Development of mouse models with restricted HLA-B*57:01 presentation for the study of flucloxacillin-driven T-cell activation and tolerance in liver injury

BACKGROUND: Flucloxacillin (FLX)-induced liver injury is immune-mediated and highly associated to HLA-B*57:01 expression. Host factors leading to drug-induced liver injury are not yet well understood. OBJECTIVE: 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. METHODS: HLA-B*57:01 transgenic mice (Tg) or Tg strains with H2-K(b)D(b) knockout (Tg/KO) or H2-K(b)D(b)/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. RESULTS: 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-K(b)D(b) 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. CONCLUSIONS: 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.