Rebound Polarization of Monocyte-derived Tumor Macrophages Under Repeated Anti-CD40 Immunotherapy II

To confirm that both pro-inflammatory and immunosuppressive subsets arise from common monocyte precursors, we employed Ms4a3-Cre Rosa26-tdTomato fate-mapping mice. Flow cytometry of dissociated tumors showed that most F4/80+ macrophages were tdTomato+, with subpopulations differing in MHC class II expression. scRNA-seq of sorted CD45+tdTomato+ cells confirmed a bifurcated differentiation trajectory that yields pro-inflammatory, MHC II–high macrophages (Cxcl9+, H2-Eb1), as well as Spp1+ Arg1+ immunocompromised phenotypes. Overall design: scRNA-seq of sorted CD45+tdTomato+ cells