Christoph Burkhart

T-cell involvement in drug-induced acute generalized exanthematous pustulosis

Acute generalized exanthematous pustulosis (AGEP) is an uncommon eruption most often provoked by drugs, by acute infections with enteroviruses, or by mercury. It is characterized by acute, extensive formation of nonfollicular sterile pustules on erythematous background, fever, and peripheral blood leukocytosis. We present clinical and immunological data on four patients with this disease, which is caused by different drugs. An involvement of T cells could be implied by positive skin patch tests and lymphocyte transformation tests. Immunohistochemistry revealed a massive cell infiltrate consisting of neutrophils in pustules and T cells in the dermis and epidermis. Expression of the potent neutrophil-attracting chemokine IL-8 was elevated in keratinocytes and infiltrating mononuclear cells. Drug-specific T cells were generated from the blood and skin of three patients, and phenotypic characterization showed a heterogeneous distribution of CD4/CD8 phenotype and of T-cell receptor Vbeta-expression. Analysis of cytokine/chemokine profiles revealed that IL-8 is produced significantly more by drug-specific T cells from patients with AGEP compared with drug-specific T cells from patients that had non-AGEP exanthemas. In conclusion, our data demonstrate the involvement of drug-specific T cells in the pathomechanism of this rather rare and peculiar form of drug allergy. In addition, they indicate that even in some neutrophil-rich inflammatory responses specific T cells are engaged and might orchestrate the immune reaction.

Recognition of Sulfamethoxazole and Its Reactive Metabolites by Drug-Specific CD4+ T Cells from Allergic Individuals

The recognition of the antibiotic sulfamethoxazole (SMX) by T cells is usually explained with the hapten-carrier model. However, recent investigations have revealed a MHC-restricted but processing- and metabolism-independent pathway of drug presentation. This suggested a labile, low-affinity binding of SMX to MHC-peptide complexes on APC. To study the role of covalent vs noncovalent drug presentation in SMX allergy, we analyzed the proliferative response of PBMC and T cell clones from patients with SMX allergy to SMX and its reactive oxidative metabolites SMX-hydroxylamine and nitroso-SMX. Although the great majority of T cell clones were specific for noncovalently bound SMX, PBMC and a small fraction of clones responded to nitroso-SMX-modified cells or were cross-reactive. Rapid down-regulation of TCR expression in T cell clones upon stimulation indicated a processing-independent activation irrespective of specificity for covalently or noncovalently presented Ag. In conclusion, our data show that recognition of SMX presented in covalent and noncovalent bound form is possible by the same TCR but that the former is the exception rather than the rule. The scarcity of cross-reactivity between covalently and noncovalently bound SMX suggests that the primary stimulation may be directed to the noncovalently bound SMX.

The PKC inhibitor AEB071 may be a therapeutic option for psoriasis

PKC isoforms tau, alpha, and beta play fundamental roles in the activation of T cells and other immune cell functions. Here we show that the PKC inhibitor AEB071 both abolishes the production of several cytokines by activated human T cells, keratinocytes, and macrophages in vitro and inhibits an acute allergic contact dermatitis response in rats. To translate these findings into humans, single and multiple ascending oral doses of AEB071 were administered to healthy volunteers and patients with psoriasis, respectively. AEB071 was well tolerated with no clinically relevant laboratory abnormalities. Ex vivo stimulation of lymphocytes from subjects exposed to single doses of AEB071 resulted in a dose-dependent inhibition of both lymphocyte proliferation and IL2 mRNA expression. Clinical severity of psoriasis was reduced up to 69% compared with baseline after 2 weeks of treatment, as measured by the Psoriasis Area Severity Index (PASI) score. The improvement in psoriasis patients was accompanied by histological improvement of skin lesions and may be partially explained by a substantial reduction of p40+ dermal cells, which are known to mediate psoriasis. These data suggest that AEB071 could be an effective novel treatment regimen for psoriasis and other autoimmune diseases, and that AEB071 warrants long-term studies to establish safety and efficacy.

Antigenicity and immunogenicity of sulphamethoxazole: demonstration of metabolism-dependent haptenation and T-cell proliferation in vivo

Sulphamethoxazole has been associated with the occurrence of hypersensitivity reactions. There is controversy as to whether the immune response is metabolism‐dependent or ‐independent. We have therefore investigated the site of antigen formation and the nature of the drug signal presented to the immune system in vivo. Male Wistar rats were dosed with sulphamethoxazole, sulphamethoxazole hydroxylamine or nitroso sulphamethoxazole. Antigen formation on cell surfaces was determined by flow cytometry using a specific anti‐sulphamethoxazole antibody. Immunogenicity was determined by assessment of ex vivo T‐cell proliferation. Administration of nitroso sulphamethoxazole, but not sulphamethoxazole or sulphamethoxazole hydroxylamine, resulted in antigen formation on the surface of lymphocytes, splenocytes and epidermal keratinocytes, and a strong proliferative response of splenocytes on re‐stimulation with nitroso sulphamethoxazole. Rats dosed with sulphamethoxazole or sulphamethoxazole hydroxylamine did not respond to any of the test compounds. CD4+ or CD8+ depleted cells responded equally to nitroso sulphamethoxazole. The proliferative response to nitroso sulphamethoxazole was seen even after pulsing for only 5 min, and was not inhibited by glutathione. Responding cells produced IFN‐γ, but not IL‐4. Haptenation of cells by sulphamethoxazole hydroxylamine was seen after depletion of glutathione by pre‐treating the rats with diethyl maleate. Splenocytes from the glutathione‐depleted sulphamethoxazole hydroxylamine‐treated rats responded weakly to nitroso sulphamethoxazole, but not to sulphamethoxazole or sulphamethoxazole hydroxylamine. Dosing of rats with sulphamethoxazole produced a cellular response to nitroso sulphamethoxazole (but not to sulphamethoxazole or its hydroxylamine) when the animals were primed with complete Freunds adjuvant. These studies demonstrate the antigenicity of nitroso sulphamethoxazole in vivo and provide evidence for the role of drug metabolism and cell surface haptenation in the induction of a cellular immune response in the rat.

Influence of reduced glutathione on the proliferative response of sulfamethoxazole-specific and sulfamethoxazole-metabolite-specific human CD4+ T-cells

Hypersensitivity to the drug sulfamethoxazole (SMX) is thought to be a consequence of bioactivation to the hydroxylamine metabolite (SMX‐NHOH) and further oxidation to the ultimate reactive metabolite, nitroso‐sulfamethoxazole (SMX‐NO). SMX‐NO covalently modifies self proteins which in turn might be recognized as neo‐antigens by T‐cells. The antioxidant glutathione (GSH) is known to protect cells from reactive metabolites by conjugation and subsequent dissociation to SMX‐NHOH and/or SMX. To study the reactivity of T‐cells to SMX metabolites and their respective role in the generation of drug‐specific T‐cells, we analysed the effect of GSH on the response of PBMC to SMX and its metabolites SMX‐NHOH and SMX‐NO. Furthermore, we monitored the proliferative response of drug‐specific T‐cell clones in the presence or absence of GSH. We found that addition of GSH to peripheral blood mononuclear cells had no effect on the SMX‐specific response but enhanced the proliferation to SMX‐metabolites. The response of SMX‐NO‐specific T‐cell clones was abrogated when GSH was present during the covalent haptenation of antigen presenting cells (APC). Conversely, SMX‐specific T‐cell clones gained reactivity through the conversion of SMX‐NO to the parent drug by GSH. While GSH had no effect on the initial activation of T‐cell clones, it prevented covalent binding to APCs, reduced toxicity and thereby led to proliferation of drug‐specific T‐cells to non‐reactive drug metabolites. Our data support the concept that in allergic individuals T‐cells recognize the non‐covalently bound parent drug rather than APC covalently modified by SMX‐NO.

Non-covalent presentation of sulfamethoxazole to human CD4+ T cells is independent of distinct human leucocyte antigen-bound peptides

Background It has been shown that drugs comprise a group of non‐peptide antigens that can be recognized by human T cells in the context of HLA class II and that this recognition is involved in allergic reactions. Recent studies have demonstrated a MHC‐restricted but processing‐ and metabolism‐independent pathway for the presentation of allergenic drugs such as lidocaine and sulfamethoxazole (SMX) to drug‐specific T cells. However, there is little information so far on the precise molecular mechanisms of this non‐covalent drug presentation.

Molecular Basis of Drug Recognition by Specific T–Cell Receptors

In recent years the involvement of T cells in allergic reactions to drugs has been well established. However, several molecular aspects of drug recognition by specific T cells remain still unclear. This review will discuss the known pathways of drug presentations by antigen presenting cells, the recognition of MHC/peptide/drug complexes by specific T–cell receptors, and the activation mechanism of drug–specific T cells.

Pharmacodynamics in the development of new immunosuppressive drugs.

Over the past 10-20 years a number of immunosuppressive drugs, such as cyclosporine A, tacrolimus, sirolimus, or mycophenolate mofetil have been approved for clinical use and have been highly successful in preventing or delaying graft rejection. Nevertheless, there is an incessant need for better and safer drugs to improve short-term and long-term outcomes following transplantation. A number of low-molecular-weight molecules that interfere with immune cell functions are in development. These include molecules that inhibit the janus protein tyrosine kinase JAK3, compounds that alter lymphocyte trafficking (the sphingosine-1-phosphate receptor antagonist FTY720), and new malononitrilamides (FK778). All seem to show promising therapeutic potential. Among the biologic agents, there are high expectations for antibodies or recombinant chimeric molecules targeting costimulatory surface molecules or pathways involved in the migration of immune cells. The list of such targets includes the ligand pairs CD28:B7, CD154:CD40, LFA-1:ICAM-1, ICOS:B7RP-1, and VLA-4:VCAM-1. However, the clinical development of drugs for transplantation has proved to be difficult, complex, and time consuming. Therefore, newly emerging drug candidates will also demand better methods for monitoring their effecacy as well as their side effects in vivo. Pharmacokinetics (PK) and pharmacodynamics (PD) are complementary approaches used to select drugs on the basis of their in vivo efficacy as well as safety. Whereas PK monitors the handling of the drug by the body, PD focuses on the biologic effect of the drug on its target. Therefore, PD studies of in vivo efficacy are useful for clinical decisions to determine the optimal dose and type of immunosuppressant. At the preclinical stage, PD is aimed at accelerating the selection of lead compounds via PD-controlled trials in animals. Moreover, PD can help to discover new mechanisms of action for a drug or a drug candidate. However, its full potential has not been used, mainly because of laborious and time-consuming methodology. This review focuses on established and novel PD/PK approaches to assess immunosuppressive compounds in the context of new evolving drugs or drug combinations.