48 well cell culture plates

RPMI and DMEM media (Invitrogen, Carlsbad, CA, USA) were used for all mouse and human cultures, respectively. Media were supplemented with 10% FCS, L-glutamine, 2ME, HEPES and Pen/Strep; all from Invitrogen (Carlsbad, CA, USA). For bone marrow cultures, cells were isolated from the femurs of EμTCL1-Tg and age-matched WT controls. Human bone marrow cryosamples were thawed and washed in sterile PBS. Bone marrow cells were seeded at 10⁵ cells/ml in 48-well cell culture plates (BD, San Diego, CA, USA). For lymphocyte cultures, cells were seeded at 10⁶-10⁷ cells/ml in 24- or 48-well cell culture plates (BD, San Diego, CA, USA). All cultures were incubated at 37°C and 5% CO₂. Stimulatory factors were used at the following concentrations: Flt3L, 100-400ng/ml (R&D Systems, Minneapolis, MN, USA); CpG B ODN 7909, 1μM and CpG C ODN 2395, 1μM (Invivogen, San Diego, CA, USA). Optimal CpG stimulations were conducted for 6-12 hrs. In vitro neutralization of TNF and transforming growth factor beta (TGF-β) was achieved using purified anti-mouse TNF (MP6-XT22), anti-mouse TGF-β (TW7-20B9), anti-human TNF (mAb11) and anti-human/mouse TGF-β (19D8) (Biolegend, San Diego, CA, USA), all used at 1μg/ml.