Fig 1: Combination of SKI‐G‐801 and anti‐PD‐1 synergistically inhibits spontaneous lung metastasis in the 4T1 model. (a) Western blot analysis for pAXL inhibition by SKI‐G‐801 in 4T1 cells. Experiments were repeated twice. (b) pAXL were measured in 4T1 tumor‐bearing syngeneic mice. Mice were treated with SKI‐G‐801 10 mg kg−1 (upper) and 30 mg kg−1 (lower) for up to 24 h. (c) Representative images of H&E‐stained lung tissue demonstrating lung metastases are shown. (d) Total number of metastatic nodules was counted, and tumor area in lung tissue was measured. Lung and percentage of tumor area were calculated with ImageJ (n = 4 or 6 per group, derived from two independent experiments). (e) The proportions of IFN‐γ‐expressing cytotoxic T cells (CD3+CD8+IFN‐γ+) and central memory CD8+ T cells (CD3+CD8+CCR7+), measured by flow cytometry, were significantly increased in the combination group compared to other treatment groups. The dendritic cells (DCs) population (F4/80−CD11c+H2+) and CD80 expression on DCs are shown. Values are indicated as means ± SEM, and P‐values were calculated using the Student’s t‐test; *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001. Flow cytometry analysis was done once.
Fig 2: High granulosity cells in non pregnant uterus are primarily eosinophils.Viable R1- or R2- gated CD45.2+ cells from NP uterus were analysed by FACS stained for APC and granulocytes markers (CD11b, CD24, CD11b, F4/80, Ly6C, Ly6G, MHC class II and CCR2) (A, B and Table 1) and for NK and T cell markers (CD11b, NK1.1, CD3ε, CD8α and CD4) (B and Table 1). The experiment was repeated twice with 6 females each time (A, B). Viable CD45.2+, CD11b+, F4/80+ R1 (C) or R2 (D) cells were sorted by flow cytometry and spun onto Super + glass microscope slides using a Cytospin cytofuge. Cells were fixed with methanol and stained with Wright-Giemsa. Pictures were taken on a Nikon H600L microscope equipped with a DS-Fi2-Nikon camera (C, D). The experiment was performed twice with at least 5 females each time.
Fig 3: Representative flow cytometric analysis of BMDM – T cell coculture(A) Representative gating strategy for analysis of CD45+CD11b+F4/80+MHCII+ macrophages, FasL abundance and CD45+CD11b-CD3+CD4+/− T cells.(B) T cells analyzed by FACS after cell–cell interaction phase in coculture, initially treated with sham or stroke serum.(C) Proportion of F4/80+MHCII+ macrophages in coculture after serum treatment (sham or stroke).(D) FasL expression on coculture macrophages analyzed by FACS after serum treatment. (n=6 per group; 3 independent experiments).
Fig 4: Dysregulated Immune and Inflammatory Responses in Mtb-Infected HO-1−/− Mice(A–M) Cytokine analysis of IL-1β (A), IL-2 (B), IL-3 (C), IL-5 (D), IL-6 (E), IL-10 (F), IL-17A (G), MIP-1α (H), MIP-1β (I), KC (J), GM-CSF (K), G-CSF (L), TGF-β (M) in the BALF of uninfected and Mtb-infected HO-1+/+ and HO-1−/− mice at 6, 12, and 18 weeks.(N and O) Percentage of neutrophils in the BALF (N) and lungs (O) of Mtb-infected HO-1+/+ and HO-1−/− mice at 6, 12, and 18 weeks post-infection.(P–S) Other monocytes and granulocytes in the lungs of Mtb-infected HO-1+/+ and HO-1−/− mice.(P and Q) Representative scatterplot for CD11b+Ly6C+F4/80+ monocytes (P) and percent differences (Q) at 6 weeks post-infection.(R and S) Representative scatterplot for CD11b+Ly6G+F4/80+Gr-1+ granulocytes (R) and percent differences at 12 and 18 weeks post-infection (S).n = 4 for each time point. Statistical testing was performed using the unpaired Student’s t test. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Fig 5: Stromal-Immune Crosstalk Supports the Development of an Immunosuppressive Niche(A) Overview of selected statistically significant interactions between stromal subsets and other cell types using a cell-cell communication pipeline based on CellPhoneDB. Size indicates p values (permutation test, see STAR Methods), and color indicates the means of the receptor-ligand pairs between 2 clusters.(B) Violin plots displaying expression log(TPM+1) of ligands Cxcl12, Csf1, and C3 and cognate receptors Cxcr4, Csf1r, and C3ar1 on respective stromal populations. n = 26 mice.(C) Confocal images of representative tumor-tissue borders. CXCR4, CSFR1, or C3aR expressing macrophages located proximally to CD34+ CAFs (green, F4/80; red, CXCR4, CSF1R, or C3aR; white, PDPN; blue, CD34). Scale bars, 50 μm.(D) Flow cytometric quantification of CXCL12 and C3 expression across compartments of the tumor microenvironment. Each point represents a tumor. CXCL12 n = 42 tumors, C3 n = 12 tumors. One-way ANOVA with Tukey post hoc test.(E) In vivo blockade of C3a in established tumors. Top left: experimental design and treatment regimen; top right: tumor volume (in cubic millimeters) of mice treated with IgG control (blue) or anti-C3a (red); bottom left: myeloid infiltration in day 6 tumors, after 24 h of treatment with IgG or anti-C3a. The number of F4/80 and Ly6C+ Ly6G− cells are shown as a percentage of Cd11b and CD45 cells, respectively; bottom right: the number of tumor-infiltrating CD8+ T cells at day 11, displayed as raw counts normalized to tumor volume (in cubic millimeters). Data presented as means ± SEMs. n = minimum 13 mice. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; t test.(F) Schematic diagram of the dynamic crosstalk identified within the tumor microenvironment.
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