CLL cells induce in vitro CD14⁺HLA-DR^lo MDSCs with T-cell suppressive and T_Reg-promoting capabilities. (A) Fold-expression of surface markers based on their mean fluorescence intensity (MFI) on CD14⁺HLA-DR^lo cells from patients (CLL [n = 10]) and healthy controls (HD [n = 10]). (B) Expression levels of HLA-DR, CD62L, CD274, and HLA-G, respectively, expressed as their MFI on purified HD-derived CD14⁺ monocytes cultured for 5 days in absence/presence of primary CLL cells and CLL cell lines (n = 4-22). (C) The dose-dependent suppressive activity CD14⁺HLA-DR^lo cells converted from healthy control monocytes (n = 4) in presence of CLL cells was evaluated in coculture experiments with VPD450-labeled autologous T cells activated using anti-CD2, -CD3, and -CD28 microbeads. Proliferation of T cells was assessed based on the VPD450 dye dilution in activated T cells after 5 days by FACS and compared with stimulated T cells alone (set as 100% T-cell proliferation). (D) Representative dot plot of a T_Reg FACS analysis from the peripheral blood of CLL patients. Naturally occurring T_Regs were defined as CD4⁺ T cells positive for CD25 and FOXP3 and low/negative for CD127. Frequency of T_Regs among CD4⁺ T cells is shown for healthy controls (HD [n = 13]) and CLL patients (n = 43). (E) Increase of the T_Reg frequency among CD4⁺ T cells of HDs after coculture with autologous CLL-induced CD14⁺HLA-DR^lo MDSCs. (F) The CD120b MFI as assessed by FACS on T_Regs from CLL patients (n = 9), T_Regs induced in the presence of MDSCs (n = 5), and T_Regs from HD (n = 14). (G) Correlation between the T_Reg frequency among CD4⁺ T cells and the proportion of HLA-DR^lo cells among CD14⁺ cells in the peripheral blood of CLL patients. The bars represent the standard error of the mean. *P < .05; **P < .01; ***P < .001.