Figure 4
Myeloid cells cosynergize with CLL cells in promoting TReg expansion. (A) Correlation of the absolute TReg number with the CLL cell number in the peripheral blood of CLL patients. (B) Fold-increase in the TReg 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 TRegs in the absence of cocultured cells). (C) Representative histogram of the dose-dependent suppressive activity of purified TRegs induced in the presence of CLL cells. TRegs 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 (TConv) and TRegs based on the VPD450 dilution after coculture with CLL cells. Ratio of the percentages of proliferating Ki67+ TRegs to Ki67+ TConv cells in relation to the number of circulating CLL cells in the patients. (E) Fold-increase in TReg 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 TRegs in the absence of cocultured cells). (F) Percentage induction of TRegs with removal of adherent cells before coculture of PBMCs retrieved from healthy controls with CLL cells (n = 8-10) (TReg 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 TRegs in the absence of monocytes). The bars represent the standard error of the mean. *P < .05; **P < .01; ***P < .001.

Myeloid cells cosynergize with CLL cells in promoting TReg expansion. (A) Correlation of the absolute TReg number with the CLL cell number in the peripheral blood of CLL patients. (B) Fold-increase in the TReg 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 TRegs in the absence of cocultured cells). (C) Representative histogram of the dose-dependent suppressive activity of purified TRegs induced in the presence of CLL cells. TRegs 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 (TConv) and TRegs based on the VPD450 dilution after coculture with CLL cells. Ratio of the percentages of proliferating Ki67+ TRegs to Ki67+ TConv cells in relation to the number of circulating CLL cells in the patients. (E) Fold-increase in TReg 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 TRegs in the absence of cocultured cells). (F) Percentage induction of TRegs with removal of adherent cells before coculture of PBMCs retrieved from healthy controls with CLL cells (n = 8-10) (TReg 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 TRegs in the absence of monocytes). The bars represent the standard error of the mean. *P < .05; **P < .01; ***P < .001.

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