CLL CD14⁺HLA-DR^lo MDSCs suppress T cells and induce T_Regs through IDO. (A) Induced (i)MDSCs converted from healthy control monocytes in the presence of CLL cells were cocultured for 5 days with autologous VPD450-labeled activated T cells with/without being separated by a semipermeable membrane (TW) (n = 10). T-cell proliferation and T_Reg induction was assessed by FACS (T-cell proliferation/T_Reg induction without TW is set as 100%). (B) FACS-sorted primary (p)MDSCs from CLL-patients were cocultured for 5 days with autologous VPD450-labeled activated T cells with/without pretreatment with 500 µM of the IDO inhibitor 1-methyl-dl-tryptophan (1-MT). T-cell proliferation (n = 3) and T_Reg induction (n = 5) was assessed by FACS (T-cell proliferation/T_Reg induction without 1-MT application is set as 100%). (C) FACS-sorted iMDSCs were cocultured for 5 days with autologous VPD450-labeled activated T cells with/without pretreatment with 500 µM 1-MT (n = 5). T-cell proliferation, T_Reg induction, and IFNγ release was assessed by FACS and enzyme-linked immunosorbent assay, respectively (T-cell proliferation/T_Reg induction/IFN-γ release without 1-MT application is set as 100%). (D) FACS-sorted (i)MDSCs were cocultured for 5 days with autologous VPD450-labeled activated T cells with/without addition of an anti-PD-1L blocking antibody (n = 5). T-cell proliferation and T_Reg induction was assessed by FACS (T-cell proliferation/T_Reg induction without the presence of an anti-PD-1L antibody is set as 100%). (E) FACS-sorted pMDSCs (n = 3) and/or iMDSCs (n = 11-16) were cocultured for 5 days with autologous VPD450-labeled activated T cells with/without addition of an anti-HLA-G blocking antibody. T-cell proliferation and T_Reg induction was assessed by FACS (T-cell proliferation/T_Reg induction without the presence of an anti-HLA-G blocking antibody is set as 100%). The bars represent the standard error of the mean. *P < .05; **P < .01; ***P < .001.