Department of Molecular and Clinical Pharmacology, MRC Centre for Drug Safety Science, University of Liverpool, LiverpoolL69 3GE, UK.
Institute for Immunology and Infectious Diseases, Murdoch University, Murdoch, Western Australia, Australia.
Toxicol Sci. 2020 Jun 1;175(2):266-278. doi: 10.1093/toxsci/kfaa034.
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.
由于缺乏合适的模型和已知的风险因素，药物过敏反应的预测较为困难。目前已经开发出了评估免疫原性的体外初始 T 细胞致敏检测方法。然而，由于需要两批自体树突状细胞（DC）且结果不一致，以及在探索化合物特异性 T 细胞频率未定义的反应时，由于只能进行单次孔读数，因此其应用受到限制。因此，我们旨在开发一种改良但简化的检测方法，称为 T 细胞多孔检测法（T-MWA），该方法可评估药物对初始 T 细胞的特异性激活作用，同时分析诱导反应的强度以及响应的培养物数量。用模型半抗原（亚硝基磺胺甲恶唑 [SMX-NO]、班多斯基碱 [BB] 或哌拉西林 [PIP]）对初始 T 细胞与 DC 进行 2 周的共培养，共培养物在最多 48 孔中进行。在半抗原再刺激时，通过[3H]-胸苷掺入法测量 T 细胞增殖。用 SMX-NO 观察到初始 T 细胞的致敏作用，再刺激时无需 DC。SMX-NO 激活了超过 65%的培养物，其中 8.0%、30.8%和 27.2%的培养物被认为是弱反应（刺激指数 [SI]=1.5-1.9）、中度反应（SI=2-3.9）和强反应（SI>4）。当比较单独的献血时，可再现培养物的数量和反应的强度。共抑制检查点阻断增加了增殖反应的强度，但未增加响应培养物的数量。BB 检测到中等至强的致敏反应，而 PIP 仅刺激少数培养物弱增殖。在药物反应性培养物中，还鉴定出可诱导的 CD4+CD25+FoxP3+CD127low Treg。总之，T-MWA 优于现有的检测方法，通过进一步开发，它可以用于在单个平板格式中研究多个 HLA 定型供体。
