Jacqueline Adam

Delayed drug hypersensitivity: models of T‐cell stimulation

Drug-induced hypersensitivity reactions can cause a variety of serious diseases by involving drug-specific T-cells. Many of these reactions have been explained by the hapten concept, which postulates that small chemical compounds need to bind covalently to proteins to be recognized by the immune system. Due to their chemical reactivity, haptens stimulate the innate immunity by binding covalently to endogenous proteins and form so called hapten-carrier complexes, which are antigenic and induce T-cell responses. In recent years, a new concept has been developed since drug-induced hypersensitivity reactions were also observed with chemically unreactive drugs. This concept implies direct and reversible interactions of the drug between T-cell receptors (TCR) and major histocompatability complex (MHC) molecules. Therefore it was termed pharmacological interactions with immune receptors (p-i concept). Early observations on drug reacting T-cell clones (TCC) let believe that drugs bind first to the T-cell receptor since HLA molecules could be exchanged without affecting the drug reactivity. However, MHC molecules were always required for full activation of TCC. According to its strong HLA-B*5701 association, recent data on abacavir suggest that a drug could first bind to the peptide binding groove of the MHC molecule. The thereby modified HLA molecule can then be recognized by specific T-cells. Consequently, two types of reactions based on the p-i mechanism may occur: on the one hand, drugs might preferentially bind directly to the TCR, whereas in defined cases with strong HLA association, drugs might bind directly to the MHC molecule.

HLA Haplotype Determines Hapten or p-i T Cell Reactivity to Flucloxacillin

Drug-induced liver injury (DILI) is a main cause of drug withdrawal. A particularly interesting example is flucloxacillin (FLUX)-DILI, which is associated with the HLA-B*57:01 allele. At present, the mechanism of FLUX-DILI is not understood, but the HLA association suggests a role for activated T cells in the pathomechanism of liver damage. To understand the interaction among FLUX, HLA molecules, and T cells, we generated FLUX-reacting T cells from FLUX-naive HLA-B*57:01+ and HLA-B*57:01− healthy donors and investigated the mechanism of T cell stimulation. We found that FLUX stimulates CD8+ T cells in two distinct manners. On one hand, FLUX was stably presented on various HLA molecules, resistant to extensive washing and dependent on proteasomal processing, suggesting a hapten mechanism. On the other hand, in HLA-B*57:01+ individuals, we observed a pharmacological interaction with immune receptors (p-i)–based T cell reactivity. FLUX was presented in a labile manner that was further characterized by independence of proteasomal processing and immediate T cell clone activation upon stimulation with FLUX in solution. This p-i–based T cell stimulation was restricted to the HLA-B*57:01 allele. We conclude that the presence of HLA-B*57:01 drives CD8+ T cell responses to the penicillin-derivative FLUX toward nonhapten mechanism.

Avidity determines T-cell reactivity in abacavir hypersensitivity.

The antiretroviral drug abacavir (abc) elicits severe drug hypersensitivity reactions in HLA‐B*5701+ individuals. To understand the abc‐specific activation of CD8+ T cells, we generated abc‐specific T‐cell clones (abc‐TCCs). Abc reactivity could not be linked to the metabolism and/or processing of the drug, since abc metabolizing enzymes were not expressed in immune cells and inhibition of the proteasome in APCs did not affect TCC reactivity. Ca2+ influx assays revealed different reactivity patterns of abc‐TCCs. While all TCCs reacted to abc presented on HLA‐B*5701 molecules, a minority also reacted immediately to abc in solution. Titration experiments showed that the ability to react immediately to abc correlated significantly with the TCR avidity of the T cells. Modifications of soluble abc concentrations revealed that the reactivity patterns of abc‐TCCs were not fixed but dynamic. When TCCs with an intermediate TCR avidity were stimulated with increasing abc concentrations, they showed an accelerated activation kinetic. Thus, they reacted immediately to the drug, similar to the reaction of TCCs of high avidity. The observed immediate activation and the noninvolvement of the proteasome suggest that, in contrast to haptens, abc‐specific T‐cell stimulation does not require the formation of covalent bonds to produce a neo‐antigenic determinant.

Human leukocyte antigens (HLA) associated drug hypersensitivity: consequences of drug binding to HLA.

Recent publications have shown that certain human leukocyte antigen (HLA) alleles are strongly associated with hypersensitivity to particular drugs. As HLA molecules are a critical element in T‐cell stimulation, it is no surprise that particular HLA alleles have a direct functional role in the pathogenesis of drug hypersensitivity. In this context, a direct interaction of the relevant drug with HLA molecules as described by the p‐i concept appears to be more relevant than presentation of hapten‐modified peptides. In some HLA‐associated drug hypersensitivity reactions, the presence of a risk allele is a necessary but incomplete factor for disease development. In carbamazepine and HLA‐B*15:02, certain T‐cell receptor (TCR) repertoires are required for immune activation. This additional requirement may be one of the ‘missing links’ in explaining why most individuals carrying this allele can tolerate the drug. In contrast, abacavir generates polyclonal T‐cell response by interacting specifically with HLA‐B*57:01 molecules. T cell stimulation may be due to presentation of abacavir or of altered peptides. While the presence of HLA‐B*58:01 allele substantially increases the risk of allopurinol hypersensitivity, it is not an absolute requirement, suggesting that other factors also play an important role. In summary, drug hypersensitivity is the end result of a drug interaction with certain HLA molecules and TCRs, the sum of which determines whether the ensuing immune response is going to be harmful or not.

Abacavir Induced T Cell Reactivity from Drug Naïve Individuals Shares Features of Allo-Immune Responses

Abacavir hypersensitivity is a severe hypersensitivity reaction which occurs exclusively in carriers of the HLA-B*57∶01 allele. In vitro culture of PBMC with abacavir results in the outgrowth of abacavir-reacting CD8+ T cells, which release IFNγ and are cytotoxic. How this immune response is induced and what is recognized by these T cells is still a matter of debate. We analyzed the conditions required to develop an abacavir-dependent T cell response in vitro. The abacavir reactivity was independent of co-stimulatory signals, as neither DC maturation nor release of inflammatory cytokines were observed upon abacavir exposure. Abacavir induced T cells arose in the absence of professional APC and stemmed from naïve and memory compartments. These features are reminiscent of allo-reactivity. Screening for allo-reactivity revealed that about 5% of generated T cell clones (n = 136) from three donors were allo-reactive exclusively to the related HLA-B*58∶01. The addition of peptides which can bind to the HLA-B*57∶01-abacavir complex and to HLA-B*58∶01 during the induction phase increased the proportion of HLA-B*58∶01 allo-reactive T cell clones from 5% to 42%. In conclusion, abacavir can alter the HLA-B*57∶01-peptide complex in a way that mimics an allo-allele (‘altered self-allele’) and create the potential for robust T cell responses.

NKp46+ cells express granulysin in multiple cutaneous adverse drug reactions.

To cite this article: Schlapbach C, Zawodniak A, Irla N, Adam J, Hunger RE, Yerly D, Pichler WJ, Yawalkar N. NKp46+ cells express granulysin in multiple cutaneous adverse drug reactions. Allergy 2011; 66: 1469–1476.

Clathrin heavy chain plays multiple roles in polarizing the Drosophila oocyte downstream of Bic-D

Bicaudal-D (Bic-D), Egalitarian (Egl), microtubules and their motors form a transport machinery that localizes a remarkable diversity of mRNAs to specific cellular regions during oogenesis and embryogenesis. Bic-D family proteins also promote dynein-dependent transport of Golgi vesicles, lipid droplets, synaptic vesicles and nuclei. However, the transport of these different cargoes is still poorly understood. We searched for novel proteins that either mediate Bic-D-dependent transport processes or are transported by them. Clathrin heavy chain (Chc) co-immunopurifies with Bic-D in embryos and ovaries, and a fraction of Chc colocalizes with Bic-D. Both proteins control posterior patterning of the Drosophila oocyte and endocytosis. Although the role of Chc in endocytosis is well established, our results show that Bic-D is also needed for the elevated endocytic activity at the posterior of the oocyte. Apart from affecting endocytosis indirectly by its role in osk mRNA localization, Bic-D is also required to transport Chc mRNA into the oocyte and for transport and proper localization of Chc protein to the oocyte cortex, pointing to an additional, more direct role of Bic-D in the endocytic pathway. Furthermore, similar to Bic-D, Chc also contributes to proper localization of osk mRNA and to oocyte growth. However, in contrast to other endocytic components and factors of the endocytic recycling pathway, such as Rabenosyn-5 (Rbsn-5) and Rab11, Chc is needed during early stages of oogenesis (from stage 6 onwards) to localize osk mRNA correctly. Moreover, we also uncovered a novel, presumably endocytosis-independent, role of Chc in the establishment of microtubule polarity in stage 6 oocytes.

The involvement of specific t cells in the pathogenesis of metamizole-induced agranulocytosis

Background Non-chemotherapy related, drug-induced agranulocytosis is a rare idiosyncratic reaction, which may be fatal. Along with betalactames, the analgesic metamizole is reported to occasionally cause agranulocytosis. The disease results in a severe reduction of granulocytes rendering affected patients susceptible to bacterial and fungal infections. The pathogenesis of drug-induced agranulocytosis is complex as non-immune (mainly toxic) and immune mechanisms might be involved. Aim The identification and characterization of metamizolespecific T cells in patients with drug-induced agranulocytosis by generating metamizole-specific T cell lines. Methods PBMCs from metamizole-allergic and metamizole-tolerant subjects were induced with 100ug/ml metamizole. Cultures were supplemented with IL-2 and were restimulated every 14 days. Drug-specific cell activation was determined by flow cytometry after a 6h restimulation phase. Results After two restimulation rounds, metamizole-specific T cells were identified in a metamizole-allergic and in a metamizole-tolerant individual. In both cases, CD8+ T cells but no CD4+ cells reacted to the drug. Reactive cells secreted IFN and upregulated CD107a upon drug exposure. Interestingly, T cells were activated exclusively by metamizole in solution. Autologous antigen presenting cells incubated overnight in 100ug/ml metamizole prior to restimulation failed to activate CD8+ T cells. Furthermore, metamizole-specific T cells from both donors, allergic and tolerant, were self-reactive, i.e. reacted to the drug even in the absence of antigen presenting cells. Conclusion