American Society of Hematology

Figure 2.

$Stimulating CLL cells through TLR9 causes an increase in CD38, CD49d, CD69, p-STAT3, p-p65 NF-κB, and migration. (A) PBMCs from 15 different patients with CLL were incubated alone or in the presence of ODN2006 for 24 hours. Cells were then harvested and labeled with antibodies against CD5, CD19, CD38, CD49d, CD69, and a viability dye. Fluorescence minus one control tubes using stimulated CLL cells defined the thresholds for positivity. The MFI of CD38, CD49d, and CD69 were assessed by using flow cytometry; all were significantly increased in viable CD5+CD19+ CLL cells after activation with ODN2006. (B) Primary PBMCs were incubated for 4 hours with or without ODN2006. Cells were harvested and labeled for CD5, CD19, and intracellular p-STAT3, p-p65 NF-κB, p-STAT5, or an isotype matched control and then assessed by flow cytometry. (i) Representative overlaid histograms showing that gated CD5+CD19+ cells have some constitutive p-STAT3 (red histogram), which is increased after stimulation with ODN2006 (light blue histogram). (ii) Figure shows the MFI fold change compared with resting cells. There is an increase in both p-STAT3 and p-p65 NF-κB for all 24 cases after ODN2006 stimulation but no increase in p-STAT5. (C) Primary PBMCs were incubated for 4 hours with or without ODN2006, autologous plasma, or autologous plasma with the TLR9 inhibitor ODN-INH-18. Cells were harvested and stained for CD5, CD19, and intracellular p-STAT3, p-p65 NF-κB, or p-STAT5 and then assessed by using flow cytometry. Autologous plasma increased both p-STAT3 (i) and p-p65 NF-κB (ii), but not p-STAT5 (iii), and this was abrogated in the presence of a TLR9 inhibitor. (D) Primary PBMCs from 24 different patients were incubated alone or in the presence of ODN2006 for 24 hours. Cells were then harvested and then transferred into 5 μm pore polycarbonate transwell migration chambers and incubated for 4 hours; cells migrated toward a CXCL12 (100 ng/mL) gradient. The migrated and nonmigrated cells were collected, stained with CD5 and CD19 for CLL cell identification, and then quantitated volumetrically. The ODN2006 prestimulated CLL cells had greater levels of migration compared with the unstimulated fraction. Due to between-patient variance in levels of migration, the results are normalized to the unstimulated. NS, not significant.$

Stimulating CLL cells through TLR9 causes an increase in CD38, CD49d, CD69, p-STAT3, p-p65 NF-κB, and migration. (A) PBMCs from 15 different patients with CLL were incubated alone or in the presence of ODN2006 for 24 hours. Cells were then harvested and labeled with antibodies against CD5, CD19, CD38, CD49d, CD69, and a viability dye. Fluorescence minus one control tubes using stimulated CLL cells defined the thresholds for positivity. The MFI of CD38, CD49d, and CD69 were assessed by using flow cytometry; all were significantly increased in viable CD5⁺CD19⁺ CLL cells after activation with ODN2006. (B) Primary PBMCs were incubated for 4 hours with or without ODN2006. Cells were harvested and labeled for CD5, CD19, and intracellular p-STAT3, p-p65 NF-κB, p-STAT5, or an isotype matched control and then assessed by flow cytometry. (i) Representative overlaid histograms showing that gated CD5⁺CD19⁺ cells have some constitutive p-STAT3 (red histogram), which is increased after stimulation with ODN2006 (light blue histogram). (ii) Figure shows the MFI fold change compared with resting cells. There is an increase in both p-STAT3 and p-p65 NF-κB for all 24 cases after ODN2006 stimulation but no increase in p-STAT5. (C) Primary PBMCs were incubated for 4 hours with or without ODN2006, autologous plasma, or autologous plasma with the TLR9 inhibitor ODN-INH-18. Cells were harvested and stained for CD5, CD19, and intracellular p-STAT3, p-p65 NF-κB, or p-STAT5 and then assessed by using flow cytometry. Autologous plasma increased both p-STAT3 (i) and p-p65 NF-κB (ii), but not p-STAT5 (iii), and this was abrogated in the presence of a TLR9 inhibitor. (D) Primary PBMCs from 24 different patients were incubated alone or in the presence of ODN2006 for 24 hours. Cells were then harvested and then transferred into 5 μm pore polycarbonate transwell migration chambers and incubated for 4 hours; cells migrated toward a CXCL12 (100 ng/mL) gradient. The migrated and nonmigrated cells were collected, stained with CD5 and CD19 for CLL cell identification, and then quantitated volumetrically. The ODN2006 prestimulated CLL cells had greater levels of migration compared with the unstimulated fraction. Due to between-patient variance in levels of migration, the results are normalized to the unstimulated. NS, not significant.

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