Cd33 pe

Extracellular staining with fluorescent antibodies on fresh or frozen PBMC was done as described previously [8 (link)]. At each time point, analyses were performed for gMDSC and mMDSC respectively. The following antibodies were used: gMDSC: CD11b-FITC, CD14-APC, CD15-PerCP, CD66b-PE [14 (link), 17 (link)]; mMDSC: CD11b-FITC, CD14-APC, CD33-PE, HLA-DR-PerCP, PerCP isotype control (all BioLegend, USA). Cells were analyzed on a FACSCalibur (BD, Germany) and analysis of data was done using FlowJo software 7.2.1 (TreeStar, Inc. Ashland, OR). Gating strategies were according to Vollbrecht et al. [8 (link)] and Rieber et al. [14 (link), 17 (link)] for gMDSC. For the gating of mMDSC, we used a PerCP isotype control in order to gate for HLA-DR and our gating strategy was according to Dumitru et al. [2 (link)] (see Additional file 1: Figure S1). For the statistics, we indicated MDSC as percentage of PBMC in all subgroups because the numbers of monocytes vary substantially in subjects with e.g. chronic viral infections. However, in the dot plots shown in Fig. 2, frequencies of the described populations are shown in percentages of monocytes.

The following fluorescence-labeled monoclonal antibodies (mAb) were used: CD3-FITC or PE/Cy5 (clone UCHT1), CD11c-FITC (3.9), CD16-APC/Cy7 (3G8), CD30-PE/Cy7 (BY88), CD33-PE, BV605 or PE/Cy5 (P67.6), CD56-PE/Dazzle™594 (N901), CD57-FITC (HCD57), CD62L-PE/Cy7 (DREG56), CD107-BV510 (H4A3), CD117-BV421 (104D2), KLRG1-APC/Fire 750 (SA231A2), PD1-APC/Cy7 (EH12-2H7), NKG2D-PE (1D11), NKp46-BV510 (9E2), NKp44-APC (P44-8), NKp30-BV785 (P30-15), Granzyme B-Pacific Blue (GB11), IFNγ PE/Cy7 (B27), Perforin-PE (dG9), TNFα PE/Dazzle™ 594 (all from Biolegend, CA, USA), CD3 PerCP-Vio700 (REA613), CD14-PerCP-Vio700 (REA599), CD33-APC, CD56 (REA196), anti-biotin-VioBright515 and 7-AAD (all Miltenyi Biotec), NKG2C-AF700 (134591) from R&D systems (MN, USA), and CD158b1,b2,j-PE/Cy5 (GL183), NKG2A-APC (Z199) purchased from Beckman Coulter (CA, USA). Flow cytometric analyses were performed on a CytoFLEX (Beckman Coulter) or MACSQuant 10 Analyzer (Miltenyi Biotec). Data analysis was performed on Kaluza 2.1.1 software.

For T-cell isolation, PBMCs were stained with CD8 Alexa Fluor 700 (Invitrogen (Waltham, MA, USA)/MHCD0829), CD4 FITC (BD (Franklin Lakes, NJ, USA)/555346), CD14 FITC (BD/555397), CD19 FITC (BD/555412), and in-house generated PE-conjugated pHLA tetramers produced as outlined previously with minor modifications [20 (link)]. T-cell clones were stained with pHLA tetramers for 15 min at room temperature. AML samples were stained with HLA class I FITC (Bio-Rad (Hercules, CA, USA)/MCA81), CD54/ICAM1 APC Fire 750 (BioLegend (San Diego, CA, USA)/353121), CD58/LFA-3 BUV395 (BD/752794), and CD102/ICAM2 BB700 (BD/746179). For the cytotoxicity assays, the AML cells were stained with CD33 PE (Biolegend/397225), CD34 APC (BD/343510), CD14 V450 (BD/561390), and CD206 BV711 (Biolegend/321136); to exclude the T-cells, CD3 BV421 (BD/562427) and CD8 BV421 (Biolegend, 344748) were used. Conventional flow cytometry was performed on a BD FACS LSR-II 4L Full (BD Biosciences, San Jose, CA, USA) using BD FACSDiva version 6 software. Spectral flow cytometry was performed on a 5L Cytek Aurora flow cytometer (Cytek Biosciences, Fremont, CA, USA). Raw spectral flow cytometry data were unmixed using SpectroFlo (Cytek Biosciences). Data were analyzed using OMIQ flow cytometry software (Dotmatics, www.omiq.ai).

To investigate the percentages of MDSCs, 200 μL of whole blood sample was stained with monoclonal antibodies, including HLA-DR-PE/Cy7, CD11b-APC, CD33-PE, CD14-FITC, and CD15-PerCp5.5 (BioLegend, San Diego, CA, USA). The cells were incubated at room temperature for 15 min and then were treated with 1 mL of lysing solution (Beckman Coulter, Miami, FL, USA) at room temperature for 10 min. Finally, samples were washed twice and analyzed with flow cytometry.
To investigate the percentages of IFN-γ-producing T cells, PBMCs (1 × 10⁶) were cultured with phorbol-12-myristate 13-acetate (50 ng/mL, Multi Sciences, Hangzhou, Zhejiang, China), ionomycin (1 μg/mL, Multi Sciences), and GolgiStop (Becton Dickinson, San Diego, CA, USA) at 37°C for 4 h. The cells were stained with CD4-FITC and CD8a-PerCP/Cy5.5 (BioLegend) and then fixed and permeabilized with Fixation and Permeabilization Solution (Becton Dickinson). After staining with IFN-γ-PE/Cy7 (BioLegend), all samples were analyzed using FACSCanto^II flow cytometer with FACSDiva software (BD Biosciences, San Jose, CA).

HSPCs were incubated with fluorescence-conjugated antibodies (CD33-PE, CD34-FITC, or CD36-PE) purchased from Biolegend (San Diego, CA, USA) for 30 min on ice. The antibody-treated HSPCs were resuspended in FACS buffer (0.1% BSA and EDTA in PBS) and analyzed using a flow cytometer (BD FACS CANTO II) and the CellQuest program (BD Biosciences, San Jose, CA, USA). A dataset of 10,000 cells was acquired.