Moflo system

293T cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (HyClone) containing 10% fetal bovine serum, 100 Um L⁻¹ penicillin, 100 µg mL⁻¹ streptomycin and 292 µg mL⁻¹ l-glutamine (Gibco). Rhesus macaque peripheral blood mononuclear cells (PBMC) were separated over Ficoll gradient and cultured at 2.5×10⁶ cells mL⁻¹ in RPMI containing 10% fetal bovine serum, 100 U mL⁻¹ penicillin, 100 µg mL⁻¹ streptomycin and 292 µg mL⁻¹ l-glutamine. 24 hours prior to infection with vector, cells were activated with 100 IU ml⁻¹ IL-2 and 5 µg mL⁻¹ Phytohaemagglutinin (PHA). 1000 TCID₅₀ JRFL pseudotyped vector was added to activated PBMC. Infected cells were sorted for mCherry expression on a Beckman Coulter MoFlo system.

8227 cells were stained with CD34–APC-Cy7 (BioLegend) and CD38–PE-Cy7 (BD Biosciences) and washed with PBS before sorting CD34⁺CD38⁻ and CD34⁻CD38⁺ cells. Cells were FACS-sorted on a MoFlo system (Beckman Coulter). Primary AML specimens were stained with BB515–CD45 (BD Biosciences, 564585) to identify the blast population, PE–CD19 (BD Biosciences, 555413) and PE-Cy7–CD3 (BD Biosciences, 557749) to exclude the lymphocyte populations, 4,6-diamidino-2-phenylindole (DAPI; EMD Millipore, 278298) as a dead-cell marker and CellROX deep red (Thermo Fisher Scientific, C10422), and sorted using a Sony SH-800 system. ROS-low LSCs were identified as the cells with the 20% lowest ROS levels and the ROS-high blasts were identified as the cells with the highest 20% ROS levels, as recently described in detail^{14 (link)}.

CD4⁺ cells were isolated from lymph nodes of C57BL/6 mice by negative selection using anti-CD8 (clone KT1.5) and anti-MHC class II (clone M5/114) antibodies followed by sheep anti rat IgG Dynabeads (Invitrogen). Th0 and iTreg cells were cultured for 5 days in RPMI, 10% FCS, 50 μM βME (Invitrogen), 1% Pen-strep in plates previously coated with anti-CD3 and anti-CD28 antibodies (0,05–5 μg/ml). For iTreg induction TGFβ1 (2,5 μg/ml, R&D) and IL2 (20 ng/ml, R&D) were added to the medium. After 5 days, cells were harvested and co-cultured with satellite cells obtained from Pax7-ZsGreen mice. Muscles from Pax7-ZsGreen mice were dissected and dissociated by enzymatic digestion with collagenase type V (0.5 mg/ml, Sigma Aldrich) and dispase (3.5 mg/ml, Invitrogen) at 37°C for 40 min. On the same day naive T cells (CD4⁺) were isolated from lymph nodes and natural Treg (nTreg) were sorted from FOXP3-GFP mice (CD3⁺ GFP⁺ cells). FACS sorting was performed using the MoFLo system (Beckman Coulter). Satellite cells and T cells were co-cultured at a 1: 25-50-100-200 ratio in proliferating medium (F10, 20% horse serum, penicillin 100 U/ml, streptomycin 100 μg/ml, gentamycin 50 μg/ml, Gibco, with bFGF 5ng/ml, Sigma, added daily).

C57BL/6 mice were i.p. injected with 10 µg (or 30 µg) αDEC205-Ova, αDCIR2-Ova, αFcγRIIB-Ova (30 µg), αFcγRIV-Ova, or 30 µg isotype-Ova control antibodies. 12 h later, mice were sacrificed, and single-cell suspensions from spleens were positively enriched for a mixture of CD11c⁺ and CD115⁺ cells, as well as CD19⁺ B cells by MACS beads (Miltenyi Biotec). CD11c⁺CD115⁺ cells were further separated by cell sorting on a FACS-Aria-II or a MoFlo system (Beckman Coulter) into lin⁻ (CD3⁻CD19⁻NKp46⁻Ly6G⁻Siglec-H⁻) CD11b^highCD11c^lowLy6C^high, lin⁻CD11b^highCD11c^int Ly6C^low, lin⁻CD11b^negCD11c^highCD8⁺, and lin⁻CD11b^posCD11c^highCD8⁻ cells. Cell sort purified cells and CD19⁺ MACS bead–enriched B cells were irradiated (3 Gy) and co-cultured with positive MACS bead–enriched CD8⁺ OT-I or CD4⁺ OT-II T cells. 16 h (OT-I) or 40 h (OT-II) later, ³H-thymidine was added to the cultures. T cell proliferation was assessed by measurement of ³H-thymidine incorporation 24 h later.