CLL CD14⁺HLA-DR^lo MDSCs express high levels of IDO. (A) The relative gene expression of MDSC-related molecules was comparatively assessed in healthy control (HD) monocytes cocultured with CLL cells (n = 7) in cell-to-cell contact, separated by a transwell system (TW) or in the presence of CLL cell supernatant (the baseline = 1 represents gene expression levels in monocytes in the absence of cocultured malignant cells). Arginase 1 and 2 (Arg1/2), inducible nitric oxide synthetase (inos), signal transducer, and activator transcription factor 3 (stat3), indoleamine 2,3 dioxygenase (ido), reduced NAD phosphate (NADPH) subunits p67 and gp91, IL-6 and IL-10, heme-oxygenase-1 (ho1), transforming growth factor β (TGF-β), and cyclooxygenase 2 (cox2) were included in the analyses. (B) Representative dot plots from FACS analyses evaluating the IDO expression in (i) CD14⁺ cells from HD and patients, as well as in (ii.-iii.) HLA-DR^hi and HLA-DR^lo CD14⁺ cells of CLL patients. IDO expression levels shown as the MFI were compared between CD14⁺ cells from HDs (n = 4) and HLA-DR^hi and HLA-DR^lo CD14⁺ cells from CLL patients (n = 4). (C) Coexpression of CD33 (red) and IDO (green) was analyzed in CLL infiltrated lymph nodes (n = 5) as shown in the representative fluorescence microscopy images. The 4′,6 diamidino-2-phenylindole (DAPI) (blue) was used as a nuclear counterstain. Five representative high-power fields (at ×400) were selected for evaluating the number of double-positive CD33⁺ IDO⁺ cells (white circles) and the proportion of IDO⁺ cells among total CD33⁺ cells. (D) Serum concentrations of tryptophan (Trp) and its degradation products 3-hydroxykynurenine (Hk), nicotinamide (Nam), kynurenine (Kyn), xanthurenic acid (Xa), kynurenic acid (Ka), indole-3-lactic acid (Ila), indole-3-propionic acid (Ipa), and indole-3-acetic (Iaa) were quantified in samples from the CLL patients with the highest MDSC (MDSC^hi [n = 10]) and the lowest MDSC (MDSC^lo [n = 10]) frequency by LC-ESI-MS/MS. The bars represent the standard error of the mean. n.d., not detected. *P < .05; **P < .01; ***P < .001.