Pe anti mouse cd11b

Flow cytometry was used to characterize the peripheral blood mononuclear cells (PBMCs) and splenocytes. Peripheral blood was collected via retro-orbital bleeding. Splenocytes were collected from spleens that were homogenized by grinding between glass slides. The cells were stained with the following antibodies conjugated to fluorophores: Alexa Fluor 700- anti-mouse CD4 (BD Biosciences, San Jose, CA, United States), PE-Cy5- anti-mouse CD25 (eBioscience, San Diego, CA, United States), PE-CF594- anti-mouse Foxp3 (BD Biosciences), PE- anti-mouse CTLA-4 (eBioscience), FITC-anti-mouse Helios (Biolegend, San Diego, CA, United States), PE-anti-mouse CD11b (BD Biosciences), APC-Cy7 anti-mouse CD8a (BD Biosciences), and Alexa Fluor 700-anti-mouse B220 (eBioscience). The cells were fixed and permeabilized before staining with the eBioscience Foxp3/transcription factor staining buffer set. Flow cytometry was performed on a LSRII flow cytometer (BD Biosciences) and data were analyzed using FlowJo software (FlowJo LLC, Ashland, OR, United States). Gating strategy is included in the Supplementary Figure 1.

To isolate cells from spleens, lymph nodes and tumors, organs were homogenized by a plunger of a syringe and then passing through a 200-mesh sieve. Isolated cells were blocked twice in PBS with 5% FBS and stained with PE-anti-mouse CD 11b and FITC-anti-mouse Ly-6G (Gr-1) obtained from BD Pharmingen (San Jose, CA, USA) for myeloid-derived suppressor cells (MDSC); or with FITC-anti-mouse CD11b, APC anti-mouse F4/80 obtained from Biolegend (San Diego, CA, USA) for macrophages; or with FITC-anti-mouse CD11b, APC-anti-mouse-CD25 and PE-anti-mouse Foxp3 obtained from eBioscience (Thermo Fisher Scientific) for regulatory T (Treg) cells or IgG isotype for 30 min at 4 °C in the dark. Then cells were washed twice with blocking buffer, fixed with 4% paraformaldehyde and analyzed by flow cytometer (FACSCanto II, Becton Dickinson, Franklin Lakes, NJ, USA). Data from 10,000 cells were collected and analyzed. Lymphocytes isolated from spleens were counted, seeded in 96-well plate at a density of 3 × 10⁶/well, activated with PHA (10 μg/mL) and cell supernatant was collected after 24 h incubation at 37 °C. Enzyme-linked immune sorbent assays (ELISA) kits of mouse IL-2, 6, TNF-α and IFN-γ (BD Biosciences, San Jose, CA, USA) were performed according to the manufacturer’s instructions.