25-Hydroxycholesterol regulates lysosome AMP kinase activation and metabolic reprogramming to educate immunosuppressive macrophages

Macrophages are critical to turn noninflamed “cold tumors” into inflamed “hot tumors”. Emerging evidence indicates abnormal cholesterol metabolites in the tumor microenvironment (TME) with unclear function. Here, we uncovered the inducible expression of cholesterol-25-hydroxylase (Ch25h) by interleukin-4 (IL-4) and interleukin-13 (IL-13) via the transcription factor STAT6, causing 25-hydroxycholesterol (25HC) accumulation. scRNA-seq analysis confirmed that CH25Hhi subsets were enriched in immunosuppressive macrophage subsets and correlated to lower survival rates in pan-cancers. Targeting CH25H abrogated macrophage immunosuppressive function to enhance infiltrating T cell numbers and activation, which synergized with anti-PD-1 to improve anti-tumor efficacy. Mechanically, lysosome-accumulated 25HC competed with cholesterol for GPR155 binding to inhibit the kinase mTORC1, leading to AMPKα activation and metabolic reprogramming. AMPKα also phosphorylated STAT6 Ser564 to enhance STAT6 activation and ARG1 production. Together, we propose CH25H as an immunometabolic checkpoint, which manipulates macrophage fate to reshape CD8+ T cell surveillance and anti-tumor response. [Display omitted] •TAMs accumulate 25HC to enhance their immunosuppressive function•Lysosomal-accumulated 25HC activates AMPKα through GPR155-mTORC1 complex•AMPKα directly binds to and phosphorylates STAT6 at Ser564, leading to STAT6 activation•Targeting CH25H improves anti-tumor efficacy together with or without anti-PD-1 therapy Cholesterol increases macrophage-mediated inflammation, but how oxysterols control tumor-associated macrophages (TAMs) remains unclear. Xiao et al. show that TAMs exhibit elevated expression of CH25H, resulting in lysosome-accumulated 25HC that activates AMPKα to promote STAT6-dependent ARG1 production. CH25H-deficient macrophages switch “cold tumors” into “hot tumors” and improve anti-PD-1-mediated anti-tumor efficacy.