Lymphosep

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood using gradient density centrifugation (Lymphosep™, Biowest, MO, USA) as previously described^{39 (link)}. Isolated PBMC were counted by an inverted microscope, and cell concentrations were accessed in cRPMI-1640 medium [RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 50 μg/ml of gentamycin]. The viability of isolated PBMC was determined by Trypan blue (Sigma-Aldrich, MO, USA) staining and more than 90% viability was used for in vitro stimulation for IL-10 production and enzyme-linked immunospot (ELISPOT) assay as described below.

Primary samples were collected after obtaining written informed consent. The study was approved by Comitato Etico della Romagna (protocol 5244/2019) and was carried out in accordance with the principles laid down in the 1964 Declaration of Helsinki. Primary leukemic cells were isolated using Lymphosep (Biowest, Nuaillé, France) from the bone marrow of adult newly diagnosed B-ALL patients (n = 3, Supplementary Table 1) and were seeded in RPMI-1640 Advance (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 20% FBS (GE Healthcare, Piscataway, NJ, USA).

The peripheral blood mononuclear cells (PBMCs) were isolated from 5 mL of venous blood collected early in the morning from five healthy donors with an age range of 40–45 years old (Ethics Committee number 8/2021, 15 September 2021). PBMCs were isolated from heparinized blood by Lymphosep (Biowest) and cultured at a concentration of 3 × 10⁵/mL cells in a 24-well, flat-bottom plate in complete RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, and 100 U/mL penicillin/streptomycin at 37 °C. PBMCs were not treated (control) or were 4-h treated with PPPwsf at a concentration of 200 µg/mL with or without lipopolysaccharide (LPS) at a concentration of 1 µg/mL. At the end of the treatment, the cells were collected for RNA extraction and the supernatant was submitted to IL1β Enzyme-Linked Immunosorbent Sandwich Assay–ELISA (Sigma–Aldrich) according to manufacturer instruction.

Flow cytometric analysis was performed using a 3-laser FacsCanto II (BD Biosciences, San Jose, CA). A minimum of 50.000 CD45⁺ lymphocytes has been recorded for each analysis. Flow cytometry data were analyzed with DiVa 6.1.1 software and FCS Express 7 Reader. Appropriate isotype controls were included for each sample.
CAR-T cell tracking was performed on fresh whole blood at all available time-points and on 100 μl of bag leftovers (18 (link)). Cells were stained with the CD19 CAR FMC63 Idiotype antibody-APC (Miltenyi Biotec, Bergisch-Gladbach, Germany) following the manufacturer’s instruction. Then, cells were labeled with the following set of antibodies: CD45, CD3, CD4, CD8, CD45RA, CD62L, CD57, CD28 (from BD Biosciences). The analysis of monocytic (M-) and polymorphonuclear (PMN-) myeloid derived suppressor cells (MDSC) was performed on peripheral blood mononuclear cells (PBMC) separated by density gradient centrifugation using Lymphosep (Biowest) within 4 h after pre-LD sample collection. The following mAbs were used for MDSC identification: CD11b, CD14, CD15, CD33, CD45, and HLA-DR (from BD Biosciences). A minimum of 100.000 events has been recorded in the PBMC-gated population.

PBMCs were isolated from heparinized blood sample by gradient density centrifugation (Lymphosep, Lymphocyte separation media, Biowest, USA) according to a previously described protocol^{38 (link)}. The isolated PBMCs were resuspended in advanced RPMI-1640 (GIBCO, CA, USA) supplemented with 10% FBS, Antibiotic–Antimycotic Solution, Glutamax, 25 mM HEPES (Sigma-Aldrich, MO, USA) and 50 μM β-mercaptoethanol (Sigma Chemical Co., USA). Viability of PBMCs were determination using Trypan blue (Sigma-Aldrich, MO, USA) staining and subjected for further analyses including the measurement of IFN-γ and Interleukine-10 (IL-10).

BM samples were obtained from patients diagnosed with MDS and from healthy donors (HD) after obtaining written informed consent, as approved by the institutional procedures of the independent ethics committee and the ‘Comité de Protection des Personnes’-Ile de France (NCT03233074/17.07.2017). HDs were aged matched and had blood counts in the normal range. Detailed characteristics of MDS patients are shown in Table 1.
BM mononuclear cells (BMMNCs) were isolated using Lymphosep (Biowest, Nuaillé, France) gradient separation. 1 × 10⁶ cells were cultured in T25 cell flasks in MesenCult® MSC basal medium (Stem Cell Technologies, Vancouver, BC, Canada) and Mesenchymal Stem Cell Stimulatory Supplement (Stem Cell Technologies, Vancouver, BC, Canada) and in 1% penicillin/streptomycin (Lonza, Basel, Switzerland) at 37 °C in 5% CO₂, with medium replacement twice a week in the beginning of the cultures and every week after date until 80% of confluence is reached.
The resulting CD45-CD73+ CD105+ CD90+ (purity >98%), low-passage BM-stromal cells were used to perform different tests.
Adherent cells were analysed by flow cytometry. The list of antibodies used for BMSC discrimination and characterisation is shown in Table 2.