Characterize in vivo immune mechanisms determining the development of CD8+ T cells reactive to FLX in animals expressing the risk human leukocyte antigen (HLA) allotype.
HLA-B∗57:01 transgenic mice (Tg) or Tg strains with H2-KbDb knockout (Tg/KO) or H2-KbDb/PD-1 double knockout (Tg/DKO) were treated with drug and/or anti-CD4 antibody. Drug-induced liver injury was evaluated on the basis of liver enzyme and histologic changes at day 10 of treatment. FLX-reactive CD8+ T cells were characterized in vitro by release of effector molecules on drug restimulation, gene expression, and flow cytometry analysis, and functionality tested for hepatic cytotoxicity.
CD8+ T-cell responses to FLX in Tg were dependent on both HLA and mouse major histocompatibility complex I presentation and in vivo priming. Eliminating H2-KbDb in Tg/KO to allow exclusive presentation of FLX by HLA resulted in a less robust drug-specific CD8+T-cell response unless CD4+ cells, including regulatory T cells, were depleted. Treatment of Tg/KO with anti-CD4 antibody and FLX led to subclinical liver inflammation associated with an increase in PD1+CD8+ T cells in the lymphoid organs and liver. Impaired PD-1 expression in Tg/DKO led to liver histopathologic and transcriptional alterations but without hepatic enzyme elevations. Moreover, effector lymphocytes accumulated in the liver and showed FLX-dependent hepatic cytotoxicity in vitro when tolerogenic liver cells were depleted.
In our in vivo models, FLX primes CD8+ T cells to recognize drug presented by HLA-B∗57:01 and murine major histocompatibility complex I. HLA-B∗57:01-dependent CD8+ T-cell reaction to FLX is limited by the presence of CD4+ cells, presumably regulatory T cells, and PD-1 expression. Tolerogenic hepatic cells limit clinical disease through PD-L1 or additional unexplored mechanisms.
目的:表征体内免疫机制,确定表达风险HLA同种异型的动物中与FLX反应的CD8+T细胞的发育。
方法:用药物和/或抗CD4抗体(aCD4Ab)处理具有H2-KbDb敲除(Tg/KO)或H2-KbDb/PD-1双敲除(Tg/DKO)的HLA-B*57:01转基因小鼠(Tg)或Tg品系。在治疗第10天基于肝酶和组织学变化评价DILI。FLX反应性CD8+T细胞在体外通过在药物再刺激时释放效应分子来表征,基因表达和流式细胞术分析,和功能测试肝细胞毒性。
结果:在Tg中对FLX的CD8+T细胞应答依赖于HLA和小鼠MHC-I呈递和体内引发。消除Tg/KO中的H2-KbDb以允许HLA独家呈递FLX导致较不稳健的药物特异性CD8+T细胞应答,除非CD4+细胞,包括调节性T细胞(Treg),耗尽了。用aCD4Ab和FLX治疗Tg/KO导致与淋巴器官和肝脏中PD1+CD8+T细胞增加相关的亚临床肝脏炎症。Tg/DKO中PD-1表达受损导致肝脏组织病理学和转录改变,但没有肝酶升高。此外,当致耐受性肝细胞耗尽时,效应淋巴细胞在肝脏中积累并在体外显示FLX依赖性肝细胞毒性。
结论:在我们的体内模型中,FLX启动CD8+T细胞识别HLA-B*57:01和鼠MHC-I呈递的药物。HLA-B*57:01依赖性CD8+T细胞对FLX的反应受到CD4+细胞存在的限制,大概是Treg,和PD-1表达。致耐受性肝细胞通过PD-L1或其他未探索的机制限制临床疾病。