Pumpens and A. Dishlers, Riga, Latvia), HBs protein (kindly provided by Rhein Biotech AG, Düsseldorf, Germany) or ovalbumin. For flow cytometry analysis, cells were stained with ethidium monoacide (Invitrogen) and anti–mCD3-V500, anti–mCD4-eFluor450, anti–mCD8-eFluor780, anti–mNK1.1-PerCP-Cy5.5,

anti–mTNFα-PE-Cy7, anti–mIL2-APC, anti–mIFNγ-PE, anti–mFoxp3-AlexaFluor647, anti–mCD62L-PE-Cy7, anti–mCD127-APC, anti–m33D1-PE, anti–mF4/80-APC, anti–mMHCII-eFluor450, anti–mNK1.1-APC, anti–mCD137-PE, respectively (eBioscience, MLN2238 price San Diego, CA). HBV multimers HBc93-100 and HBs190-197 were produced as described.17 For intracellular staining, cells were permeabilized and fixed after surface staining using the BD Cytofix/Cytoperm Kit (BD Biosciences, Heidelberg, Germany). Flow cytometrical analysis was performed on a FACSCanto II (BD Biosciences). Data are expressed as the mean and SD. Results are analyzed using the Student t test. A P value of ≤0.05 was considered significant. Infection with AdHBV but not with control AdHBV k/o induced a rapid increase of Treg frequencies (day 3) in the Alisertib datasheet liver

and subsequently (day 7) an increase in Treg numbers (Fig. 1A). Increased Treg frequencies were first observed in the liver and only at day 21 postinfection in the spleen (Fig. 1B) where we detected no antigen expression (data not shown). This indicated a local expansion of Tregs in the liver as the site of antigen expression before recruitment of additional Tregs. Enzalutamide To study Treg function during experimental AdHBV-infection, we injected DEREG mice intraperitoneally

with DTX shortly before and on 2 days following intravenous infection with AdHBV (Fig. 1C) efficiently depleting Tregs from liver and spleen in AdHBV-infected DEREG mice (Supporting Fig. 1A). Shortly after depletion (day 7), Tregs started to re-expand (Supporting Fig. 1B) and frequently lost green fluorescent protein expression, indicating selection of transgen-negative Tregs (Supporting Fig. 1C). We systematically analyzed other time points of Treg elimination, but neither depletion 1 week before nor 1 to 5 weeks after infection significantly altered any of the parameters studied here (data not shown). This led us to choose depletion of Tregs during infection with AdHBV as shown in Fig. 1C for all experiments shown. Whereas systemic Treg frequencies normalized after week 3 (data not shown), frequencies in the liver remained elevated for more than 2 months if HBV antigens were expressed (data not shown). HBV-specific T cell responses against virus-infected hepatocytes result in inflammatory liver disease and hepatocyte death, which can be detected by increased ALT activity in the serum of infected individuals. Around day 7 postinfection, serum ALT levels peaked in AdHBV-infected mice and remained elevated until day 21 (Fig. 1D).