Pathological analysis of the liver and Ishak score grading showed a significant (P = 0.0004) increase in hepatitis grade of Ad-hFTCD at 30 weeks compared to the 12-week timepoints (Fig. 2E; Supporting Fig. 4B). In this late stage of disease we observed liver fibrosis
by silver staining of liver sections. Reticular fibers of connective tissue (Gomori), periportal fibrosis bridging to neighboring portal tracts, and reticular fibers leading to the dissociation of hepatocytes were observed in 4 of 8 Ad-hFTCD-infected NOD mice (50%) up to fibrosis score 3. In contrast, no meaningful fibrosis was seen in Ad-GFP-infected (Fig. 2C). Nutlin-3a clinical trial Immunofluorescence analysis 12 weeks after infection revealed that the cellular infiltrates consisted predominantly of CD4+ T cells
and B cells. In contrast, only a few CD8+ T cells were found (Fig. find more 3A). In addition, flow cytometry analyses of intrahepatic leukocytes (IHLs) showed no differences in the gdT and abT cell compartment, including CD4+ and CD8+ subpopulations, and the natural killer T (NKT) cells (Fig. 3B-D; Supporting Fig. 5). Only NK cell numbers were significantly elevated in Ad-FTCD animals with emAIH compared to their Ad-eGFP controls (P = 0.0072). Total IHL numbers and absolute and relative numbers of the above subsets were not different between groups. In our model the average portal infiltrate size represents just 1%-2% of the liver area. As even the healthy liver is very rich in intrahepatic lymphocytes, the portal inflammation seen in our model was not sufficient to lead to a significant increase of total IHLs, which has so far just been reported in transgenic models or models with fulminant or fatal AIH due to ablation of several tolerance mechanisms. The break of humoral tolerance was demonstrated in various animal models for AIH, but T-cell responses with a break of cellular tolerance were not reported outside this website of transgenic systems. Therefore, we attempted to adoptively transfer the emAIH by different immune cells of Ad-hFTCD-infected, autoimmune hepatitis-bearing
mice. Purified CD4+ and CD8+ T-cell splenocytes as well as total splenocytes were activated with ConA and transferred into NODscid mice. Eight weeks after transfer all mice receiving activated cells from Ad-FTCD mice developed hepatitis by histopathological analysis (Fig. 4A) while no hepatitis was seen after transfer of activated cells from Ad-eGFP mice (data not shown). Encouraged by these results, naïve T-cell subpopulations were sorted from Ad-hFTCD infected NOD mice and transferred without in vitro activation. Even under these conditions animals that received CD4+ T cells developed hepatitis characterized by periportal infiltrates, which was not observed after transfer of CD8+ T (Fig. 4B).