rNAMPT induces monocytes to differentiate into macrophages with M2 features. (A) PBMC preparations from CLL patients were plated in complete medium with or without rNAMPT (200 ng/mL). After 5 days, cells were fixed and stained for CD11b. Original magnification ×20. Middle panel represents a zoomed area of the same sample. CD11b staining was also confirmed by immunofluorescence. The panels on the right show Giemsa staining to evaluate cellular morphology. (B) Cumulative data showing the number of CD11b⁺ cells in at least 4 ×20 fields of 9 different samples. (C-D) Box plots reporting the mRNA (C) or the protein (D) expression levels of CCL18, IL-10, IL-6, IL-8 and CCL3, in monocytes sorted from leukemic PBMCs (n = 9) cultured with or without rNAMPT (24 hours). (E) Confocal microscopy analysis of CD163 (red) and CD206 (green) expression in sorted CLL monocytes differentiated for 5 days with or without rNAMPT. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; blue); actin filaments were visualized using phalloidin (white). Original magnification ×63. (F-G) Cumulative analysis of CD206 (F) and CD163 (G) pixel intensity (arbitrary units, a.u.), scoring at least 10 different cells for 3 different samples.