3R-Project 80-01

Induction of a primary T cell mediated immune response against drugs and drug metabolites in vitro

Werner J. Pichler
Department for Rheumatology and Clinical Immunology/Allergology, Inselspital, University of Bern, 3010 Bern. Switzerland
werner.pichler@insel.ch

Keywords: human; t-cell; immunology; allergy; cell cultures: organ-specific; cell cultures: primary; reduction; replacement

Duration: 2 years Project Completion: 2005

Background and Aim
Side effects mediated by the immune system account for about 1/6 of all drug-related side effects. They are responsible for >60% of drug withdrawals, and thus represent a substantial health and economical problem. T-cells are involved in most allergic side effects to drugs. New data indicate that not only hapten like drugs, which covalently bind to peptides/proteins are immunogenic, but that also chemically inert drugs can under certain circumstances activate T cells via direct binding of drugs to the T-cell receptor for antigen (TCR) without previous hapten-carrier formation. The following questions were addressed:
a) can one generate an immune response in vitro to a chemically inert drug ?
b) what are the conditions of T cell activation by a drug and
c) how many T cells can react with a drug in sensitized individuals.

Method and Results
a) The data of this project demonstrate the successful induction of a primary T-cell immune response in vitro against a chemically inert drug using blood from healthy individuals, previously not exposed to the drug. Blood lymphocytes were stimulated by the chemically inert drug sulfamethoxazole and the protein-reactive drug-metabolite sulfamethoxazole-nitroso in the presence of IL-2. 3/10 reacted to the chemically inert compound sulfamethoxazole, while 10/10 reacted with the hapten sulfamethoxazole-nitroso. Drug reactive T-cells could be detected after 14-35d of cell culture by drug-specific proliferation or cytotoxicity, which was MHC-restricted. These cells were CD4, CD8 positive or CD4/CD8 double positive. The reactivity to the inert compound was confirmed by cloning sulfamethoxazole-specific T cells. These data confirm the ability of chemically inert drugs to stimulate certain T-cells by their TCR and may provide the opportunity to screen new drugs for their ability to interact with TCRs (Engler O. et al, 2004).
b) while previous attempts to induce a drug specific T cell response to sulfamethoxazole and other inert drugs failed if activated dendritic cells and drugs were used, the activation of T cells seemed to be the decisive difference enabling a response of T cells to inert drugs.
c) The above mentioned results raised the question how many cells are reactive with a drug in healthy and drug allergic individuals? Five patients with severe drug hypersensitivity reactions to amoxicillin, vancomycin, sulfamethoxazole and carbamazepine were analysed for the presence of circulating memory T-cells able to be stimulated by the drug. In addition 5 healthy controls without known drug hypersensitivity were analysed. Using ELISPOT with 4 cytokine determinations and CSFE staining for identifying drug reactive cells we found that between 1:250 - 1:3000 CD4 +T-cells of patients with well documented drug allergy reacted with the incriminated drug – which was equal or higher than the simultaneously detected reactivity of T-cells with tetanus toxoid. This reactivity could be found up to 12 years after the original reaction and persisted in spite of the strict avoidance of the drug. No detectable reactivity was found to unrelated drugs or in non allergic individuals (Beeler A et al, 2006).

Conclusions and Relevance for 3R
The stimulation of T-cells via TCR seems to be an important cause of unexpected drug hypersensitivity reactions and are usually not detected by animal experiments. The data show a possible in vitro method to detect such reactions. For future in vitro tests it is important that only pre-activation of T-cells (stimulated with IL-2) and a rather long cell culture duration is necessary to lead to a detectable response to this pharmacological stimulation of T-cells via TCR. In future, this system might be able to replace certain tests aimed to characterize the sensitizing potential of a drug that are currently conducted in animals (e.g. local or popliteal lymph node assays).
The analysis of precursor frequencies of drug reactive T-cells illustrates that non allergic donors do not harbour such cells in a detectable amount. However, drug allergic reactions have lead to a massive expansion of such T-cells – which seem to persist for a long time. Tests to improve the detection of drug-specific T-cells may therefore rely initially on the characterization of such T-cells from drug allergic donors.

References
1) Gerber B, Pichler WJ. (2004) Cellular mechanisms of T cell mediated drug hypersensitivity. Curr. Op. Immunol.: 16:732-737 (Review article).

2) Engler, O.B., Strasser I., Naisbitt, D.J., Cerny A. and Pichler W.J. (2004) A chemically inert drug can stimulate T cells in vitro by their T cell receptor and induce cytokine secretion, proilferation and cytotoxicity. Toxicology, 197: 47-56.

3) Beeler A, Engler O, Gerber B, Pichler WJ. (2006) Long lasting reactivity and high frequency of drug-specific T-cells after severe systemic drug hypersensitivity reactions. J Allergy Clin Immunol, 117(2): 455-62.