Biocoll cell separation solution

Hematopoietic donor cell chimerism was performed as previously described [15 (link)]. Immune reconstitution was monitored in peripheral blood mononuclear cells (PBMCs) by flow cytometry in between week 0–38. PBMCs were isolated by density gradient centrifugation using Biocoll cell separation solution (Biochrom, Berlin, Germany). Immune cells were stained using the directly fluorescence labeled mAbs CD138-PE (clone: MI15, BD Biosciences, Franklin Lakes, NJ, USA), CD138-PE (clone: MI15, BD Biosciences), CD38-PerCP (clone: HB-7, BD Biosciences), CD19-PacificBlue (clone: SJ25C1, BD Biosciences), CD27-FITC (clone: M-T271, BD Biosciences), IgA-APC (clone: 97924, R&D Sytems, Minneapolis, MN, USA), IgG-APC (clone: 97924, R&D Systems), IgM-APC (clone: IL/41, ThermoFisher), HLA-A2-APC (clone: BB7.2, ThermoFisher), HLA Class I B7-PE (clone: BB7.1, abcam, Cambridge, UK), HLA-Class1 BW6-PE (Miltenyi Biotec, Bergisch Gladbach, Germany) and HLA-Class1 B8-APC (Miltenyi Biotec). Dead cells were excluded from analysis by LIVE/DEAD™ Fixable Aqua Dead Cell Stain (ThermoFisher, Waltham, MA, USA). Measurements were performed using a FACS Canto II or FACS Fortessa (BD Biosciences) and data analyzed using the software FlowJo V10 (FlowJo LCC, Ashland, OR, USA).

All experiments were carried out in accordance with the Helsinki protocol and the Ethics Committee of the University of Tübingen vote (13/2007V) between 2007 and 2020. All experimental protocols were approved by the Ethics Committee of the University of Tübingen (13/2007V). Informed consent was obtained from all patients. Gene nomenclature was carried out in accordance with the HUGO Gene Nomenclature Committee (HGNC) recommendations for the designation of gene fusions (Bruford et al. 2021 (link)).
Peripheral blood samples of healthy donors or B-ALL patients were collected and PBMCs were isolated using density gradient centrifugation with Biocoll Cell Separation Solution (Biochrom, Berlin, Germany). The B-ALL cell lines Nalm-6, Nalm-16, SD-1 and TOM-1 were purchased from the German Collection of Microorganisms and Cell Cultures (DMSZ, Braunschweig, Germany) and were routinely tested negative for mycoplasma. PBMCs and cell lines were kept in RPMI 1640 (LifeTechnologies, Darmstadt, Germany) supplemented with 10–20% heat-inactivated fetal calf serum (Biochrom), 1 mmol/l sodium pyruvate (Biochrom), non-essential amino acids (Biochrom), 2 mmol/l l-glutamine (Lonza Group, Basel, Switzerland), 100 U/ml penicillin (Sigma-Aldrich, St.Louis, USA), 100 μg/ml streptomycin (Sigma-Aldrich), and 50 μmol/l beta-mercaptoethanol (Merck, Darmstadt, Germany) at 37 °C and 5% CO₂.

Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by density gradient centrifugation with Biocoll cell separation solution (Biochrom AG, Berlin, Germany) and frozen in FCS + 10% DMSO. For analysis, PBMCs were thawed and washed twice with PBS. One million cells were stained with smCD3-APC-H7 (clone SK7, BD), CD19-PC7 (clone J3-119, Beckman Coulter), CD20-V450 (clone 2H7, BioLegend), CD27-BV605 (clone L128, BD) and CD38-APC-R700 (clone HIT2, BD for 30 min at room temperature. Flow cytometry was performed on a BD FACSLyric^TM and data was analyzed using FlowJo software (version 10).