Development of an Improved T-cell Assay to Assess the Intrinsic Immunogenicity of Haptenic Compounds

Abstract

The prediction of drug hypersensitivity is difficult due to the lack of appropriate models and known risk factors. In vitro naïve T-cell priming assays that assess immunogenicity have been developed. However, their application is limited due requirements for 2 batches of autologous dendritic cells (DC) and inconsistent results; a consequence of single well readouts when exploring reactions where compound-specific T-cell frequency is undefined. Hence, we aimed to develop an improved, but simplified assay, termed the T-cell multiple well assay (T-MWA), that permits assessment of drug-specific activation of naïve T cells, alongside analysis of the strength of the induced response and the number of cultures that respond. DC naïve T-cell coculture, depleted of regulatory T cells (Tregs), was conducted in up to 48 wells for 2 weeks with model haptens (nitroso sulfamethoxazole [SMX-NO], Bandrowski's base [BB], or piperacillin [PIP]). Cultures were rechallenged with hapten and T-cell proliferation was measured using [3H]-thymidine incorporation. Priming of naïve T cells was observed with SMX-NO, with no requirement for DC during restimulation. Greater than 65% of cultures were activated with SMX-NO; with 8.0%, 30.8%, and 27.2% characterized as weak (stimulation index [SI] =1.5-1.9), moderate (SI = 2-3.9), and strong responses (SI > 4), respectively. The number of responding cultures and strength of the response was reproducible when separate blood donations were compared. Coinhibitory checkpoint blockade increased the strength of the proliferative response, but not the number of responding cultures. Moderate to strong priming responses were detected with BB, whereas PIP stimulated only a small number of cultures to proliferate weakly. In drug-responsive cultures inducible CD4+CD25+FoxP3+CD127low Tregs were also identified. To conclude, the T-MWA offers improvements over existing assays and with development it could be used to study multiple HLA-typed donors in a single plate format.

Keywords: drug hypersensitivity; human; immune system.