Fig. 3

The lack of CD244 led to an increase in M1 macrophage populations. (A) After inoculating B16F10 cells into CD244^fl/fl and CD244^fl/flLysM^cre mice, the percentage of Ly6C^high macrophages (CD11b⁺Ly6G⁻Ly6C^highF4/80^low) and Ly6C^low macrophages (CD11b⁺Ly6G⁻Ly6C^lowF4/80^high) within tumor was assessed 14 days later. Representative flow cytometry plots (left) and relative proportion of Ly6C^high macrophages (middle) and Ly6C^low macrophages (right) in CD45⁺ cells were shown. (B-C) Bone marrow cells (B) or tumor-infiltrating CD11b⁺ cells (C) were cultured with M-CSF for 3 days. Presented are a representative flow cytometry plot (1st), proportion of Ly6C^low macrophages (2nd), Mean fluorescence intensity (MFI) of F4/80 (3rd) and absolute Ly6C^low macrophage count (4th). (D-E) Monocytes were isolated from bone marrow of CD45.1 WT and CD45.2 CD244^−/− mice, stained with CellTrace Far Red (CTFR), and mixed in a 1:1 ratio. This monocyte mixture was then injected directly into B16F10 tumor mass of WT CD45.2 recipient mice. Tumors were harvested after 48 h, and the ratio of CTFR⁺ CD45.1 and CD45.2 Ly6C^low macrophages was determined. (D) Schematic representation of in vivo differentiation experiment. (E) Demonstrated are a representative flow cytometry plots (left) and the proportion (right) of CTFR⁺ CD45.1 and CD45.2 Ly6C^low macrophages within tumors. (F) WT and CD244^−/− bone marrow cells were cultured for 72 h with M-CSF and treated either with an isotype control or an anti-CD48 antibody. The proportion of Ly6C^low macrophages was determined and presented. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; unpaired Student’s t-test (A, B, C) or two-way ANOVA (B, C) or paired Student’s t-test (E, F). Data are representative of three (B, C) or two (E, F) or compiled from three (A) independent experiments