Before phenotyping, cells were incubated with Fc-Block for 15 minutes (BD Biosciences, Heidelberg, Germany). After staining of dead cells with EMA (Life Technologies) and cell surface molecules, intracellular cytokines were stained using the Cytofix/Cytoperm Kit (BD Biosciences). Cells were analyzed using a FACSCanto II flow cytometer (BD Biosciences), and data were analyzed Galunisertib manufacturer using FlowJo 9.2 software (Tree Star, Inc, Ashland, OR). T cells

were isolated, stimulated, and transduced for 3 days before transfer. The cells were then harvested and washed 2 times with ice-cold PBS (180g, 4°C, 8 minutes). CAR expression was determined by flow cytometry. The cell number was adjusted to 4 × 106 CAR+ cells per animal dissolved in PBS and injected intraperitoneally. Mice were bled at indicated time points. Recipient mice were 16- to 24-week-old male animals. Groups of mice were matched for age and hepatitis B e antigen titers. Data are reported as mean values ± SEM. Groups were compared with the nonparametric Kruskal–Wallis test using Prism 5.0 (GraphPad Software, Inc, La Jolla, CA). A P value less than .05 was considered statistically significant. Additional methods are described

in Supplementary Materials and Methods. The HBV-specific chimeric antigen receptor (S-CAR) used in this study to redirect T cells contains a single-chain antibody fragment (scFv) that binds to the S domain of all 3 HBV envelope proteins (S, M, and L protein, combined as HBsAg). The scFv selleck chemicals is linked to the CD3ζ and costimulatory CD28 signaling domains (Figure 1A), providing combined activation signals to T cells when recognizing cell surface–bound HBsAg. The aim of this is

to overcome local hepatic coinhibitory signals. 11 A human carcinoembryonic Cytidine deaminase antigen (CEA)-specific CAR served as a control for antigen-independent activation of grafted T cells. After transduction of T cells with CARs using retroviral vectors ( Figure 1B), only S-CAR–transduced T cells produced high amounts of interferon (IFN)-γ and proliferated in an antigen-specific manner, that is, when cocultured with HBV-replicating human hepatoma cells but not with HBV-negative parental cells ( Figure 1C and D). We observed mobilization of the lysosomal-associated membrane protein 1 on binding of S-CAR–grafted T cells to plate-bound HBsAg ( Figure 1E), indicating release of cytotoxic granules. Notably, S-CAR–redirected T cells recognized surface antigen of the 2 most prevalent subtypes of HBV: adw and ayw ( Figure 1F). Critical for the success of adoptive cell therapy is the proper functionality of transferred T cells, ensuring that these cells survive and accumulate at the site of antigen expression.16 We compared classic IL-2 stimulation with IL-12 stimulation of T cells during in vitro expansion and retroviral CAR transduction.