Human serum

The bidirectional SIN.LV.PGK.BACH2 and SIN.LV.PGK.STAT5B plasmid constructs were generated using Gateway Technology (for details refer to Supplementary Methods). All concentrated VSV.G-pseudotyped LV stocks were produced by transient transfection of 293 T cells (CRL-3216, ATCC), as previously reported^{7 (link)}.
For transduction, Naïve T lymphocytes were pre-activated for 24 h in X-VIVO (Lonza), 5% human serum (Euroclone), in presence of anti-CD3/CD28 activating beads (Dynabeads Human T cell activator CD3/CD28, Life Technologies). Treg and Teff cells were pre-activated for 24 h in X-VIVO (Lonza), 5% human serum (Euroclone), in presence of Human Treg Expander beads (Life Technologies). Cells were infected with the indicated LV at MOI 20.

Primary MLL-AF9-expressing cells were obtained from BM samples of diagnosed AML pediatric patients stored in the BioBank of the laboratory of Pediatric Hematology of the University Hospital of Padua, (Italy) according to the guidelines of the local ethics committee. Initial AML xenografts were established by tail vein injection with 8 × 10⁶ primary cells suspended in 300 μl of PBS in 6- to 8-week-old NSG mice, which were purchased from Charles River (Wilmington, MA, USA). All animal experiments were performed in accordance with institutional guidelines and established protocols [53 (link)]. Engraftment was monitored by weekly blood collections and flow cytometry analysis with antihuman CD45 (BD Biosciences). The engraftment rate was defined by the number of days required for the transplanted human CD45+ cells to reach at least 20% in the peripheral blood. Human leukemic cells from the spleens of engrafted mice were collected and cultivated in RPMI supplemented with 10% Human serum (Euroclone), antibiotics, and cytokines SCF, FLT-3L and TPO (40 ng/ml for each), IL-3 and IL-6 (20 ng/ml for each). (All cytokines were obtained from Inalco, Milan, Italy). For ex vivo experiments, two independent biological replicates were performed.

Leukemic cell lines NALM-6 and BV173 were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 (BioWhittaker) supplemented with 10% FBS (Lonza), 100 IU/mL penicillin/streptomycin, and glutamine. The ALL-CM cell line was provided by Fred Falkenburg (Leiden University Medical Center, Leiden, The Netherlands) and maintained in culture in X-VIVO (Lonza) with 3% human serum (Euroclone) and 100 IU/mL penicillin/streptomycin. For in vivo experiments, the NALM-6 cell line was transduced with a lentiviral vector encoding the secreted Lucia luciferase (Lucia⁺NGFR⁺ NALM-6), as previously reported (57 (link)).