Cd80 apc

Gli1-tdTomato⁺ Mice were euthanized as described above, and aseptically removed kidneys were fixed in 4% paraformaldehyde in PBS for 1 h at 4°C, incubated overnight in 30% sucrose in PBS at 4°C, then embedded in OCT (Fisher Scientific, Hampton, NH). Embedded kidneys were cryosectioned into 5–8-μm sections and mounted onto Superfrost Plus slides (Fisher Scientific). For immunofluorescence staining, sections were washed with PBS, blocked with 10% fetal bovine serum (FBS) in PBS, then stained with fluorescently conjugated primary antibodies against CD206-Alexa Fluor 488 (1:200; Biolegend #141709) and CD80-APC (1:200; Biolegend #104713). Sections were then washed with PBS, stained with 1:5,000 4′,6-diamidino-2-phenylindole (DAPI) and mounted with ProLong Gold (both from Life Technologies, Carlsbad, CA). Images were captured digitally with a Zeiss LSM 880 Airyscan confocal microscope (Oberkochen, Germany).

To investigate if SPION^Dex inhibit DC maturation, iDCs were incubated in the presence of SPION^Dex. Therefore, SPION^Dex at iron concentrations of 50, 100 and 200 µg/mL were incubated with iDCs at a cell density of 10⁶/100 µL in 6 well plates at 37 °C for 48 h. The DC medium containing IL-1β (200 U/mL), IL-6 (1000 µ/mL), tumor necrosis factor (TNF)-α (10 ng/mL, all from ImmunoTools, Friesoythe, Germany) and prostaglandin E2 (PGE2) (1 µg/mL, Pfizer, New York, USA) served as positive control, pure DC medium served as negative control. After 48 h of incubation, the matured DCs (mDCs) were harvested from the 6 well plates by washing with ice-cold PBS-EDTA. The cells were centrifuged at 200 rcf for 12 min, the supernatant removed and the cell number adjusted to 1 × 10⁶/mL with PBS containing 2 vol% fetal calf serum (FCS). To analyze the expression of surface maturation markers, 50 µL cells were stained with fluorescence labelled antibodies (CD83-PE/Cy7, CD40-PerCP/Cy5.5, CD80-APC, CD86-PerCP/Cy5.5 (all from BioLegend, San Diego, CA, USA) in PBS containing 2% FCS for 30 min. Isotype antibodies were used as controls. Subsequently, cells were washed and fixated with 100 µL PBS containing 1% paraformaldehyde. Then, cells were analyzed in flow cytometry.

Bone marrow derived dendritic cells (BMDCs) of C57BL/6 mice were cultured with 20 ng/mL GM-CSF (Peprotech, USA) and 10 ng/mL IL-4 (Peprotech, USA). Media was replaced on day 3; non-adherent and loosely adherent immature dendritic cells (iDCs, routinely 60–80% CD11c⁺) were collected on day 6. Then, iDCs were incubated with free peptide (Tyrp1, M20 or M27, 10 μg/ml) and equivalent nanoparticles (Tyrp1-NP, M20-NP or M27-NP). Cells were collected 48 h later and stained with CD11c-FITC, CD80-APC and CD86-PE antibodies (Biolegend, USA). BD Accuri C6 (BD Bioscience, USA) were used to detect activated DCs.

Cells were incubated 10 min with the TruStain fcX (clone 93) washed and incubated with the antibodies during 20 minutes followed by two washes with PBS. Monoclonal antibodies specific for mouse molecules were purchased from Biolegend: CD3-FITC (clone 17A2), CD3-APC (clone 17A2), CD3-PerCp/Cy5.5 (clone 17A2), CD8-Brillant Violet 421 (clone 53-6.7), CD45-PE (clone 30-F11), CD45-PErCP(clone 30-F11), CD45.1-PE/Cy7 (clone A20), CD45.1-FITC (clone A20), CD103-APC (clone 2E7), CD103-PerCP (clone 2E7), CD69-APC/Cy7 (clone H1.2F3), CD69-APC (clone H1.2F3), CD44-PerCP (clone IM7), IFN-γ-PE (clone XMG1.2), IFN-γ-APC (clone XMG1.2), TNF-α-APC/Cy7 (clone MP6-XT22), CD11b-FITC (clone M1/70), CD207-PE (clone 4C7), XCR1-APC (clone ZET), XCR1-PerCP-Cy5.5 (clone ZET), CD11c-PE/Cy7 (clone N418), MHCII-APC/Cy7 (clone M5/114.15.2), CD24-PerCP-Cy5.5 (clone M1/69), CD80-APC (clone 16-10A1), CD80-PE/Cy7 (clone 16-10A1), CD86 Brilliant Violet 421 (clone GL-1), CCR7-PE/Cy7 (clone 4B12), IL-2-PE/Cy7 (clone JES6-5H4) IL-12/23-APC (clone C15.6), granzyme B-APC (clone GB11) and viability dye Zombie Aqua (ref 423101). Samples were acquired in a FACSCanto II cytometer (BD Bioscience) and data were analyzed using FlowJo version X.0.7 (Tree Star, Inc.). Gating strategies for all flow cytometry experiments are shown in Supplementary Fig. 4.

One week after the last treatment, lymph nodes, spleens and tumors from murine hepatocellular carcinoma models were collected to detect the immune responses initiated by R837/Nab-PTX/ALG. Lymph nodes and spleens were ground, filtered to make single cell suspensions (0.5–1 × 10⁶ cells mL⁻¹). Tumors were firstly cut into small pieces and incubated with collagenase type IV (1 mg/mL, Sigma, USA) at 37 ℃ for 3–4 h, before filtered to make single cell suspensions (0.5–1 × 10⁶ cells mL⁻¹). All tested antigens are expressed on cell membranes, so all samples were stained with specific antibodies for 20 min in 4 °C in dark, and then washed before analysis. The following monoclonal antibodies (mAbs) were used for flow cytometry and purchased from Biolegend: CD11c-FITC (5 μg/mL), CD80-APC (2 μg/mL), CD86-PE (2 μg/mL), CD3-FITC (5 μg/mL), CD4-PerCP/Cy7 (2 μg/mL), CD8-PE-Cy5 (2 μg/mL), CD44-PE (2 μg/mL), CD62L-APC (2 μg/mL).