One group of mice received 0.2 g/L doxycycline hyclate (Sigma-Aldrich) in the drinking water R788 cell line starting 2 days before transplantation and up to 8 weeks after transplantation. Doxycycline
was exchanged every 3–6 days. All animal procedures were conducted in compliance with the German animal protection laws with the protocol approved by the Landesamt für Gesundheit und Soziales, Berlin (G0099/08). Four weeks after transplantation of 5×106 transgenic pre-BI cells into irradiated Rag1−/− mice, the spleens were extracted and crushed between two frosted glass slides. About 3×105–5×105 CD19+ cells (either purified by MACS (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) or in bulk culture) or MACS-sorted IgM+ cells/well in 3 mL medium were cultivated in SF-IMDM medium supplemented with 2% FCS +/−1 μg/mL doxycycline hyclate and optionally supplemented with 1.5% IL-5 supernatant 41; 5 μg/mL anti-CD40 antibody (clone FGK-45) and/or anti-IgM antibody (M41 42); or 10 μg/mL LPS. Cells were subpassaged every 3–4 days. Cells were incubated with 2.5 μM CFSE in PBS+0.1% BSA for 7 min at 37°C and subsequently quenched with 10 vol of ice-cold medium +2% FCS for 5 min on ice. The cells were then washed twice and resuspended
in fresh medium ±1 μg/mL doxycycline or 10 μg/mL LPS. After 4 days, FACS analysis was performed. Staining of pre-B cells with AnnexinV-Cy5 one day after removal of IL-7 in the presence GSK3 inhibitor or absence of doxycycline was performed as suggested by the supplier (BD Pharmingen). Cells were stained with anti-mouse CD19 (clone ebioID3); CD93 (aa4.1); c-kit (ACK4); CD25 (eBio3C7); IgM (M41 (in-house) or II41) in the presence of antiFCγRII (in-house). All antibodies were acquired from eBioscience (San Diego CA, USA), except where otherwise stated. Cells were analyzed using an LSRII FACS (BD Biosciences) in the presence of DAPI (Carl Roth GmbH). Aggregates and doublets were gated out. Acquisition was performed using the DiVa software 6.1 (BD Biosciences). Analysis was performed using the FlowJo software (Tree Star, Ashland OR, USA). The authors thank Hermann Bujard, ZMBH,
Heidelberg, Germany for helpful advice in the use of his rtTA/tetO gene control system. Many thanks Ureohydrolase to Simon Fillatreau (DRFZ, Berlin), and Thomas Blankenstein (MDC, Berlin) for critical reading of our manuscript. The work was supported by a Reinhard Koselleck-Grant of the Deutsche Forschungsgemeinschaft ME 2764/1-1 to F.M. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“The Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is generally expressed in all EBV-associated tumours and is therefore an interesting target for immunotherapy.