Flt3l

Cell lines were obtained from American Type Culture Collection and tumor cells from bone marrow (BM) aspirates following written informed consent under an Institutional Review Board approved protocol. Cells were cultured in RPMI1640 (Life Technologies) with 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 μg/ml) and cytokines (10 ng/ml IL7, FLT3-L and SCF; R&D Systems, Minneapolis, MN). Cell lines were cultured similarly without cytokines. OP9 murine stromal cells were expanded in DMEM with growth additives before co-culture with primary ALL blasts.

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.