Supplementary Material for: Enhancement of Drug-Specific Lymphocyte Proliferation Using CD25^hi-Depleted CD3⁺ Effector Cells

<b><i>Background:</i></b> The lymphocyte transformation test (LTT) is used for in vitro diagnosis of drug hypersensitivity reactions. While its specificity is over 90%, sensitivity is limited and depends on the type of reaction, drug and possibly time interval between the event and analysis. Removal of regulatory T cells (Treg/CD25^hi) from in vitro stimulated cell cultures was previously reported to be a promising method to increase the sensitivity of proliferation tests. <b><i>Objective:</i></b> The aim of this investigation is to evaluate the effect of removal of regulatory T cells on the sensitivity of the LTT. <b><i>Methods:</i></b> Patients with well-documented drug hypersensitivity were recruited. Peripheral blood mononuclear cells, isolated CD3⁺ and CD3⁺ T cells depleted of the CD25^hi fraction were used as effector cells in the LTT. Irrelevant drugs were also included to determine specificity. ³H-thymidine incorporation was utilized as the detection system and results were expressed as a stimulation index (SI). <b><i>Results:</i></b> SIs of 7/11 LTTs were reduced after a mean time interval of 10.5 months (LTT 1 vs. LTT 2). Removal of the CD25^hi fraction, which was FOXP3⁺ and had a suppressive effect on drug-induced proliferation, resulted in an increased response to the relevant drugs. Sensitivity was increased from 25 to 82.35% with dramatically enhanced SI (2.05 to 6.02). Specificity was not affected. <b><i>Conclusion:</i></b> Removal of Treg/CD25^hi cells can increase the frequency and strengths of drug-specific proliferation without affecting specificity. This approach might be useful in certain drug hypersensitivity reactions with borderline responses or long time interval since the hypersensitivity reaction

Supplementary Material for: Multiple Drug Hypersensitivity

<p>Multiple drug hypersensitivity (MDH) is a syndrome that develops as a consequence of massive T-cell stimulations and is characterized by long-lasting drug hypersensitivity reactions (DHR) to different drugs. The initial symptoms are mostly severe exanthems or drug rash with eosinophilia and systemic symptoms (DRESS). Subsequent symptoms due to another drug often appear in the following weeks, overlapping with the first DHR, or months to years later after resolution of the initial presentation. The second DHR includes exanthema, erythroderma, DRESS, Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN), hepatitis, and agranulocytosis. The eliciting drugs can be identified by positive skin or in vitro tests. The drugs involved in starting the MDH are the same as for DRESS, and they are usually given in rather high doses. Fixed drug combination therapies like sulfamethoxazole/trimethoprim or piperacillin/tazobactam are frequently involved in MDH, and 30-40% of patients with severe DHR to combination therapy show T-cell reactions to both components. The drug-induced T-cell stimulation appears to be due to the p-i mechanism. Importantly, a permanent T-cell activation characterized by PD-1+/CD38+ expression on CD4+/CD25^low T cells can be found in the circulation of patients with MDH for many years. In conclusion, MDH is a drug-elicited syndrome characterized by a long-lasting hyperresponsiveness to multiple, structurally unrelated drugs with clinically diverse symptoms.</p