α cd11b

4T1 mouse breast cancer model was constructed. Tumor‐bearing mice were treated with PBS, Dox‐aPD‐1 (5 mg kg⁻¹ Dox, single injection), Dox@HFn Gel (5 mg kg⁻¹ Dox), and Combo (5 mg kg⁻¹ Dox), respectively. aPD‐1 (100 µg per mice) was intraperitoneal administered on days 1, 3, and 5. Lymph nodes were harvested in tumor‐bearing mice on the 9th day after various treatments. The lymph nodes were ground into single cells. The cells in lymph node were blocked with CD16/32 antibodies and then labeled with fluorescent‐conjugated antibodies (α‐CD11c (Biolegend, 506904), α‐IA/IE (Biolegend, 107622), α‐CD80 (Biolegend, 107406), α‐CD86 (Biolegend, 105012), α‐CD45.2 (Biolegend, 109838) and α‐CD11b (Biolegend, 101259)) for 30 min. The cells in lymph node were detected by flow cytometry (BD Biosciences, USA).

For analysis of mouse cell phenotypes, the following monoclonal antibodies were used: α-CD3 (clone: 145-2C11), α-CD4 (RM4-5), α-CD8α (53–6.7), α-CD25 (PC61), α-CD44 (IM7), α-CD62L (MEL14), α-CD90.1 (OX-7), α-CD40L (MR-1), α-CD11c (HL3), α-CD11b (M1/70), α-Gr1 (RB6-8C5), α-Ly6C (HK1.4), α-Ly6G (1A8), α-F4/80 (BM8), α-IFN-γ (XMG1.2), α-IL-17A (TC11-18H10), α-GM-CSF (MP1-22E9; all from BioLegend) and α-IL-22 (AM22-3; provided by J.C. Renauld). For staining of lipid rafts, biotin-conjugated cholera toxin subunit B (Molecular Probes) was used together with fluorochrome-conjugated streptavidin (BioLegend). Dead cells were always excluded by staining with 7-AAD (BioLegend) or 4,6-diamidino-2-phenylindole (Sigma). All antibodies were used at 1 μg per 10⁶ cells. For intracellular cytokine staining, cells were stimulated for 5 h with phorbol 12-myristate 13-acetate (10⁻⁷ M; Sigma) and ionomycin (1 μg ml⁻¹; Sigma), in the presence of brefeldin A (10 μg ml⁻¹; Sigma) for the last 3 h of culture. Cells were fixed with 4% (wt/vol) paraformaldehyde and permeabilized with 0.5% (wt/vol) saponin (Sigma). Eight-colour staining was performed with the appropriate combinations of antibodies conjugated to fluorochromes. Samples were acquired on a FACSCanto II (BD Biosciences) and analysed with FlowJo software (TreeStar) and when necessary using the Boolean gating strategy65 (link).

Tumor singlets and MGFP suspensions in FACS buffer were treated with α-CD16/32 (clone 93, 1:200; BioLegend) at 4 °C for 20 min to block Fc receptors, followed by incubation with fluorescent-labeled antibodies at 4 °C in the dark for 25 min in FACS buffer. The following antibodies were used: α-CD11b (clone M1/70, 1:100; BioLegend), α-F4/80 (clone CI:A3-1, 1:20; Bio-Rad Laboratories) for MGTRMs, α-CD11c (clone N418, 1:100; eBioscience, San diego, CA), α-MHC-II (clone M5/114.15.2, 1:100, BD Bioscience) for dendritic cells, α-CD11b (1:100), α-Ly-6c (clone HK1.4, 1:200; BioLegend) for tumor-infiltrating monocytes, α-CD8a (clone 53-6.7, 1:100; eBioscience) for T-cells, and α-NK1.1 (clone PK136, 1:100; eBioscience) for natural killer cells. The antibody-labeled cells were sorted and analyzed using an SH800Z fluorescence activated cell sorter and EC800 flow cytometry analyzer (Sony, Tokyo, Japan), respectively.