Fig 2: DU325 treatment hampered the viability of splenocytes, both CD11b+/Ly6C+ and CD11b+/Ly6G+ splenic MDSCs. Cells were treated with 5 µM, 1 µM, 200 nM, 40 nM, and 20 nM DU325 for 72 h and assayed by the resazurin viability test (A). The percentage of AnnV+/PI+ late apoptotic cells was increased after treatment with 200 nM DU325 for 72 h (B). The immunofluorescent staining showed a reduction of both monocytic CD11b+/Ly6C+ and granulocytic CD11b+Ly6G+ MDSCs treated with 200 nM DU325 for 72 h ex vivo (C). Data are shown as arithmetic mean values ± standard deviation from triplicate experiments. Statistical significance was set to *** p < 0.001.

Fig 3: The a-PD-L1 treatment reduces the PD-L1+ cells in TME.B6 mice were intraperitoneally inoculated with 5 × 105 MC38-luc cancer cells and treated with VV and/or a-PD-L1 as described. Tumour-bearing mice were killed at day 5 post first treatment and primary tumours were collected and analysed to determine the PD-L1+ CD45- cells (a,b), PD-L1+ DC (defined as CD45+ CD11b+CD11c+Ly6G-PD-L1+) (c), PD-L1+G-MDSC (defined as CD45+CD11c-CD11b+Ly6G+Ly6clowPD-L1+) (d), PD-L1+ M-MDSC (defined as CD45+CD11c-CD11b+Ly6G-Ly6chiPD-L1+) (e), PD-L1+ TAM1 (defined as CD45+CD11c-CD11b+Ly6G-F4/80+CD206-PD-L1+) (f), PD-L1+ TAM2 (defined as CD45+CD11c-CD11b+Ly6G-F4/80+CD206+PD-L1+) (g). Of note, the anti-PD-L1 antibody clone 10F.9G2 was used for therapy while clone MHI5 was used for subsequent analysis. Data were analysed using Student's t-test (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

Fig 4: Identification and quantitative analysis of neutrophils in the airways and the lung vessels. (A) The representative three-dimensional image of the lung of mouse that received i.p. RBD injections and the o.ph. application of 100 nm particles in the RBD solution. The large vessels (CD106, magenta), the airways (Streptavidin, grayscale), and 100 nm particles agglomerates (FluoSpheres, red) are represented via volume rendering. Scale bar 1000 µm. (B) The higher magnification of the region boxed in (A) demonstrating the vessel (magenta) and CD11b+ cells (light blue) as the frontal (left image) and the lateral (right image) projections. Scale bar 100 µm. (C) The image of the region boxed in (B) showing the vessel and CD11b+ cells via surface rendering. Scale bar 40 µm. (D,E) Representative three-dimensional images of the regions of conducting airway mucosa of the mouse with induced RBD-specific antibodies (D) and of the PBS-injected mouse (E) 24 h after the application of 100 nm particles in the RBD solution. 100 nm particles (FluoSpheres, red), epithelial and smooth muscle cells (phalloidin, grayscale), neutrophils (Ly6G, light blue) and CD11c+ cells (green) are represented as the frontal (upper images) and the lateral (lower images) projections. Scale bar 30 µm. (F,G) The quantitative analysis of neutrophils in the airway mucosa (F) or BAL (G) of mice that were injected with RBD (gray bars, black circles) or with PBS (open bars, black circles) 24 h after the application of 100 nm particles in the RBD solution, and in intact mice (NM, open bars, open circles). (H,I) The levels of IgA (H) and IgG (I) in the BAL fluids of mice that were injected with RBD (gray bars, black circles) or with PBS (open bars, black circles) 24 h after the application of 100 nm particles in RBD solution, and in the intact mice (open bars, open circles). The data are shown as medians and i.q.r. for n = 4 mice per group. A significant difference between the indicated group and the intact mice was detected using the Mann–Whitney test *: p = 0.05.

Fig 5: ZQT induced apoptosis in G-MDSCs in TME and inhibited its immunosuppressive activity by activating the STAT3/S100A9/Bcl-2/caspase-3 signaling pathway. Mice were treated with anti-Ly6G neutralizing antibody or isotype control by intraperitoneal injection for 14 days before surgery, after which model mice were given maintenance treatment every other day. (a, b) The bioluminescence images of model mice on day 7 and day 28 after surgery. (c) Survival curves. (d, e) The percentage of G-MDSCs, CD3+ T cells, CD4+ T cells, and CD8+ T cells in tumor tissue were analyzed by flow cytometry. (f, g) CD3+ T cells isolated from the tumor tissue of tumor-bearing C57BL/6 mice in different groups were cocultured with LLC cells for 4 h before they were incubated with CD3, CD8, and CD107a antibodies. Events shown were finally gated on CD3 and CD8. (h) ELISA analysis for the expression of IFN-? in cells from tumor tissue. (i) RT-qPCR analysis of Arg-1, iNOS, and S100A9 gene expression and flow cytometry for ROS production. (j) IHC staining for Ly6G and CD8 in tumor tissue (scale bar: 50 µM). n = 5. Data are expressed as the mean ± SD. n/s: nonstatistical significance. *p < 0.05; **p < 0.01; ***p < 0.001.