loading . . . Harnessing Inflammatory Monocytes to Overcome Resistance to Anti-PD-1 Immunotherapy Background Resistance to immune checkpoint inhibitors represents a major therapeutic challenge, as less than 50% of patients with melanoma achieve long-term response to immune checkpoint inhibitor therapy. One mechanism of acquired resistance involves somatic mutations, such as loss of beta-2 microglobulin ( B2m ), that enable tumor cells to evade T cell-mediated killing. Methods This study used single-cell RNA-seq, flow cytometry, and ex vivo functional assays to characterize tumor-infiltrating immune cells in antigen presentation-deficient tumors. Tumor-bearing mice were treated with anti-PD-1 or CD40 agonist antibodies and cell depletion or cytokine blocking antibodies to define mechanisms of action. Analysis of published human RNA-seq datasets was performed to dissect the contributions of inflammatory monocytes to patient outcomes. Results We found an increase in immunosuppressive macrophages in B2m -null tumors. We hypothesized that repolarizing myeloid cells may restore control of tumor growth. Treatment with CD40 agonist antibody, which promotes differentiation of monocytes and macrophages towards a proinflammatory phenotype, reduced tumor growth and improved survival in B2m -null melanoma and colorectal cancer models. Unexpectedly, both CD8+ T cells and NK cells, but not CD4+ T cells, were required for the efficacy of CD40 agonist, even though CD8+ T cells cannot directly recognize antigen presentation-deficient tumor cells. Instead, these lymphocytes control tumor growth via secretion of IFNฮณ, as depletion of IFNฮณ inhibited the therapeutic effect of CD40 agonist. IFNฮณ receptor ( Ifngr1 ) expression was required on host cells, not tumor cells, for CD40 agonist-mediated tumor control. Single-cell analysis identified a distinct population of inflammatory monocytes that were enriched for an IFNฮณ response signature in CD40 agonist-treated tumors, suggesting that these cells may be important for tumor control. Analysis of human bulk and single-cell RNA-seq datasets demonstrated that an inflammatory monocyte signature derived from our data was associated with improved patient outcomes and response to immune checkpoint inhibitors. Conclusions These data demonstrate that CD8+ T cells contribute to tumor control even in the absence of direct antigen presentation by tumor cells. More broadly, our work suggests that strategies to activate the effector functions of inflammatory monocytes may limit tumor growth and overcome acquired resistance to immune checkpoint inhibitors. ### Competing Interest Statement PKS is a co-founder and member of the BOD of Glencoe Software, member of the SAB for RareCyte, Reverb Therapeutics and Montai Health, and consultant for Merck. He holds equity in Glencoe and RareCyte. WNH is an employee and holds equity in Arsenal Biosciences. AHS has patents or pending royalties on the PD-1 pathway from Roche and Novartis. AHS is on advisor boards for Elpiscience, Alixia, Monopteros, GlaxoSmith Kline, Janssen, Amgen, Corner Therapeutics, Bioentre, AltruBio, ImmVue and MabQuest. AHS also is on scientific advisory boards for the Massachusetts General Cancer Center, Program in Cellular and Molecular Medicine at Boston Childrens Hospital, the Human Oncology and Pathogenesis Program at Memorial Sloan Kettering Cancer Center, Perlmutter Cancer Center at NYU, the Gladstone Institutes and the Johns Hopkins Bloomberg-Kimmel Institute for Cancer Immunotherapy. She receives research funding from TaiwanBio, unrelated to this paper. She is an academic editor for the Journal of Experimental Medicine. BCM has consulted for Cellarity, LifeOmic, and Telix Pharmaceuticals. * B2m : beta-2 microglobulin MHC-I : major histocompatibility complex I TNF : tumor necrosis factor TAM : tumor-associated macrophage IFN : interferon LPS : lipopolysaccharide agCD40 : CD40 agonist scRNAseq : single-cell RNA-sequencing TME : tumor microenvironment TIL : tumor-infiltrating lymphocyte National Institute of General Medical Sciences, https://ror.org/04q48ey07, T32 GM133364, T32 GM007753 National Cancer Institute, K08CA248960 Burroughs Wellcome Fund, https://ror.org/01d35cw23, CAMS Gene Lay Institute Ludwig Center at Harvard The Mark Foundation for Cancer Research, https://ror.org/00v7th354, ASPIRE award American Cancer Society, https://ror.org/02e463172, Research Scholar Grant PF-24-1316850-01-CD https://www.biorxiv.org/content/10.64898/2026.02.05.704029v1.full
