Cytexpert for dxflex version 1

MDSC subsets, M-MDSCs (CD14⁺CD15^-CD11b⁺CD33⁺HLA-DR^-/low) and G-MDSCs (CD15⁺CD33⁺CD11b⁺CD14^-HLA^-DR^-/low) were identified by FACS using a CytoFLEX (Beckman Coulter, Inc.) with a panel of fluorescent-labeled monoclonal antibodies (all eBioscience; Thermo Fisher Scientific, Inc.) as follows: Anti-CD15 (cat. no. 11-0159-42), anti-CD33 (cat. no. 56-0338-42), anti-CD14 (cat. no. 61-0149-42), anti-CD45 (cat. no. 47-0459-42), anti-CD11b (cat. no. 46-0118-42) and anti-HLA-DR (cat. no. 25-9952-42). The frequency of cells expressing PD-2 and PD-1 was determined in MDSC subsets by flow cytometry using fluorescent-labeled monoclonal antibodies CD274-APC (cat. no. 17-5983-42, eBioscience, Thermo Fisher Scientific, Inc.) and CD273-PE (12-5888-42, eBioscience, Thermo Fisher Scientific, Inc.), allowing the identification of PD-L1⁺G-MDSCs, PD-L1⁺M-MDSCs, PD-L2⁺G-MDSCs and PD-L2⁺M-MDSCs. Samples were added to 100 µl PBS, and the cell precipitates were blown and mixed well. A total of 5 µl antibodies was added according to the manufacturer's instructions, blown and mixed well. They were incubated at 4˚C for 30min. CytExpert for DxFLEX version 1.0 (Beckman Coulter, Inc.) was used for flow cytometry analysis.

Flow cytometry was performed to detect phenotypes of T cells. The functional compartments of CD4⁺ and CD8⁺T cells were phenotypically characterized as CD3⁺CD4⁺ and CD3⁺CD4, respectively. The phenotypic characteristic of Tregs were determined according to their differential expression of CD25 and CD127 (CD4⁺CD25⁺brightCD127^-/⁺low expression). The subpopulations expressing PD-1 and CTLA4 were determined in each of the T cell subsets as follows: CTLA4⁺CD4, PD-1⁺CD8, CTLA4⁺CD8, PD-1⁺Treg, PD-1⁺CD4, CTLA4⁺Treg. Phenotypical analyses of T cells were characterized by fluorescence-activated cell sorting (FACS) using a CytoFLEX (Beckman Coulter, Inc.) with anti-human-specific antibodies against surface CD markers (all eBioscience; Thermo Fisher Scientific, Inc.) as follows: CD3 (cat. no. MHCD0327; APC-Alexa Fluor^™ 750), CD4 (cat. no. 25-0049-42; PE-Cyanine), CD25 (cat. no. 12-0257-42; PE), CD127 (cat. no. 17-1278-42; APC), CTLA-4 (cat. no. 85-46-1529-42; PerCP-eFluor^® 710) and PD-1 (CD279; cat. no. 61-2799-42; PE-eFluor^™ 610). A total of 5 µl antibodies was added according to the manufacturer's instructions and incubated at 4˚C for 30 min. CytExpert for DxFLEX version 1.0 (Beckman Coulter, Inc.) was used for flow cytometry analysis.