Cd45 alexa fluor 700

CD34⁺ HSPCs were stained with CD45-AlexaFluor700 (BioLegend, clone H130), CD34-PE/Cy7 (BioLegend, clone 581) and CD44-BV421 (BioLegend, clone IM7). All antibodies were used in the concentration recommended by the manufacturer. Debris and dead cells were omitted by forward scatter (FSC), side scatter (SSC), DAPI or 7-AAD staining (1:100 dilution prior to analysis). The flow cytometry analyses were performed using the BD LSRFortessa instrument (BD Biosciences) and cells were sorted using BD FACSAria IIu (BD Biosciences). The software FlowJo (FlowJo, LLC) was used to analyse the data.

Heart single-cell suspension and flow cytometry was prepared as previously described [30 ]. Briefly, hearts were perfused, extracted, finely minced, and then incubated with digestive enzymes at 37 °C on a rocking shaker at 50 rpm for 45–60 min. Samples were homogenized with a 40 μm cell strainer. Red blood cells were lysed using ammonium-chloride-potassium (ACK) lysis buffer. Next, samples were centrifuged at 400× g for 5 min at 4 °C, and the pellet was then suspended with FACS buffer. Cells were immune-stained with the following anti-mouse antibodies: CD45-Alexa Fluor^®® 700 (BioLegend, 103128, San Diego, CA, USA), CD11b-PerCP (BioLegend, 101228, CA, USA), F480-PE (BioLegend, 123110, CA, USA), CD206-BV421 (BioLegend, 141717, CA, USA), CD64-APC (BioLegend, 161006, CA, USA), Ly-6G- Brilliant Violet 510™ (BioLegend, 127633, CA, USA), Ly-6C-PE/Cyanine7 (BioLegend, 128018, CA, USA), Propidium iodide (Sigma, 25535-16-4, Darmstadt, Germany). Cells were incubated (30 min, 4 °C) with the antibody mixture in a staining buffer (PBS containing 1% bovine serum albumin and 0.05% sodium azide) and then washed twice with a staining buffer. Cells were acquired using a LSRFortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The data were analyzed using FlowJo V.10 software (FlowJo, Ashland, OR, USA).

For analysis of PD-1, cells were washed in serum-free PBS and stained with a fixable viability dye, eFluor506, CD4-PerCPCy5.5, PD1-PECY7, CD8-APCeFluor780 (eBioscience), incubated for 15 min at room temperature in the dark, and then washed in PBS buffer containing 1% FBS and sodium azide. Cells were stained for Treg cell markers using a FoxP3 staining buffer set (eBioscience) and accompanying protocol. Treg cell markers included CD39-FITC, FoxP3-PE, CD73-PerCPeFluor710, CD25-PECY7, CTLA-4-APC, CD127-APCeFluor780, Ki67-eFluor450 (eBioscience), and CD4-V500 (BD Biosciences). An intracellular staining kit (Fix and Perm kit, Invitrogen) was used to analyze cytokine production after restimulation with PMA/ionomycin. Cells were stained with IL-17A-AlexaFluor488, IL-10-PE, TNF-α-PerCPCy5.5, CD45RA-PECY7, CD8-APCeFluor780, FoxP3-eFluor450 (all eBioSciences), CD45 AlexaFluor700 (BioLegend), IFN-γ-APC, CD3-V500, and IL-2-PE-CF594 (BD Biosciences). Due to PMA/ionomycin-mediated reduction in CD4 expression, CD4⁺ T cells were identified as CD3⁺CD8⁻ T cells for cytokine analysis. Cells were acquired on a BD LSRFortessa flow cytometer and analyzed using FlowJo software (Flowjo LLC).

Total blood cells or leukocytes from the liver or spleen were incubated with Fc block (eBiociences, Thermo Fisher Scientific) for 10 minutes at 4-8°C. Surface staining was performed for 20 minutes at 4-8°C and always included Fixable Viability Dye (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) for exclusion of dead cells. The following conjugated anti-mouse mAbs (and respective clones) were used: CD3 PerCP-Cy5.5 (145-2C11), CD4 APC or Brilliant Violet (BV) 510 (GK1.5), CD8 BV711 (53-6.7), CD62L FITC (MEL-14), CD19 PE (1D3), CD25 APC (3C7), CD44 PE-Cy7 (IM7), CD45 Alexa Fluor 700 (30-F11), CD69 BV650 (H1.2F3), CD127 PE-Dazzle 594 (A7R34), NK1.1 PE-Cy5 (PK136), KLRG1 PE (MAFA), CXCR3 APC (CXCR3-173), TCR γδ BV421 (GL3), all from either BioLegend (San Diego, CA, USA) or Sysmex (Kōbe, Hyōgo, Japan). Cells were acquired in a BD LSR Fortessa X-20 cytometer and analyses were performed within live, single (based on FSC-A vs. FSC-W parameters) CD45+ leukocytes using FlowJo v10 (FlowJo, BD). For the clustering and visualization of high-dimensional data, equivalent numbers of live, single CD45+ cells from each condition were concatenated. Clustering was performed using X-shift (number of clusters determined by the algorithm) and data are presented using the dimensionality reduction method TriMAP (large-scale dimensionality reduction using triplets).

CD45 Alexa Fluor 700 (30-F11, Biolegend), CD19 PerCP/Cy5 (6D5, Biolegend), CD3ε Brilliant Violet 650 (17A2, Biolegend), CD4 PerCP-Cy5.5 (GK1.5, Biolegend), CD49b PE-dazzle 594 (DX5, Biolegend), CD8a Brilliant Brilliant Violet 510 (53–6.7, Biolegend), FoxP3 Alexa 647 (3G3, Thermo-Fisher), and CD285 PE (TLR-5) (ACT5, BD Biosciences) (Supplementary Table 10).

Eight- to ten-week-old NOD/SCID IL2Rγ^−/− mice were intravenously injected with 1*10⁵ MOLM-13 cells. Beforehand, AML cells were cultured alone or in the presence of either purified ECAR or control vector modified T cells at an E:T ratio of 5∶1 for 4 h. Prior to the experiment, T cells were irradiated at 5 Gy to prevent xenograft reaction in recipient mice [50] . Mice were sacrificed when visible tumors developed at injection site and single-cell suspensions from bone marrow obtained from femur and tibia of the left hind leg were prepared. Erythrocytes were removed by lysis and nucleated cells were stained with anti-mouse CD45.1/PE-Cy7 (eBioscience, clone A20), anti-human CD3/APC-eFluor780 (eBioscience, clone SK7), CD19/APC (BD Bioscience, clone HIB19), CD33/PE (eBiosience, clone HIM3-4), and CD45/AlexaFluor700 (Biolegend, clone HI30) mabs. Doublet discrimination was routinely carried out and dead cells were excluded by 4, 6 diamidino-2-phenylindole (DAPI)-staining (Sigma-Aldrich, Taufkirchen, Germany). All measurements were performed on a BD LSRII FACS machine (BD Biosciences). Data analysis was realized using FlowJo-software (Tree Star Inc., Ashland, USA).