Polarization profile of TAMs in DLBCL. (A) Sections of DLBCL biopsies were stained by immunofluorescence for CD68 and CD163. Thin and thick arrows show an M2 and non-M2 macrophage, respectively. Photographs are representative of 10 patients. Scale bar, 10 µm. (B) Signal intensity (40−Ct value) of the indicated genes in CD163⁺ (left panel) and CD86⁺ (right panel) single cells. The percentage of CD163⁺ M2 and CD86⁺ M1 macrophages coexpressing the indicated M1 and M2 genes is shown on top of the panels. (C) The number of M1/M2 genes expressed in CD163⁺ M2 and CD86⁺ M1 single cells. (D) Phenotypic description of CD86⁺CD163⁺ noncanonical M1M2 macrophages at the mRNA level. (E) Expression levels (40−Ct value) of M1 genes in CD86⁺ M1 macrophages compared with CD86⁺CD163⁺ M1M2 cells (left panel) and expression levels of M2 genes in CD163⁺ M2 macrophages compared with CD86⁺CD163⁺ M1M2 cells (right panel). (F) Frozen DLBCL cell suspensions were analyzed by flow cytometry for expression of canonical CD86 M1 and CD163 M2 markers on gated CD68⁺ TAMs (left panel). Staining with control antibodies to determine the gating is also shown. Mean ± standard deviation (SD) fluorescence (fluo.) intensity for CD86 on the 3 TAM subtypes (middle panel). Surface expression of receptors in noncanonical M1M2 macrophages (right panel). Shaded line graphs represent isotype control. Data are mean ± SD and are representative of the 3 DLBCL patients shown in Figure 5B. *P < .05. IL, interleukin; TGF-β1, transforming growth factor β1; VEGFA, vascular endothelial growth factor A.