J Hepatol

J Hepatol. that transfected B cells offered mainly endogenously synthesized core peptides. Presentation of secreted antigens from adjacent antigen-expressing cells was not enough to stimulate a core-specific T-cell response. Only poor T-cell proliferative responses were generated by activation with B-LCLs that had been pulsed beforehand with at least a 10-fold-higher amount of transfected COS cells in the form of cell lysate, suggesting that presentation of antigens released from lifeless cells in the B-LCL cultures had a minimal role. Titrating numbers of APCs, we showed that as Rabbit Polyclonal to RGAG1 few as 104 transfected B-LCL APCs were sufficient to stimulate T cells. This presentation pathway was found to be leupeptin sensitive, and it can be blocked by antibody to HLA class II (DR). In addition, expression of a costimulatory transmission by B7/BB1 on B cells was essential for T-cell activation. Hepatitis C computer virus (HCV) has been known as a major etiologic agent of posttransfusion and sporadic community-acquired non-A, non-B hepatitis. Like the other users in the family DNA or RNA, was checked by agarose gel electrophoresis. Transfection. B-LCLs were activated by activation with 10 ng of phorbol 12-myristate 13-acetate (PMA)/ml and 0.5 M calcium ionophore (A23187) for 24 h prior to Ledipasvir (GS 5885) transfection (13). After three washes with RPMI 1640 medium, 107 cells were mixed with 40 g of pCMV980 or pcDNA3/CAT in 0.8 ml of the same medium, then transferred to a 0.40-cm cuvette for electroporation using a Gene Pulser (Bio-Rad, Richmond, Calif.). The optimal conditions of 0.3 kV and 960 F were predetermined to produce maximal CAT activity at 48 h posttransfection (by an ELISA; Boehringer Mannheim). After electric pulsing, the cell suspension was transferred to 0.8 ml of the growth medium in which the cells had been growing previously. Transfected cells were allowed to grow for 48 h; then they were used either for transient expression assays by immunofluorescence (IF) and immunoblotting or for establishing stable expression cell lines. In the latter case, the cells were cultured in total medium for an additional 2 weeks in the presence of 400 g of geneticin (GIBCO BRL)/ml. The positive selected cell lines were maintained in total medium made up Ledipasvir (GS 5885) of 200 g of geneticin/ml. All transfectants were restimulated with antibody to human immunoglobulin M (IgM) (chain) [F(ab)2] (Caltag Laboratories, Burlingame, Calif.) at 10 g/ml for 24 h before they were used as APCs. COS7 cells were transfected with the same plasmids by using Lipofectin (GIBCO BRL) according to the manufacturers instructions. The Ledipasvir (GS 5885) cells were harvested at 48 h posttransfection for analyses by IF and immunoblotting or were cultured constantly Ledipasvir (GS 5885) in the selection medium in order to generate a stable expression cell collection, as mentioned above. IF and immunoblotting. Mouse monoclonal antibody (MAb) specific for HCV core protein (H-29) was a kind gift from A. Takamizawa. Mouse MAbs specific for HCV E1 (A4) and E2 (A11) were generously provided by S. M. Feinstone. For IF, the transfected cells were harvested at 48 h posttransfection, washed with phosphate-buffered saline (PBS), and fixed with chilly acetone (?20C) on glass slides. Nonspecific binding sites were blocked with normal goat serum at 20% (for B-LCLs) or 5% (for COS7 cells) in PBS made up of 0.1% saponin. The cells were incubated with anti-core antibody for 1 h, and specific binding was detected by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG F(ab)2 (Leinco Technologies Inc., Ballwin, Mo.). All procedures were carried out at room heat. Observation was performed with an Orthoplan universal large-field microscope (Leica GmbH, Wetzlar, Germany) or an LSM-GB200 confocal laser microscopy (Olympus Corp., Tokyo, Japan). For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses, the transfected cells were harvested Ledipasvir (GS 5885) at 48 h posttransfection and washed as explained above. The cell pellet was resuspended.