Eight weeks after HSC transplantation, blood was taken from HIS mice to verify the level of engraftment by flow cytometry, as described elsewhere [18] . HIS mice with a good level of human reconstitution (.20% hCD45 + cells) were immunized by intramuscular route (biceps femoris) using a 29G needle, three times on weeks 14, 16 and 18 with either 100 ml of the HBV vaccine (Engerix-B, GlaxoSmithKline) or 50 ml of tetanus toxoid (TT) containing vaccine (Tetanus vaccine, The Netherlands Vaccine Institute). These amounts correspond to 1/10 of the normal human dose. Negative controls received the same volume of PBS buffer. Two weeks after the last immunization, HIS mice were exsanguinated under isofluran/oxygen narcosis. Spleens and mLN were removed aseptically and cellular suspensions were prepared. The BM cells were isolated from the femur and tibia. from Beckman Coulter; CD3 (SK7), CD4 (SK3), CD8 (SK1), CD19 (HIB19), CD38 (HIT2), CD45 (2D1 and HI30), CD45RA (HI100), CD138 (MI15), IgM (G20-127), IgD (IA6-2), IgG (G18-145) and CCR7 (3D12) from BD Biosciences; CD27 (LT27) from AbD-Serotec; CD27 (LG.7F9) from eBioscience. TT-specific B cells were also occasionally stained with PE-coupled TT, kindly provided by Dr. Andreas Radbruch (German Rheumatism Research Center, Berlin, Germany). Dead cells were excluded based on DAPI incorporation. All washings and reagent dilutions were done with PBS containing 2% FCS and 0.02% NaN 3 . Stained cells were analyzed with an LSR-II interfaced to a FACS-Diva software system (BD Biosciences). Cell sorting of B cell subsets were performed on HIS mouse BM and spleens using a FACS-Aria cell sorter interfaced to a FACS-Diva software system (BD Biosciences). For these experiments, all washings and reagent dilutions were done with 2% FCS supplemented PBS without NaN 3 .