Kaluza 1.5a software

Immunophenotyping was performed on peripheral blood for the identification of T, B, and NK cells. Intracytoplasmatic staining of cytotoxic granules in CD8 and NK cells was performed using FACSLysing and PermII buffers (BD Bioscience, Madrid, Spain). Analysis of NK-cell surface markers in GATA2-deficient patients was done in ≥200 NK cells per patient. Conjugated anti-human monoclonal antibodies are listed in Table S2 in Supplementary Material. Flow cytometry data were collected using a Beckman Coulter Navios cytometer and analyzed with Kaluza 1.5a software (Beckman Coulter, Madrid, Spain).

Cryopreserved peripheral blood‐derived mononuclear cells (PBMCs), derived from five age‐ and sex‐matched end‐stage IHD patients and five matched end‐stage DCM patients, were collected. Peripheral blood‐derived mononuclear cells were thawed and washed with RPMI (61870010, Gibco) supplemented with GlutaMax (room temperature) containing 25 nM HEPES, 1% penicillin/streptomycin and 2% foetal bovine serum (FBS; 10270‐106, Gibco). Peripheral blood‐derived mononuclear cells were filtered over a 40‐μm cell strainer (542040, Greiner Bio‐One). The single cell suspension was added to an antibody mixture containing different cell surface markers to identify B‐cell subtypes as described before.32 Cells were stained with a fixable viability dye (eBioscience, eFluor‐506, 65‐0866‐14). Viable CD19⁺CD3⁻ B lymphocytes were selected for further gating of C24⁻CD38⁺ plasmablasts and CD27⁻, IgG⁺, CD24⁺, and CD38⁺ transitional/regulatory B cells using gating strategy as described by Meeuwsen et al.32 All appropriate controls were included in the experiments, including isotype/subclass‐matched primary antibody of irrelevant specificity. After flow cytometry, data were analysed using Kaluza 1.5a software (Beckman Coulter).

Cells in complete growth medium were seeded into 12-well culture plates. The day after, the medium was changed to serum-free medium with compounds at various concentrations. After 48 h, supernatants were transferred into Eppendorf tubes. Cells were detached with 500 µL Accutase^® solution (Sigma Aldrich, St. Louis, MO, USA) and added to the respective supernatant. The suspension was centrifuged for 5 min at 1500 rpm. After removal of the supernatant, the cell pellet was washed twice with 1 mL DPBS and re-suspended in 98.5 µL of 1X Annexin V Buffer with 1 µL Annexin V-APC (Immuno Tools, Friesoythe, Germany) and 0.5 µL of 1 mg/mL propidium iodide (PI; Sigma Aldrich, St. Louis, MO, USA). After 15 min of incubation at room temperature in the dark, 200 µL of 1X Annexin V Buffer was added, and samples were measured on a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). Analyses from at least three independent experiments were performed with the Kaluza 1.5a software (Beckman Coulter, Brea, CA, USA).

MCF-7 cells (1 × 10⁵ cells/well) were plated overnight in 12-well culture plates. Subsequently, the medium was removed and serum-free medium was added, containing the compounds at various concentrations. After 48 h, supernatants were transferred to Eppendorf tubes. Cells were harvested with 500 μL of Accutase^® solution (Sigma-Aldrich, St. Louis, MO, USA) and mixed with the supernatant. The suspension was centrifuged at 1500 rpm for 5 min and washed twice with 1 mL of DPBS. The obtained pellet was stained for 15 min in 98.5 µL of 1X Annexin V Buffer with 1 µL of Annexin V-APC (Immuno-Tools, Friesoythe, Germany) and 0.5 µL of 1 mg/mL propidium iodide (PI, Sigma-Aldrich, St. Louis, MO, USA). Subsequently, 200 µL of 1X Annexin V Buffer were added and measured with the CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). At least three different experiments were conducted and analyzed with the Kaluza 1.5a software (Beckman Coulter, Brea, CA, USA).

CD4 T cell-derived EVs and CEM-EVs were analyzed using high-sensitivity flow cytometry as previously described^{49 (link)}. Thirty microliters of EV samples was incubated with the appropriate amount of specific antibodies and 10 μL of FITC-bound Annexin V reagent (Tau Technologies, Netherlands). Each stained sample was analyzed on a 3-laser Navios flow cytometer (Beckman-Coulter), based on a protocol standardized with Megamix-Plus FSC beads (BioCytex, Marseille, France). The following fluorescently-tagged antibodies were procured from Beckman Coulter: PE-tagged anti-TCRαβ, APC-tagged anti-CD45, PE-tagged anti-CD3, and PE-tagged anti-CD4 and their respective controls. The presence of EVs derived from different cellular origins was determined using PC7-tagged anti-CD41, APC-tagged anti-CD11b, Alexa 750 APC-tagged anti-CD235, PE-tagged anti-CD31 and their respective control antibodies. The presence of apoptotic bodies was determined by colabeling with FITC-bound Annexin V and DAPI (4′,6′-diamidino-2-phenylindole). Before analysis, CytoCount beads (Dako, Copenhagen, Denmark) were added as internal standards to samples to determine the concentration of EVs. Flow cytometry data were analyzed using Kaluza 1.5a Software (Beckman Coulter).

Proportions and lymphocyte count of T-, B-, and NK-cells were determined in blood samples using conjugated mouse anti-human monoclonal antibodies and data were collected by flow cytometry using a Navios Cytometer (Beckman Coulter, Madrid, Spain) and analyzed with Kaluza 1.5a software (Beckman Coulter, Indianapolis IN, US).
PBMCs from patient and healthy controls were irradiated with 10Gy, fixed and stained for CD3, CD19, and phospho-histone H2AX. Mean fluorescence intensities (MFI) of γH2AX were evaluated on gated CD3+ lymphocytes.

Cells grown overnight in 6-well plates were treated with compounds at various concentrations for 72 h. Afterwards, the supernatants were harvested and transferred into Eppendorf tubes. Detachment of cells was performed with 500 μL Accutase^® solution. After centrifugation at 1500 rpm for 5 min, the pellet was washed with 1 mL DPBS. The supernatant was removed, and the cell pellet was re-suspended in 100 μL DPBS. For the fixation of cells, 1 mL of ice-cold 70% ethanol was added slowly to the cells, under constant agitation. After freezing for at least 1 h at −20 °C, the cell suspension was washed twice with 2 mL DPBS. The obtained pellet was stained for 15 min with 5 μL PI (0.4 mg/mL), 5 μL RNase solution (1 mg/mL, Sigma Aldrich, St. Louis, MO, USA) and 90 μL DPBS. Subsequently, cells were measured on a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). At least three independent experiments were conducted and analyzed with the Kaluza 1.5a software (Beckman Coulter, Brea, CA, USA).