Galunisertib

Mouse or human T cells were labeled with CellTrace Violet (Thermo Fisher Scientific), plated in 96‐well plates, and activated with mouse‐ or human‐specific CD3/CD28‐Dynabeads (Thermo Fisher Scientific). Mouse T‐cell medium consisted of RPMI1640 + GlutaMAX (GIBCO), 10% fetal bovine serum (GIBCO), 10 μg/ml Gentamycin (GIBCO), 10 mM HEPES (BioConcept), 1 mM Sodium Pyruvate (GIBCO), 1× MEM‐NEAA (GIBCO), and 50 μM beta‐mercaptoethanol (GIBCO). Human T‐cell medium consisted of RPMI1640 + GlutaMAX, 8% human serum (Biowest, France), and 10 μg/ml Gentamycin. Where specified, small molecule inhibitors (see below), neutralizing antibodies (Appendix Table S3), and myeloid cells (neutrophils, monocytes, or macrophages) were added within 1 h after plating of T cells. Small molecule inhibitors were Acetylsalicylic acid (Sigma), Adenosine receptor 2A inhibitor (SCH58261; Cayman Chemicals), Galunisertib (LY2157299; Cayman Chemicals), L‐NMMA (Sigma), NorNOHA (Merck‐Millipore), MMP inhibitor (GM6001; Merck‐Millipore), and SB431542 (Cayman Chemicals). Cells were harvested after 3 days of co‐culture and stained with anti‐TCRβ or anti‐CD3‐specific antibodies (Appendix Table S3) and analyzed on a LSR Fortessa FACS machine. T cells with CellTrace Violet intensity below freshly stained controls were scored as proliferated T cells.

Antibodies: mouse anti-vimentin, mouse anti-cytokeratin, mouse anti-CD45, mouse anti-alpha smooth muscle actin (αSMA); rabbit anti-CD11b, rabbit anti-N-cadherin (Abcam, Cambridge, MA, USA); mouse anti-FoxP3-AF647, mouse IgG-AF647 isotype antibodies (BD Biosciences, San Diego, CA, USA); rabbit anti-T-bet (Cell Signaling, Danvers, MA, USA); chicken anti-mouse AF-488-conjugated, goat anti-rabbit AF-594-conjugated (Thermo Fisher Scientific, Waltham, MA, USA). Cytokines: human recombinant interleukin-2 and -12 (IL-2, IL-12), transforming growth factor beta-1 (TGFβ1) (Peprotech EC, London, UK). Drugs: LY2157299 (galunisertib, Cayman Chemical Company, Ann Arbor, MI, USA).

Galunisertib (LY2157299, Cayman Chemical Company) was administered at 10 mg/kg twice daily by gavage after the induction of the model until day 2, when we administered DDC diet as stated. LY2157299 was prepared in 10% polyethylene glycol, 5% DMSO and 85% saline vehicle. Control mice were administered only with the vehicle (PEG, DMSO, Saline).