The Development of In Vitro Culture Methods to Characterize Primary T-Cell Responses to Drugs
The Development of In Vitro Culture Methods to Characterize Primary T-Cell Responses to Drugs
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DOI：
10.1093/toxsci/kfs080
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发表时间：
2012-05-01
影响因子：
3.8
通讯作者：
Park, B. Kevin
中科院分区：
文献类型：
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作者：
Faulkner, Lee;Martinsson, Klara;Park, B. Kevin
药物不良反应是药物开发的主要绊脚石，那些有免疫病因学的人最难预测。我们开发了一种使用健康志愿者外周血的体外T细胞启动培养方法，以评估药物的致敏潜力。以药物代谢物亚硝基磺胺甲恶唑(SMX-NO)为模型药物过敏原，建立最佳检测条件。初始T细胞与单核细胞来源的树突状细胞以25：1的比例在药物存在下共培养8天，以扩大药物反应性T细胞的数量。然后将T细胞与新鲜的树突状细胞孵育，并使用增殖、细胞因子分泌和细胞表型读数分析药物及其抗原反应性。通过5-(和6)-羧基荧光素二乙酸酯琥珀酰亚胺酯含量和H-3-胸腺嘧啶核苷摄取量的测定，所有5名志愿者都表现出剂量依赖性的增殖。在SMX-NO存在下分裂的CD4T细胞由幼稚表型(CD45RA+)转变为记忆表型(CD45RO+)。这些记忆T细胞表达趋化因子受体CCR2、CCR4和CXCR3，表明反应人群中有T(H)1和T(H)2细胞的混合，并有归巢到皮肤的倾向。ELISpot检测到，药物刺激还与T(H)1细胞因子(干扰素-γ)和T(H)2细胞因子(IL[IL]-5和IL-13)的混合分泌有关。我们目前正在开发这种方法来研究其他药物的过敏潜力，包括那些已经建立了人类白细胞抗原等位基因与免疫反应易感性之间的关联的药物。
Adverse drug reactions represent a major stumbling block to drug development and those with an immune etiology are the most difficult to predict. We have developed an in vitro T-cell priming culture method using peripheral blood from healthy volunteers to assess the allergenic potential of drugs. The drug metabolite nitroso sulfamethoxazole (SMX-NO) was used as a model drug allergen to establish optimum assay conditions. Naive T cells were cocultured with monocyte-derived dendritic cells at a ratio of 25:1 in the presence of the drug for a period of 8 days, to expand the number of drug-responsive T cells. The T cells were then incubated with fresh dendritic cells, and drug and their antigen responsiveness analyzed using readouts for proliferation, cytokine secretion, and cell phenotype. All five volunteers showed dose-dependent proliferation as measured by 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester content and by H-3-thymidine uptake. CD4 T cells that had divided in the presence of SMX-NO had changed from a naive phenotype (CD45RA+) to a memory phenotype (CD45RO+). These memory T cells expressed the chemokine receptors CCR2, CCR4, and CXCR3 suggesting a mixture of T(H)1 and T(H)2 cells in the responding population, with a propensity for homing to the skin. Drug stimulation was also associated with the secretion of a mixture of T(H)1 cytokines (interferon gamma) and T(H)2 cytokines (interleukin [IL]-5 and IL-13) as detected by ELISpot. We are currently developing this approach to investigate the allergenic potential of other drugs, including those where an association between specific human leucocyte antigen alleles and susceptibility to an immunological reaction has been established.