Myeloid cells cosynergize with CLL cells in promoting T_Reg expansion. (A) Correlation of the absolute T_Reg number with the CLL cell number in the peripheral blood of CLL patients. (B) Fold-increase in the T_Reg frequency in HD-derived PBMCs cocultured in the presence/absence of an increasing number of autologous B-cells (n = 6), primary (p)CLL-cells (n = 5), and the 2 CLL cell lines (used in n = 12 independent experiments) (the baseline = 1 represents the frequency of T_Regs in the absence of cocultured cells). (C) Representative histogram of the dose-dependent suppressive activity of purified T_Regs induced in the presence of CLL cells. T_Regs suppressed VPD450-labeled autologous T cells activated by anti-CD2, -CD3, and -CD28 microbeads (n = 4). Proliferation of T cells was assessed after 5 days by FACS and compared with activated T cells alone (set as 100% T-cell proliferation). (D) Representative (for n = 4 independent experiments) histogram of the proliferation of conventional CD4⁺ T cells (T_Conv) and T_Regs based on the VPD450 dilution after coculture with CLL cells. Ratio of the percentages of proliferating Ki67⁺ T_Regs to Ki67⁺ T_Conv cells in relation to the number of circulating CLL cells in the patients. (E) Fold-increase in T_Reg frequencies among PBMCs cocultured in the absence/presence of CLL cells (n = 4) or 2 CLL cell lines (used in n = 7 independent experiments) being separated by a Transwell polyester membrane (TW) or in close cell-to-cell contact (the baseline = 1 represents the frequency of T_Regs in the absence of cocultured cells). (F) Percentage induction of T_Regs with removal of adherent cells before coculture of PBMCs retrieved from healthy controls with CLL cells (n = 8-10) (T_Reg induction without removal of adherent cells is set as 100%). (G) CD3⁺ T cells purified from HD PBMCs were cocultured with CLL cell lines in 4 independent experiments in absence/presence of autologous purified CD14⁺ monocytes (the baseline = 1 represents the frequency of T_Regs in the absence of monocytes). The bars represent the standard error of the mean. *P < .05; **P < .01; ***P < .001.