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Background Immunosuppressive tumor-associated macrophages (TAMs) dampen the anti-tumor immune response and drive resistance to immune checkpoint blockade (ICB). 1 Previous studies have identified folate receptor β (FRβ) as a biomarker of activated TAMs typically associated with worse prognosis.2–6 FRβ (gene: Folr2) is a high affinity folate receptor expressed uniquely on myeloid cells.7 Folate is a requisite coenzyme for one-carbon (1C) metabolism, which is critical for the maintenance of cellular redox balance, nucleotide synthesis, and global methylation reactions.8 9 The importance of FRβ-mediated folate uptake for pro-tumor TAM biology is unknown. Here we show FRβ-mediated 1C metabolism supports immunosuppressive macrophage functions through the control of reactive oxygen species (ROS).Methods We generated Folr2 -/- mice on the C57BL/6J genetic background. B16-OVA melanoma tumors were subcutaneously implanted into mice and immune infiltrates were characterized via flow cytometry. Bulk RNA-seq was conducted on flow-sorted TAMs. For in vitro experiments, bone-marrow derived macrophages (BMDMs) were cultured in M-CSF and physiological folate concentrations found in tumor-interstitial fluid. BMDMs were functionally profiled using intracellular cytokine staining, ELISA, bulk RNA-seq, ROS dye staining (CellRox, MitoSox), metabolomics, and CD8+ T-cell coculture studies. Analysis of single-nuclei RNA-seq and spatial transcriptomics was performed on myeloid cells from metastatic melanoma patient biopsies.Results FOLR2 + TAMs were enriched in melanoma biopsies from ICB-unresponsive patients, supporting an immunosuppressive role for FOLR2+ TAMs. In Folr2-/- mice, melanoma tumor growth was reduced by 70% compared with controls, with increased pro-inflammatory TAM differentiation and tumor infiltration by cytotoxic CD8+ T-cells. In vitro, Folr2 -/- BMDMs produced more pro-inflammatory cytokines and were unable to inhibit T cell activation compared to wild-type BMDMs. These phenotypes can be rescued by increasing folate in the media, suggesting Folr2 is required for immunosuppressive macrophage function due to increased folate uptake. Interestingly, Folr2 -/- BMDMs had increased intracellular ROS, were enriched for oxidative stress gene signatures, and possessed lower reduced glutathione metabolite pools, indicating a dysregulation of redox homeostasis. Transcriptionally, Folr2 -/- BMDMs upregulated NFkB signaling and other pro-inflammatory pathways that are positively regulated by ROS accumulation.10 Inhibiting ROS via antioxidant supplementation reversed the increased pro-inflammatory cytokine production and decreased T-cell suppression phenotypes observed in Folr2 -/- BMDMs.Conclusions Together, these data demonstrate that FRβ-mediated 1C metabolism is critical for the immunosuppressive functions of TAMs through mitigation of oxidative stress ( figure 1). Our studies underscore the importance of 1C metabolism as a pivotal regulator of TAM biology and potential target for TAM-directed cancer immunotherapies.Acknowledgements We would like to thank the following core facilities for their expertise and experimental assistance: UNC-CH Flow Cytometry Core, UNC-CH Animal Models Core, UNC-CH LCCC Translational Genomics Lab, and UNC-CH Microscopy Services Laboratory. This work was supported by Burroughs Wellcome Fund Career Award for Medical Scientists, NIH K08CA248960, V Foundation Scholar Award, and Melanoma Research Alliance Young Investigator Award.References Guerriero J. Macrophages: the road less traveled, changing anticancer therapy. Trends Mol Med. 2018;24(5):472-489.Puig-Kröger A, Sierra-Filardi E, Domínguez-Soto A, et al. Folate receptor β is expressed by tumor-associated macrophages and constitutes a marker for M2 anti-inflammatory/regulatory macrophages. Cancer Res. 2009;69(24):9395-9403.Tie Y, Zheng H, He Z, et al. Targeting folate receptor β positive tumor-associated macrophages in lung cancer with a folate-modified liposomal complex. Sig Transduct Target Ther. 2020;5(1):6.Rodriguez-Garcia A, Lynn R, Poussin M, et al. CAR-T cell-mediated depletion of immunosuppressive tumor-associated macrophages promotes endogenous antitumor immunity and augments adoptive immunotherapy. 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Cell Res. 2011;21(1):103-115.Ethics Approval All mouse studies were approved by University of North Carolina at Chapel Hill Division for Comparative Medicine and Institutional Animal Care & Use Committee (Protocol #24-230). Human specimens were collected under IRB #LCCC13-2225.Abstract 799 Figure 1Folate receptor beta-mediated folate uptake suppresses pro-inflammatory macrophage functions