis and thyroiditis, and abnormal activation of Th2 responses in atopic diseases and bacterial infections, are only some of the several examples where the immunopathology is determined by the overproduction of a polarized pattern of cytokines (Romagnani, 1994Romagnani S. Lymphokine production by human T cells in disease states.Annu Rev Immunol. 1994; 12: 227-257Crossref PubMed Google Scholar). The functional Th1/Th2 dichotomy is likely to play a role also in the allergic phenomena induced by penicillins, in particular with a prevalence of Th2 responses in immediate hypersensitivity reactions and of Th1 responses in delayed allergic phenomena. Evidence for this has been found by measuring the cytokine profiles of T cell lines from the peripheral blood of patients with penicillin-induced exanthems. Cell lines from subjects suffering from bullous exanthems, one of the clinical features of delayed type reactions, secreted high levels of IFN-g, whereas cells from patients with drug-induced urticaria produced IL-4 (Merk and Hertl, 1996Merk H.F. Hertl M. Immunologic mechanisms of cutaneous drug reactions.Seminars in Coutaneous Med Surg. 1996; 15: 228-235Crossref PubMed Scopus (40) Google Scholar). As part of the Th1/Th2 paradigm the selective differentiation of either subset takes place during T cell priming, with the contribution of several factors, e.g., antigen dose, cytokine environment, and the nature and activation status of antigen-presenting cells (Constant and Bottomly, 1997Constant S.L. Bottomly K. Induction of TH1 and TH2 CD4+ T cell responses: the alternative approches.Annu Rev Immunol. 1997; 15: 297-322Crossref PubMed Scopus (1255) Google Scholar). Once this differentiation is achieved the status is believed to be irreversible (Murphy et al., 1996Murphy E. Shibuya K. Hosken N.A. et al.Reversibility of T helper 1 and 2 populations is lost after long-term stimulation.J Exp Med. 1996; 183: 901-913Crossref PubMed Scopus (379) Google Scholar). Biochemical differences of the two subsets contribute to the irreversibility of this process. In particular, it has been shown that loss of expression of the IL-12 receptor chain in Th2 but not Th1 cells renders these cells unresponsive to IL-12 signaling, which drives Th1 proliferation (ββ et al, 1995). For some of our Pen G-specific T cell clones, we found an inducible shift between Th0 and Th2 phenotypes, even after continuous culture for over 1 y. The pattern of cytokines secreted by these clones was flexible and depended on the dose of Pen G used for stimulation. Thus, clone BAM25 was raised with a dose of Pen G of 1 mg per ml and behaves, in this condition, as a Th2 clone (Fig 3A, B ); however, the same clone simultaneously secreted IL-4 and IFN-, in the presence of lower doses (< 0.5 mg per ml) of Pen G, whereas IL-4, but not IFN-γ, was detected in the culture supernatant upon stimulation with higher Pen G concentrations (Fig 3A, B). It thus appears feasible to drive the cytokine profile even of established Pen G-reactive T cell clones, in vitro. At this stage, it is difficult to envisage simple correlations of these phenomena with therapeutic applications. Even if the effective in vivo concentration ofthe antibiotic could be determined, it will be dictated by the sensitivity of the invading microorganism rather than by immunologic side-effects. A closer correlation to possible therapeutic application might be envisaged from another kind of experiment. We have used two different penicillin derivatives, Pen V and Amp, which were not able to induce IL-4 or IFN-γsecretion of clone BAM25 (Fig 3A, B) to antagonise the effect of Pen G on the same clone. Although Amp did not influence the activity of Pen G, the Pen V derivative completely shut off the secretion of IFN-g, without modify- ing the IL-4 secretion induced by Pen G (Fig 3C, D). The capacity of this clone to proliferate and secrete IL-2 in the culture supernatant in response to Pen G, was not dependent on the concentration of antigen and was not affected in the presence of the nonantigenic derivatives Pen V and Amp (Padovan and Weltzien, manuscript in preparation). These data cannot be interpreted simply as another example of hapten-antagonism as recently described for TNP and dinitrophenyl in the mouse (Preckel et al., 1997Preckel T. Grimm R. Martin S. Weltzien H.U. Altered hapten ligands antagonize trinitrophenyl-specific cytotoxic T cell and block internalization of hapten-specific receptors.J Exp Med. 1997; 185: 1803-1813Crossref PubMed Scopus (42) Google Scholar). In our case the effect is not only specific for very defined combinations of penicillins, but also results in a highly selective inhibition of only some of the antigen- induced cellular functions. As penicillins form the major structural entity of the antigenic determinants contacting the TCR, we believe that the effects described in Fig 3(C, D) derive from changes in the early events of TCR-mediated signaling. Our finding might open new applications in immune intervention, because, theoretically, the possibility of administrating Pen G in a cocktail with other penicillins could represent a way to exploit the immense therapeutic capacity of this drug by limiting its adverse effects. The problem of drug allergy is certainly relevant for either clinical or basic science. In the clinical environment allergic phenomena to drugs are unpredictable, can induce serious diseases, and clearly restrict the choice of medication. For basic science, it is certainly fascinating to understand the molecular mechanism involved in these responses, which are of low frequency but have very serious consequences. There is a constant need for diagnostic tests to predict the individual risk of drug allergy, as well as for assays to predict allergic properties of drugs (Coleman, 1996Coleman J.W. Keeping up with drug allergy.Clin Exp Allergy. 1996; 26: 1341-1342Crossref Scopus (8) Google Scholar).