Fig 2: Robust antitumor immune responses triggered by IPI549@HMP-augmented RT.a–f Representative flow cytometric analysis and relative quantification of CTLs (CD8+CD3+CD45+) (a, b), MDSCs (CD11b+Gr-1+CD45+) (c, d) and TAM-M2 (CD206hiCD11b+F4/80+CD45+) (e, f). g, h Quantification by flow cytometry of CD8/Treg (g) and M1/M2 ratios (h). i Representative polychromatic immunofluorescent staining images of tumors from three biologically independent samples showing DAPI (blue), CD206+ (green), Foxp3+ (orange) and CD8+ (red) cells infiltration for Control and IPI549@HMP + RT groups. j, k Cytokine levels of TNF-a (j) and IFN-? (k) in the serum after varied treatments. G1, Control; G2, IPI549@HMP; G3, RT; G4, HMP + RT; G5, IPI549@HMP + RT. RT, radiotherapy; CTLs, cytotoxic T lymphocytes; Tregs, regulatory T cells; MDSCs, myeloid-derived suppressor cells; TAMs-M2, M2-like macrophages; TAMs-M1, M1-like macrophages. Data were expressed as means ± SD (n = 3 biologically independent samples in b, d, f, g, h, j, and k). Statistical difference was calculated using two-tailed unpaired student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig 3: Characterization of immune cell infiltrates into I/R injury. a Example maximum intensity projections (MIPs) of Ly-6G positive (Ly-6Gpos) neutrophils after 24 h (left) as well as Ly-6Gpos neutrophils and F4/80 positive (F4/80pos) macrophages after 5 d (middle; right). Depicted are CD31 (green, top row) and Ly-6G (turquoise) or F4/80 (purple) signals (middle row) together with a merge (bottom). Dashed white line marks the border of the CD31curly region. Magnification: ×3.2 (24 h), 4 × (5 days). b Immune cell quantities (top neutrophils, bottom macrophages) determined by flow cytometry (white, filled dots) or based on LSFM (white, empty dots) analysis after 24 h and 5 d of reperfusion, as well as under baseline condition (n = 4–5; median ± interquartile range; Kruskal–Wallis test with Dunn’s correction). c Representative 3D visualization of Ly-6Gpos cell distribution across whole murine hearts. Heart structure (grey,) and knot (white) are shown for orientation. Ly-6Gpos cell populations are colored in respect to their localization in either AAR (red), I/R injury (yellow), CD31curly (green) or outside the above-mentioned structures (turquoise). d Relative cell density ± 30 µm from the respective volume border (n = 5; median; Kruskal–Wallis test with Dunn’s correction). e 3D visualization of F4/80pos signals depicted as a surface rendering (purple) in relation to heart surface (left; heart volume in grey) or CD31curly surface (right; CD31curly in green) after 5 d of reperfusion. The knot (white) is visualized for orientation. Scale bar values in µm. This finding was verified in 5 mice. Source data are provided as a Source Data file

Fig 4: A, Absolute number of AMs in bronchoalveolar lavage fluid (BALF). B, Representative flow cytometry plots of the gating strategy, (upper panel), live CD45+ and Gr1- were then analyzed for CD11b and F4/80 expression (lower panel). Gates were set based on background staining in fluorescence minus one controls. C, Data from three independent experiments are summarized as mean ± SEM and plotted in the bar graph (control, n = 5; vehicle, n = 8; clodronate IV n = 8; clodronate IT, n = 7). *P < .05, **P < .01, ****P < .001 (one-way ANOVA, Tukey's multiple comparisons). D, Representative histological sections of the lungs showing Ki67 positive cellular staining. Scale bar = 100 µm, insert 20 µm. E, Percentage of Ki67+ cells. Ten regions of interest of 1 mm2 per mouse were analyzed with a minimum of 100.000 cells counted per condition. Values represent mean ± SEM; n = 4 mice per group. AM, alveolar macrophage; ANOVA, analysis of variance; IT, intratracheal; IV, intravenously

Fig 5: Loss of Lats1/2 induces M2 macrophage polarization.P and PL mice were treated with 180 mg/kg/d of TAM for 5 consecutive days; cells were isolated from both groups 4 days later (n = 3). (A) F4/80 IHC staining in P and PL mice on Day 4 after TAM injection. (B) M2 macrophage-specific genes Ym-1, Ccl17, Tgf-ß and IL10 were elevated in sorted macrophages of PL mice. (C) M1 macrophage-specific genes Tnf-a, Socs1, and Nos2 were not significantly changed. **P < 0.01, *P < 0.05. Underlying numerical values can be found in S1 Data. IHC, immunohistochemistry; LATS1, large tumor suppressor 1; P, control; PL, double knockout; TAM, tamoxifen.