Figure 2 from Tumoral Interferon Beta Induces an Immune-Stimulatory Phenotype in Tumor-Associated Macrophages in Melanoma Brain Metastases

YUMM5.2 Ifnb1eGFP-derived IFNβ modulates macrophage polarization and function. A–D, Quantitative flow cytometry analysis of myeloid populations in the YUMM5.2 Ifnb1eGFP TME after control diet (light pink) or Dox diet (dark red). Displayed as percentage of parental population (n = 6 for each group). E, Ratio of TAM polarization states in the YUMM5.2 Ifnb1eGFP TME after control diet or Dox diet. Displayed as mean percentage per group (n = 6 for each group). F, Quantitative flow cytometry analysis of TAM polarization states in YUMM5.2 eGFP tumors after Dox diet compared to control diet. Displayed as log₂ fold change (Log₂FC; n = 6 for each group). G and H, Quantitative flow cytometry analysis of CD206 (G) and CD86 (H) in bone marrow-derived and M2-like polarized macrophages after 72 hours of indicated treatments. Displayed as mean fluorescence intensity (MFI) in G or percentage of macrophages in H. sn, supernatant, collected after Dox pretreatment (n = 3). I–L, M2-like polarized BMDM generated from C57BL6/J mice were pretreated as indicated and subsequently co-cultured 1:1 (J and K) or 1:10 (L) with CTFR-stained T cells isolated from BALB/c mice. T cells were analyzed on day 5 by flow cytometry. Displayed as percentage of CD8⁺ T cells (J and K) or of CD4⁺ T cells (L). For gating strategies see Supplementary Figs. S2 and S3G. M1-like macrophages were defined as MHCII⁺CD206⁻, intermediate macrophages as MHCII⁺CD206⁺ and M2-like macrophages as MHCII⁻CD206⁺ in D–F. Data are expressed as median + IQR and individual values for A–D, G, and J–L, mean for E and individual values and mean for F and H. Statistical significance was determined by paired t test for A–D, by Mann–Whitney U test for G and by one-way analysis of variance (ANOVA) with Tukey post hoc testing for H and J–L. ES, effect size. (I, Created with BioRender.com.)