Cd33 percp cy5

The cells were stained with monoclonal antibodies to T-cell phenotype CD3 APC, CD4 PerCP Cy 5.5, CD8 APC Cy7, CD27 FITC, CD45RA PE (eBioscience, CA, USA). The cells were also stained with monoclonal antibodies to MDSC phenotype CD3 APC, CD19 APC, CD56 APC, HLA-DR APC e-fluor 780, CD33 PerCP Cy5.5, CD11b PE, CD14 PE Cy7, CD15 FITC (eBioscience, CA, USA). After 30 min of incubation with monoclonal antibodies, in the dark and at 4°C, the cells were washed with PBS and centrifuged. Living cells (based on forward and side scatter) were acquired in the FACS Canto II using the DIVA software (Becton Dickinson, USA). Further analyses of FACS data were performed using the 9.3 FLOWJO software (Tree Star, USA).
T lymphocytes were characterized as described previously (36 (link)).

Naïve: CD3⁺CD4⁺CD45RA⁺CD27⁺ or CD3⁺CD8⁺CD45RA⁺CD27⁺ (Naïve).

Central memory: CD3⁺CD4⁺CD45RA⁻CD27⁺ or CD3⁺CD8⁺CD45RA⁻CD27⁺ (CM).

Effector memory: CD3⁺CD4⁺CD45RA⁻CD27⁻ or CD3⁺CD8⁺CD45RA⁻CD27⁻ (EM).

Effector memory re-expressing CD45RA: CD3⁺CD4⁺CD45RA⁺CD27⁻ or CD3⁺CD8⁺CD45RA⁺CD27⁻ (EMRA).

Myeloid-derived suppressor cells were characterized as:

CD3⁻CD19⁻CD56⁻HLA⁻DR^−/lowCD33⁺CD11b⁺CD15⁺ granulocytic or

CD3⁻CD19⁻CD56⁻HLA⁻DR^−/lowCD33⁺CD11b⁺CD14⁺ monocytic.

Cell surface staining with antibodies conjugated with fluorochromes was performed as previously described.^{26 (link)} The following anti-human antibodies conjugated with fluorochromes were purchased from eBiosciences (San Diego, CA): CD14-FITC, CD4-FITC, CD11b-PE, CD25-PE, CD3-PerCP, CD33-PercpCY5.5, HLA-DR-APC, FoxP3-APC, and isotype-matched control antibodies conjugated with fluorochrome. Intracellular staining (ICS) with anti-human FoxP3-PE was performed using the FoxP3 staining buffer set (eBiosciences, San Diego, CA) according to the manufacturer's instructions. As a heterogeneous cell population, human MDSCs could be further divided into 2 subsets, monocytic (M-MDSC, CD14⁺) and granulocytic (G-MDSC, CD14⁻/CD15⁺).^{12 (link),18 (link),20 (link)–23 (link)} Given that G-MDSCs are unavailable in Ficoll-prepared PBMCs, we set the gating strategy for M-MDSCs: CD33⁺CD11b⁺/CD14⁺HLA-DR^Low. Meanwhile, the gating strategy for Tregs was CD3⁺CD4⁺CD25⁺FoxP3⁺. Cells were collected on a FACSCalibur (BD). The data were analyzed using FlowJo software (TreeStar, San Carlos, CA). Appropriate isotype controls were used at the same protein concentration as the test antibodies, and control staining was performed during every FACS.