Fig 2: Podoplanin, CD44 and CD9 are required for FRCs to respond to CLEC-2+ DCs. (A) Immunofluorescence of 3D cultures of indicated FRC cell lines with LPS-stimulated bone marrow-derived DCs (CD45+; magenta). Maximum Z-stack projections of representative images from n=2 biological replicates are shown. Scale bars: 20 μm. (B) Morphology index (perimeter2/4π area) of DCs in interaction with an FRC. Dots represent single DCs. n=40-72 DCs collated from two biological replicates. Error bars represent median with interquartile range. *P=0.0149; ns, not significant (Kruskal–Wallis test with Dunn's multiple comparisons). The y-axis has a log10 scale. (C) Immunofluorescence of 3D cultures of FRC cell lines without (upper row) or with (middle and bottom rows) LPS-stimulated bone marrow-derived DCs (magenta). Maximum Z-stack projections of representative images from n=2 biological replicates are shown. Scale bars: 50 μm. (D,E) F-actin intensity (mean grey value of phalloidin–TRITC staining; D), and morphology index (perimeter2/4π area; E) of indicated FRCs alone (open circles) or in interaction with a DC (closed circles). Dots represent single FRCs. n=36–104 FRCs collated from two biological replicates. Error bars represent median with interquartile range. ****P<0.0001; ns, not significant (two-way ANOVA with Tukey's multiple comparisons).

Fig 3: Combined treatment with Q702 and anti-PD-1 antibody inhibits EMT6 tumor growth in vivo. EMT6 (1 × 106) cells were implanted into the left flank of BALB/c mice. When the tumor size reached 55 mm3, mice were randomized and treated with vehicle or anti-PD-1 antibody (10 mg/kg) intraperitoneally twice a week. Four days later, mice were treated with either Q702 (30 mg/kg) orally once daily or with a combination of Q702 and anti-PD-1 antibody. (A) Dose schedule and tumor growth in vivo are shown. (B) Graph shows changes in mouse body weight over time after treatment. (C,D) Tumor samples were collected on day 21 post-dosing and immune profiling was performed. (C) Graphs show the percentages of tumor-infiltrating CD3 T (CD3+), CD8 T (CD3+CD8+), CD4 T (CD3+CD4+), and myeloid (CD11b+) cells. (D) Graphs show PD-L1 and MHC-I expression levels in tumors (CD45-negative cells). * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA.

Fig 4: Q702 induces changes in immune cell populations in the tumor microenvironment. EMT6 tumor-bearing BALB/c mice were treated with Q702 at 30 mg/kg orally once daily. (A) Tumor volumes were measured 4 and 21 days after the start of treatment, and the results are shown as mean ± SEM. (B,C) Tumor samples were collected on days 5 and 22. (B) The graph shows the percentage of tumor-infiltrating cells (CD45+), myeloid cells (CD11b+), M-MDSCs (CD11b+Ly6C+), macrophages (CD11b+F4/80+), M1 macrophages (MHC IIhighCD206low), M2 macrophages (MHC IIlowCD206high), CD3 T cells (CD3+), CD8 T cells (CD3+CD8+), and CD4 T cells (CD3+CD4+) on days 5 and 22. (C) Graphs show MHC-I and E-cadherin expression levels in tumors (CD45-negative cells). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired Student’s t-test.

Fig 5: Hip1 impedes DLL4 expression on lung DCs via a CD40-dependent mechanismC57BL/6 (CD40+/+) or CD40-/- mice were infected via the aerosol route with a low-dose of Mtb or hip1mutant. Following 2 weeks post-infection, mice were euthanized and lung ex vivo responses were measured using flow cytometry.(A) Fold of infected mice over uninfected mice (from the same mouse strain) for DLL4+ and DLL4+Jagged1+ in the CD103+ DC population.(B) Fold over uninfected for cytokine-positive CD4 T cells.(C) Fold over uninfected for NOTCH receptor-positive CD4 T cells. All CD11b+ DC populations are singlets/live cells/CD45+/CD3-/CD64-F4/80-/MHCII+CD11c+/CD11b+CD103-. All CD103+ DC populations are singlets/live cells/CD45+/CD3-/CD64-F4/80-/MHCII+CD11c+/CD11b-CD103+. All T cell populations are singlets/live cells/CD3+/CD4+. Data were analyzed using a one-way ANOVA with a correction for multiple comparisons. Data are presented as fold over uninfected mean ± SD. Data are representative of 2 independent experiments. Statistical significance p value key is the following: * = = 0.05, ** = = 0.01, *** = = 0.001.