Hil 3

Rag^−/−γc^−/− mice (3–5 days old) received sublethal irradiation and were injected intra-hepatically with 2 × 10⁵ human CB CD34⁺ HSC only or an equal number of Tregs. Between week 1 and 3, mice were injected i.p. weekly with hIL-15 (15 ng; R&D Systems), hIL-7 (30 ng, Prospec), hSCF (30 ng; Prospec), hFLT-3 (15 ng; Prospec), and hIL-3 (75 ng; only week 1–2; R&D Systems). hIL-15 (75 ng) was added between week 4–5. hIL-15 (2.5 μg) and hIL-15Rα-Fc (7.5 μg; R&D Systems) between weeks 6–9. NK cell differentiation was then analyzed in the spleen and BM of the mice 10 weeks after injection. Animal experiments were performed according to the recommendations of the UK Home Office Regulations, protocols were approved by the UK Home Office (project license 80/2456).

MDS-L cells were maintained in IMDM/Ham's F-12 (50:50) medium supplemented with 12% fetal bovine serum (Atlanta Biologicals), 5 μg/ml apotransferrin, 50 μM 2-mercaptoethanol (2-ME) and 20 ng/ml of human IL-3 (PeproTech). The cell line was authenticated by expression of cell surface markers as previously described [19 (link)]. Human primary bone marrow cells were obtained according to Institutional IRB guidelines. Bone marrow mononuclear cells were isolated by Ficoll-paque^™ plus (GE Healthcare) and maintained in RPMI medium supplemented with 50 μM 2-mercaptoethanol, 1 μM sodium pyruvate and 10% fetal bovine serum (FBS). Human primary cells were obtained in accordance with the Declaration of Helsinki and approval from the institutional review board, protocol # 88-00241. MDS/AML samples were obtained from Leukemia Tissue Bank of the Ohio State University Comprehensive Cancer Center (OSU CCC). MDS/AML cells were thawed and maintained in RPMI medium supplemented with 10% fetal bovine serum and 10 ng/ml hIL-3, GM-CSF and stem cell factor (R&D Systems). All media were purchased from GIBCO-Life Technologies.

Patient samples, containing at least 80% of blasts, were co-cultured with human stromal cells (HS-5 (ACC-441) kindly provided by Helena Boutzen, CRCT Team 18, Toulouse, France) in IMDM (Gibco) supplemented with 15% BIT (Stem Cell Technologies, Vancouver, BC, Canada), 100 units/ml penicillin and streptomycin (Invitrogen), 5 μM β-mercaptoethanol (Invitrogen), 1 mM pyruvate (Sigma), MEM 1x (Sigma), 100 ng/mL DNase (MP Biomedicals, Solon, OH, USA), 10 ng/mL hIL-3, 100 ng/mL hSCF, and 10 ng/ml hTPO (all from R&D Systems Inc, Minneapolis, MN, USA). All the samples were then processed for treatment with the different reagents (IRC-083864 or NSC-95397).