Fig 2: TGF-β/PGE2 imbalance post-RT immobilized DCs within liver tumors and worsened prognosis. (A) Bioinformatic analysis concerning the prognostic significance (overall survival) of the TGFB1/COX2 gene ratio in different types of human cancers based on the Kaplan-Meier plotter database. (B) The secretion levels of TGF-β and PGE2 in the supernatant of irradiated Hepa1-6 cells based on ELISA; N = 3; *p < 0.05. (C) The expression of CD40, CD80, and CD86 in CD11c+ cells was detected using flow cytometry. (D) The effects of TGF-β/PGE2 balance on homing of DCs to tumor-draining LNs determined using bioluminescence imaging. N = 5 in each group; *p < 0.05. The above data are representative results from two independent experiments.

Fig 3: IL-1β establishes an anti-fibrinolytic gene expression profile in 4G4G primary endothelial cells (ECs). (A) Transcriptional activation of the human PAI-1 promoter by recIL-1β. HUVEC cells were transiently transfected with the PAI-1 4G and 5G luciferase reporter plasmids. Cells were treated with or without IL-1β for 24 hours before lysis. Firefly luciferase activity was normalized to Renilla luciferase activity and is expressed as fold change to controls. Data shown are the mean ± SEM of triplicates from a representative experiment (n=3/group). (B–G) Fold change in PAI-1 (B), tPA (C), and uPA (D), KLF2 (E), NFκB (F), and TGFb (G) expression in cultured 4G4G, 4G5G, or 5G5G ECs stimulated with rec IL-1β (two independent experiments using two different cell origins per genotype were performed; data shown are from one experiment; n=4/group). The expression of the indicated genes is normalized to the endogenous reference β-actin and presented as a relative fold change to expression in the control expression of each genotype according to the comparative Ct method (2−ΔΔCt). (H) Immunoblot of plasmin/α2-antiplasmin complex (PAP) in an equal volume of supernatants of EC cultures. (I) Band intensity quantified of the G blot, whereby each recIL-1β sample was normalized to its control samples. (J–L) A representative immunoblot of Plg using supernatants from cultures treated with or without (control) IL-1β showed angiostatin fragments (J) after loading an equal volume of supernatants from EC cultures. Band intensity quantified of the I blot, whereby each recIL-1β sample was normalized to its control samples, showing the Plg cleavage fragment angiostatin at 38 kDa (K) and 50 kDa (L). All western blots were performed at least twice with similar results. # p<0.05; ## p<0.01; * p<0.05; ** p<0.01; *** p<0.005; n/s, not significant. In vitro data were presented as box plots to discriminate in vivo data from the following in vitro data. All experiments were done in triplicate, and two cell lines for each genotype were used.

Fig 4: IL-35-triggered autocrine TGF-β drives NK cell dysfunction and conversion into ILC1-like cells.a TGFB1 expression in total NK cells cultured for 2 and 4 days in IL-2 with or without IL-35 (analyzed from our scRNA-seq dataset). b Supernatants from NK cells cultured for 24 h in the presence of medium versus IL-12 and IL-18 with or without IL-35 were collected to quantify active TGF-β1 by ELISA. Mean values ± S.D are shown (n = 7 individual donors). c Representative images of NK cells after 7 days of culture in IL-2 with or without IL-35 and a TGF-βR inhibitor (Galunisertib). d Representative flow cytometry plots (upper) and quantification of IFN-γ, T-BET, and EOMES expression (lower) in NK cells at 24 h of culture in IL-12 + IL-18 with or without IL-35, TGF-βR inhibitor (Galunisertib) or anti-TGF-β1/2/3 neutralizing antibody. Results are expressed as relative MFI for each marker compared to control condition without IL-35. Mean values ± S.D are shown (n = 4 to 5 individual donors). e Representative flow cytometry plots (left) for IFN-γ expression in NK cells after 5 days in culture with IL-2 with or without IL-35 and TGF-βR inhibitor (Galunisertib) and quantification (right) of % IFN-γ+ cells in the same culture conditions. Mean values ± S.D are shown (n = 4 individual donors). f Representative flow cytometry plots (left) for CD9 and CD103 expression in NK cells after 8 days in culture with IL-2 with or without IL-35, TGF-βR inhibitor (Galunisertib) or anti-TGF-β1/2/3 neutralizing antibody and quantification (right) of % CD9+ CD103+ cells in the same culture conditions. Mean values ± S.D are shown (n = 3 to 6 individual donors). Statistical significance was determined using paired T test. Source data are provided as a Source Data file.