Lyric

Peripheral blood (10 mL) will be collected in ethylenediaminetetraacetic acid–coated tubes and processed within 24 hours of collection. Whole blood (100 µL) will be mixed directly with the fluorescence-conjugated antibodies, including CD45, CD3, CD4, CD8, CD19, CD56, NKG2D, CD14, CD11c, and PD-1 (Table 3), protected from the light, and incubated for 20 minutes at 4 ℃. Blood cells will be washed twice with 2 mL staining buffer (phosphate buffered saline+2% fetal bovine serum), centrifuged at 300×g for 5 minutes at room temperature, and further incubated with Red Blood Cell Lysis Buffer (Thermo Fisher Scientific) for lysing the erythrocytes for 10 minutes at room temperature. This step may repeat until the red blood cell is completely lyzed. The cells will be further fixed in 2% paraformaldehyde for 10 minutes at room temperature. Subsequently, the cells will be subjected to 2 additional washes with 2 mL staining buffer and centrifuged 300×g for 5 minutes at room temperature. Then, the cells will be preserved at 4 ℃ and analyzed using flow cytometry (Lyric, BD) within 3 days.

To allow intracellular labelling of cytokines, cell suspensions were stimulated 4 h at 37°C with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma), 1 µg/mL ionomycin (Sigma), and Golgi Stop™ (BD Biosciences). For extracellular labelling, cells were incubated with 200 ng of each antibody. Antibodies targeting extracellular proteins were CD45 (30-F11), PD-1 (RMP1-30) (BD Biosciences), CD3 (17A2), CD4 (GK1.5), CD44 (IM7), CCR9 (9B1), CD11c (N418), CD8a (53-6.7), PD-L2 (24F.10C12), PD-L1 (10F.9G2), MHC-class II (M5/114.5.2), CD80 (16-10A1), CD40 (3/23) (Biolegend), ɣδ-TcR (eBioGL3 (GL-3, GL3), and OVA-dextramer (H-2 Kb) (Immudex) (Table S2).
To allow intracellular labelling, cells were first permeabilized using a FoxP3 staining buffer kit (eBioscience). Antibodies targeting intracellular cytokines were IFNɣ (XMG1.2) (Biolegend), IL-10 (JES5-16E3) (Thermo Fisher Scientific), and IL-17A (TC11-18H10) (BD Biosciences) (Table S2). After intracellular labelling, cells were fixed and stored using FACS Lysing Solution 1X (BD Biosciences). All data were collected using a BD Biosciences FACSCanto II or Lyric and analyzed using FlowJo software.

Isolated MDSCs were stained with anti-CD16/CD32 (Cat# 564219, BD Biosciences, San Jose, CA, USA) for Fc receptor blocking on ice and then incubated with the anti-human antibodies. The expression of MDSCs was evaluated by monoclonal antibodies specific to markers, including CD33 FITC (Cat# 11-0339-042, Invitrogen, Waltham, MA, USA), CD11b PE (Cat# 12-0118-42, Invitrogen), and CD14 PE-Cy7 (Cat# 25-0149-42, Invitrogen). For intracellular staining of iNOS FITC (Cat# SC-7271, FITC, Santa Cruz Biotechnology, Dallas, TX, USA), IDO PE (Cat# IC6030P, R&D Systems, Bio-Techne, Minneapolis, MN, USA), and ARG1 PerCP (Cat# IC8026C, R&D Systems). MDSCs were incubated for fixed and permeabilized using BD Cytofix buffer and BD Cytoperm buffer (Cat# 554714, BD Biosciences). All samples were acquired with BD Lyric (BD Biosciences) and then analyzed with FlowJo software (v10.8.1, FlowJo LLC, Ashland, OR, USA).