Chemokine Cx3cl1 mediates the CIT9-RFP–driven immunosuppressive TME. (A) ELISA-based quantification of CX3CL1 using tumor lysates of CIT6-YFP and CIT9-RFP single-population tumors (n = 3). (B) Tumor growth curves of CIT6-YFP tumors overexpressing CX3CL1 (CIT6-YFP^Cx3cl1) and empty vector (CIT6-YFP^Empty). n = 9–10. (C and D) Infiltration of total macrophages (C; n = 20–21) and macrophage subsets (D; n = 11) in CIT6-YFP^Empty and CIT6-YFP^Cx3cl1 tumors. (E) Expression of CX3CR1 on macrophage subsets in CIT6-YFP^Cx3cl1 tumors, measured as MFI from flow cytometric analysis. n = 11. (F) Frequency of neutrophils and monocytes in CIT6-YFP^Cx3cl1 tumors (n = 20–21). Data in C–F are a representations or combinations of two independent experiments. (G) CD4 and CD8 T cell suppression assay using bone marrow–derived monocytes treated with supernatants of CIT6-YFP^Empty or CIT6-YFP^Cx3cl1 cell lines (n = 3–6). Representation of three independent experiments. (H) Tumor growth curves of CIT9-RFP tumors deficient in CX3CL1 (Cx3cl1 KO) and controls (scramble). n = 14–16. (I) Quantification of CX3CL1 using tumor lysates of CIT9-RFP scramble and Cx3cl1 KO tumors (n = 8). (J–M) Frequency of total macrophages (J), macrophage subsets (K), T cells (L), and T cell subsets (M) infiltrating CIT9-RFP scramble and Cx3cl1 KO tumors (n = 14). (N) Treg to Th1 and IFNγ⁺ CD8 T cell ratios in CIT9-RFP scramble and Cx3cl1 KO tumors (n = 14). Experiments for H–N were performed once. Statistical significance was determined by Student’s t test (A, C, D, and I–N), one-way ANOVA (E), and two-way ANOVA with Tukey’s correction posttest (B and G). *P < 0.05, **P < 0.01, and ***P < 0.001. MFI, median fluorescence intensity.