Lymphoprep

Peripheral blood mononuclear cells (PBMCs) were separated using density gradient method (Lymphoprep^TM, Alere Technologies, Norway) from EDTA-anticoagulated blood. Flow cytometry was performed using a Becton Dickinson (BD) LSRII flow cytometer and analysis using BD FACSDiva software. A six-way FACS was performed on a BD Influx™ to pure lymphocyte subsets. Purity of sorted populations was confirmed before RNA extraction using flow cytometry (>98% purity for each subset) as well as expression of lineage marker genes. Antibodies are shown in Table S1.

De-identified bone marrow aspirates were collected from the iliac crest of healthy human donors for allogeneic transplantation under written consent and the approval of the local institutional Helsinki Committee (0626-15-HMO). Aspirates were passed through a nylon cell strainer, separated by a Ficoll–Hypaque density gradient (LymphoprepTM, Alere Technologies), and resuspended in low-glucose Dulbecco's Modified Eagle Medium (DMEM; Biological Industries) supplemented with 1% L-glutamine (Biological Industries), 1% penicillin-streptomycin (Biological Industries), and 10% fetal bovine serum (Biological Industries). Cells were seeded into 75 cm² culture flasks (30 × 10⁶ cells per flask) and cultured at 37 °C in a humidified atmosphere with 5% CO₂. Cells were expanded and passaged once before seeded on the elastic matrices (P1). Medium was replaced twice weekly, and cell density was maintained <80 to 85% confluence. The positive (CD73 and CD90) and negative (HLA-DR, CD56, CD3, and CD45) surface marker repertoire was validated by fluorescence-activated cell sorting (FACS) using targeting antibodies (eBioscience).

Peripheral blood mononuclear cells were isolated from patients at diagnosis, with at least 80% AML cells among blood leukocytes and a high peripheral white blood cell count (>5.0 × 10⁹/L). The AML cell population was enriched with density gradient separation (Lymphoprep^TM, Alere Technologies, Oslo, Norway) and included >90–95% primary AML cells. The AML cells were cryopreserved in RPMI-1640 (Merck KGaA, Darmstadt, Germany) with 10% dimethyl sulfoxide (DMSO, Merck KGaA, Darmstadt, Germany) and 20% heat-inactivated fetal bovine serum, stored in liquid nitrogen until thawed and used in experiments. The patients gave written informed consent to participation and collection of biological material in accordance with the Declaration of Helsinki. The regional ethical committee granted approval from local governance (REK III no 060.02 and 059.02, REK Vest 2015/1759, REK Vest 2015.03, REK Vest 2031.06 and REK Vest 2017/305). For our experiments, cells were thawed from 79 consecutive patients. Their characteristics are shown in Table 1.

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using a Lymphoprep^TM (Alere Technologies, Oslo, Norway) density gradient. CD14+ cells were purified using human antibody-CD14-labeled magnetic microbeads and LS columns (Miltenyi Biotec, Auburn, CA, USA). CD14+ cells were seeded in RPMI 1640 medium (Corning, New York, NY, USA) supplemented with 10% fetal calf serum (HyClone, New Hampshire, UT, USA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 200 mM l-glutamine (Sigma-Aldrich, St. Louis, MO, USA). As previously described [12 (link)], monocytes were cultured with GM-CSF (10 ng/mL) (R&D System, Minneapolis, MN, USA) to obtain Mɸ, and with GM-CSF (53 ng/mL) and IL-4 (25 ng/mL) (R&D System, Minneapolis, MN, USA) to obtain DCs.
Mɸ were stimulated with LPS (100 ng/mL) for 6 and 24 h and DCs with a maturation cocktail (MC) (10 ng/mL TNF-α, 10 ng/mL IL-1β, and 1 μg/mL PGE2 (Biolegend, San Diego, CA, USA) for 2, 6, 12, and 24 h. Mɸ and DCs were also stimulated with heat-killed Mycobacterium tuberculosis H37Ra strain (HKMtb) at a multiplicity of infection (MOI) of 1:10 (1 cell per 10 bacilli) for 2, 6, 12, and 24 h; HKMtb were previously incubated in a medium containing 10% FCS at 37 °C to favor type II phagocytosis.

Bone marrow samples containing leukemia cells were obtained from pediatric patients with BCP-ALL. All patients were treated at Kagoshima University Hospital between 2005 and 2020. Mononuclear cells were isolated using sucrose density-gradient centrifugation (density, 1.077 g/mL; Lymphoprep^TM, Alere Technologies, Waltham, MA, USA) and stored at −130 °C. For each assay, leukemia cells were resuspended in RPMI-1640 (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 20% fetal bovine serum (FBS), 5 μg of insulin per mL, 5 μg of transferrin per mL, and 5 ng of sodium selenite per mL (ITS media supplement; Sigma-Aldrich, St. Louis, MO, USA) [14 (link)]. Leukemia cells were not isolated and purified from bone marrow samples before being used for each assay. This study was approved by the ethics committee of the Kagoshima University Graduate School of Medical and Dental Sciences (approval number: 170242) and was conducted in accordance with the principles of the Declaration of Helsinki.

Isolation of PBMCs from fresh carcass of EEHV4-infected and non-EEHV-infected calves was done, as previously described^{36 (link)}, with minor modification. Briefly, 20 mL of the blood obtained in the ethylenediamine tetraacetic acid (EDTA)-containing tubes was diluted to an equal volume of PBS. Then, the PBMCs were isolated by density gradient centrifugation on Lymphoprep^TM (Alere Technologies AS, Oslo, Norway) at 400 × g for 30 min at 4 °C. The interphase cells containing the PBMCs were collected and washed twice with PBS supplemented with 1% fetal bovine serum (FBS; Gibco; Thermo Scientific), and then resuspended in the Roswell Park Memorial Institute (RPMI)-1640 medium (Thermo Scientific) supplemented with 10% FBS, 100 U/mL penicillin G, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B. The cells were seeded onto the coverslip-inserted 24-well-microtiter plates (SPL Life Sciences, Gyeonggi-do, Korea) at a concentration of 2 × 10⁶ cells/mL and cultivated at 37 °C with 5% CO_2. After 2 hr of cultivation, the cells were fixed and immunofluorescent stained, as described below.

Peripheral blood samples were layered on Ficoll Hypaque gradients (LymphoprepTM; Alere Technologies, Norway) for density gradient centrifugation (453 × g for 30 min). Mononuclear cells were isolated and washed twice with phosphate-buffered saline (PBS). Decidual tissues were rinsed thoroughly with PBS and minced into 1–2 mm pieces by a pair of scalpel blades in Dulbecco's Modified Eagle Medium. Then, the suspensions were filtered through a 32 μm nylon mesh as reported elsewhere (24 (link)). All samples were cryopreserved until single-cell analysis.