Figure 6. TAMs from human clear cell RCC patients express IRF8 and IRF8-activated genes.

(A) Representative flow plots and quantification of tumor-infiltrating DC1s (green, CD45⁺Lin⁻CD14⁻HLA-DR⁺CD141⁺), DC2s (orange, CD45⁺Lin⁻CD14⁻HLA-DR⁺CD1c⁺), classical monocytes (pink, CD45⁺Lin⁻CD14⁺HLA-DR^low), and TAMs (red, CD45⁺Lin⁻CD14⁺HLA-DR^high) in human clear cell RCC tumor samples as determined by flow cytometric analysis (Lin = CD15, CD19, CD3, CD56, dead cells). Fold differences in abundance between TAMs and DC1s, TAMs and DC2s are shown. Each circle represents one patient tumor sample, data shown as mean +/− SEM, n=56 (one-way ANOVA with multiple comparisons, **** = p<0.0001).

(B) Representative plot showing expression of IRF8 in tumor-infiltrating DC1s (green), DC2s (orange), monocytes (pink), TAMs (red), and fluorescence minus one (FMO) TAM sample (gray), as determined by flow cytometric analysis.

(C) IRF8 MFI fold change in TAMs relative to tumor-infiltrating monocytes. IRF8 MFI is relative to monocyte MFI (n=21), data shown as mean +/− SEM (paired student’s t test, two-tailed, ** = p<0.01).

(D) Enrichment of human orthologs of IRF8-activated mouse TAM genes among human TAM-specific genes relative to human monocytes. Tumor monocytes (n=4) and TAMs (n=5) were sorted from human RCC samples, underwent RNA sequencing and differential gene expression analysis. The IRF8-activated genes with significantly higher expression identified in mouse TAMs were converted to their human orthologs, and the enrichment of these IRF8-activated genes was tested among human TAM-specific genes.