Figure 3
CLL cells induce in vitro CD14+HLA-DRlo MDSCs with T-cell suppressive and TReg-promoting capabilities. (A) Fold-expression of surface markers based on their mean fluorescence intensity (MFI) on CD14+HLA-DRlo 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-DRlo 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 TReg FACS analysis from the peripheral blood of CLL patients. Naturally occurring TRegs were defined as CD4+ T cells positive for CD25 and FOXP3 and low/negative for CD127. Frequency of TRegs among CD4+ T cells is shown for healthy controls (HD [n = 13]) and CLL patients (n = 43). (E) Increase of the TReg frequency among CD4+ T cells of HDs after coculture with autologous CLL-induced CD14+HLA-DRlo MDSCs. (F) The CD120b MFI as assessed by FACS on TRegs from CLL patients (n = 9), TRegs induced in the presence of MDSCs (n = 5), and TRegs from HD (n = 14). (G) Correlation between the TReg frequency among CD4+ T cells and the proportion of HLA-DRlo 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.

CLL cells induce in vitro CD14+HLA-DRlo MDSCs with T-cell suppressive and TReg-promoting capabilities. (A) Fold-expression of surface markers based on their mean fluorescence intensity (MFI) on CD14+HLA-DRlo 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-DRlo 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 TReg FACS analysis from the peripheral blood of CLL patients. Naturally occurring TRegs were defined as CD4+ T cells positive for CD25 and FOXP3 and low/negative for CD127. Frequency of TRegs among CD4+ T cells is shown for healthy controls (HD [n = 13]) and CLL patients (n = 43). (E) Increase of the TReg frequency among CD4+ T cells of HDs after coculture with autologous CLL-induced CD14+HLA-DRlo MDSCs. (F) The CD120b MFI as assessed by FACS on TRegs from CLL patients (n = 9), TRegs induced in the presence of MDSCs (n = 5), and TRegs from HD (n = 14). (G) Correlation between the TReg frequency among CD4+ T cells and the proportion of HLA-DRlo 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.

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