An Inducible Transgenic Mouse Model for Immune Mediated Hepatitis Showing Clearance of Antigen Expressing Hepatocytes by CD8+ T Cells

Abstract

The liver has the ability to prime immune responses against neo antigens provided upon infections. However, T cell immunity in liver is uniquely modulated by the complex tolerogenic property of this organ that has to also cope with foreign agents such as endotoxins or food antigens. In this respect, the nature of intrahepatic T cell responses remains to be fully characterized. To gain deeper insight into the mechanisms that regulate the CD8+ T cell responses in the liver, we established a novel OVA_X_CreERT2 mouse model. Upon tamoxifen administration OVA antigen expression is observed in a fraction of hepatocytes, resulting in a mosaic expression pattern. To elucidate the cross-talk of CD8+ T cells with antigen-expressing hepatocytes, we adoptively transferred Kb/OVA257-264-specific OT-I T cells to OVA_X_CreERT2 mice or generated triple transgenic OVA_X CreERT2_X_OT-I mice.OT-I T cells become activated in OVA_X_CreERT2 mice and induce an acute and transient hepatitis accompanied by liver damage. In OVA_X_CreERT2_X_OT-I mice, OVA induction triggers an OT-I T cell mediated, fulminant hepatitis resulting in 50% mortality. Surviving mice manifest a long lasting hepatitis, and recover after 9 weeks. In these experimental settings, recovery from hepatitis correlates with a complete loss of OVA expression indicating efficient clearance of the antigen-expressing hepatocytes. Moreover, a relapse of hepatitis can be induced upon re-induction of cured OVA_X_CreERT2_X_OT-I mice indicating absence of tolerogenic mechanisms.This pathogen-free, conditional mouse model has the advantage of tamoxifen inducible tissue specific antigen expression that reflects the heterogeneity of viral antigen expression and enables the study of intrahepatic immune responses to both de novo and persistent antigen. It allows following the course of intrahepatic immune responses: initiation, the acute phase and antigen clearance.

Effective intrahepatic CD8+ T-cell immune responses are induced by low but not high numbers of antigen-expressing hepatocytes

“…29 Ova × CreER T2 mice carry a single copy of a loxP-flanked, inversely oriented cassette that encodes an antigenic Ova fragment fused to GFP under control of the ubiquitously active Rosa26 promoter. CreER T2 is expressed from the endogenous albumin promoter.…”

“…In the Ova × CreER T2 mice, low expression of ova sense mRNA was observed in the absence of Tam, which corresponded to a small fraction of hepatocytes expressing the antigen and appeared to result from basal Cre activity (Supplementary Figure S1b and Ref. 29). We further determined the level of sense mRNA expression in the Ova × CreER T2 mice.…”

“…28,29 Importantly, no OT-I cell activation was observed upon in vitro co-culture with non-parenchymal cells (Supplementary Figure S3a). Furthermore, early T-cell activation was restricted to the liver (Supplementary Figure S3b).…”

“…The Tam application (50 µg; Ratiopharm, Ulm, Germany), alanine aminotransferase (ALT) measurements and OT-I transfer are described elsewhere. 29 For the adoptive transfer experiments, spleen-derived naïve OT-I CD8 T+ cells were isolated from OT-I transgenic donor mice (C57Bl/6 background) using a CD8a+ T-cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol; however, phosphate-buffered saline (PBS)/1% fetal bovine serum buffer was used instead of MACS buffer. The quality and purity of the isolated OT-I cells was assessed by fluorescence-activated cell sorting analysis (Supplementary Figure S2a).…”

“…29 Quantification of Ova was performed with primer pairs 1a (5′-CAGGCACTCCTTTCAAGACC-3′) and 4a (5′-GCGGTTGAGGACAAACTCTT-3′) and normalized to albumin expression (Forward-primer: 5′-GACAAGGAAAGCTGCCTGAC-3′/Reverse-primer: 5′-TTCTGCAAAGTCAGCATTGG-3′).…”