Rhgm csf

Buffy coats from healthy donors (Centro de Transfusión de la Comunidad de Madrid, Spain) were used to obtain peripheral blood mononuclear cells by density gradient centrifugation with Lymphocytes Isolation Solution (Rafer). Monocytes were isolated by positive magnetic separation using CD14 immunomagnetic beads (Miltenyi Biotec). To generate DCs, CD14⁺ cells (10⁶/mL) were cultured for 5–6 days in RPMI-1640 with 10% FBS plus 20 ng/mL rhGM-CSF and 20 ng/mL rhIL-4 (Invitrogen, Termo Fisher Scientific), and half of the medium was replaced by fresh medium with cytokines every 2 days. CD14⁺ monocytes (5 × 10⁵/mL) were also cultured for 3–5 days with 5 ng/mL rhGM-CSF or 10 ng/mL of rhM-CSF (ImmunoTools GmbH) to obtain M1 or M2 MØs, respectively, and in both cases the same concentration of cytokines was added every 2 days. To study the effects of leukaemia-derived soluble factors, monocytes were differentiated to DCs and M1 MØs in the presence of 50% conditioned media from control and BMP4-transduced Nalm-6 cells.

Monocytes (CD14+ cells) were isolated from buffy coats of healthy donors after informed consent. Blood was diluted in HBSS 1× (Gibco, ThermoFisher Scientific) and centrifuged. The middle layer was diluted in HBSS 1×, and peripheral blood mononuclear cells (PBMCs) were isolated using Histopaque^®-1077 (Sigma-Aldrich) density gradient centrifugation. Isolated PBMCs were resuspended in RPMI (ThermoFisher Scientific) supplemented with 10% FBS, 1 mM pyruvate, 2 mM glutamine and 10 μg mL⁻¹ ciprofloxacin (all from Sigma-Aldrich), and were allowed to adhere on culture plates for 1–2 h. Non-adherent cells were washed, and adherent monocytes were then cultured in RPMI. Monocytes were differentiated to Mφ1 or Mφ2 macrophages by adding 5 ng mL⁻¹ of recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF, Invitrogen, Waltham, MA, USA) or 20 ng mL⁻¹ recombinant human macrophage-colony stimulating factor (rhM-CSF, Invitrogen), respectively. Cytokine stimulation was repeated on day 3. On day 5, 10 ng mL⁻¹ of LPS (Invitrogen) and 20 ng mL⁻¹ of IFNɣ (R&D Systems, Minneapolis, MN, USA) were added to the Mφ1 macrophages, whereas 10 ng mL⁻¹ of LPS and 40 ng mL⁻¹ of IL4 (PeproTech, London, UK) were added to the Mφ2 macrophages. Under Mφ1 conditions, 250 nM or 50 μM of LIPINOVA^® were added on days 0, 3, and 5 of the differentiation protocol.

For 35 patients, 1 x 10⁵/ml low-density mononuclear cells were obtained from the bone marrow by density centrifugation over a Ficoll-Hypaque gradient. The cells were plated in multiwell plates in Iscove’s modified Dulbecco’s medium (Sigma Aldrich, St. Louis MO, USA) containing 30% fetal calf serum (Sigma Aldrich), 0.3% noble agar, 10 ng/ml rhIL-3 (Invitrogen CA, USA), and 20 ng/ml granulocyte-macrophage colony-stimulating factor (rhGM-CSF, Invitrogen) or 10 ng/ml granulocyte colony-stimulating factor (rhG-CSF). After 14 days, single aggregates of more than 40 cells were scored as CFU-GM or colony forming units–granulocyte (CFU-G). The results were compared with the historical pediatric controls of our laboratory (healthy bone marrow donors), cultured under the same conditions.

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood (Biology Speciality Corporation) by Ficoll-Paque Plus (Amersham Biosciences) density gradient centrifugation. Monocytes obtained by the adherence method from PBMCs were cultured in 1% normal human plasma (Sigma-Aldrich) in the presence of rhGM-CSF (100 IU/ml; PeproTech) and rhIL-4 (300 IU/ml; PeproTech) for 5 days. Cells were provided with fresh cytokines every other day. pDCs and mDCs isolation from PBMCs was carried out by magnetic separation with a MACS Separator (Miltenyi Biotec) according to manufacturer’s protocol.