Fascanto 2

Bone marrow specimens, collected according to international and institutional guidelines, were obtained through BM aspiration from 26 pediatric patients referred to the “IMIEM” Children’s Hospital (Toluca, Mexico) and to the “Moctezuma” Children’s Hospital (Mexico City, Mexico) and diagnosed with ALL, before any treatment (Table S1 in Supplementary Material). Control BM specimens were obtained from six healthy children undergoing minor orthopedic surgery. Mononuclear cells (MNC) were obtained by Ficoll-Paque (GE Healthcare) density gradient centrifugation and then enriched in the CD34⁺ fraction using the human CD34 MicroBead Kit UltraPure human (MiltenyiBiotec) according to the manufacturer’s instructions. Cell purity and yield were measured by flow cytometry using a BD FASCanto II. All procedures were approved by the Ethics, Research, and Biosafety Committees at the participant hospitals and by the National Committee of Scientific Research at the Mexican Institute for Social Security (CIEICE-007-01-13, R-2012-3602-29, and R-2015-785-120). All the samples were collected after written informed consent from the parents.

We evaluated IL-2, IL-4, IL-5, IL-10, IFN-γ and TNF-α production in coculture supernatants (days 1, 3 and 5), using Cytometric Bead Array (CBA Th1/Th2) (BD Company, USA), the FASCanto II (BD Company,USA), the BD FACS DIVA^TM program and the BD CBA Software (BD Company,USA). Concentration was calculated using the standard curves (FCAP Array Software BD Company,USA). Detection limits: 5 to 5000 pg/ml. For samples showing concentrations below the lower detection limit, we considered the value just below this limit to quantify cytokine production change in cocultures.

Interleukin concentrations in NPA were determined using a cytometric bead array (CBA) kit (BD-Biosciences, San Jose) in accordance with the manufacturer's protocol. We evaluated TNF-α, IL-10, IL-6, IL-1β, and IL-8, concentration by flow cytometry (FASCanto II with DIVA software, BD Biosciences, San Jose).

To evaluate the percentage of MPs compared to total cells at the third passage, samples containing approximately 5 × 10⁵ cells/sample were counted and suspended in PBS containing 1% BSA for 20 min at room temperature. Next, the cells were incubated with anti-CD90-PerCP, anti-CD45-PE-Cy7, anti-CD11b-FITC (BioLegend, San Diego, CA, USA), and anti-CD34-PE (Abcam, Cambridge, UK) antibodies for 40 min at 4°C. All flow cytometric analyses were performed with FASCanto II (BD Biosciences, San Jose, CA, USA).

Surface staining for CD4 and CD25 was performed by exposing the cells to the anti-CD4 (SK3, BD Biosciences, CA) and anti-CD25 (M-A251, BD Biosciences) monoclonal antibodies for 15 min in the dark using a 2% bovine serum albumin phosphate-buffered saline (PBS) mixture on freshly isolated PBMCs. Cells were washed in PBS and fixed for 30 min at 4°C using the eBioscience fixation/permeabilization kit, according to manufacturer's instructions. After washing in PBS and permeabilization (15 min at 4°C), the cells were incubated overnight at 4°C with anti-human FOXP3 antibody (259D, BioLegend, San Diego CA), washed, examined by flow cytometry (FASCanto II) and analyzed with FACSDiva Software (BD Biosciences). FOXP3+ positive signal was evaluated by comparing to the isotype signal. Gating strategy was performed by selecting lymphocytes based on physical parameters; CD4+ cells were selected from the lymphocyte population, and FOXP3+CD25+ cells from the CD4+ cell subpopulation. Values of CD25+FOXP3+Tregs are given as percentage within the CD4+ population.