CLL TLR9 levels correlate with CD38, CD49d, p-STAT3, and p-p65 NF-κB and ability to migrate. Unstimulated primary PBMCs from 11 patients with CLL were stained with antibodies to CD5, CD19, CD38, CD49d, and a viability dye and then permeabilized and stained for eTLR9 or an isotype matched control. Cells were assessed by using flow cytometry and positivity determined by fluorescence minus one for surface antigens or an isotype matched control for eTLR9. (A) Representative figure showing that all viable CD5⁺CD19⁺ gated CLL cells have eTLR9, but within a patient, a much larger proportion of the CD38⁺ and CD49d⁺ populations have exceptionally high TLR9 (TLR9^bright) compared with their negative counterparts. (B) CD5⁺CD19⁺CD38⁺ (i) and CD5⁺CD19⁺CD49d⁺ (ii) CLL cells from 11 different patients were gated on and the MFI of CD38, CD49d, and TLR9 established. There is a clear positive correlation between the MFIs of both CD38 and CD49d with TLR9. (C) Basal intranuclear p-STAT3 (Tyr 705) and p-p65 NF-κB levels were established on CLL cells from 12 patients using the same panel as above and including an anti–p-STAT3 or p-p65 NF-κB antibody. (i) Representative figure shows higher expression of p-STAT3 in CLL cells in a small population of CLL cells (shaded green), which also have higher expression of CD38, CD49d, and TLR9. (ii) The MFIs for both p-STAT3 and p-p65 NF-κB were quantified on both the CD38^hi CD49d^hi TLR9^bright and the CD38^lo CD49d^lo TLR9^dim population. The MFI of both p-STAT3 and p-p65 NF-κB was significantly higher in the CD38^hi CD49d^hi TLR9^bright CLL cells. (D) Basal eTLR9 levels of CLL cells from 20 patients were compared with their level of migration (as described in Figure 2C, but in the absence of any stimulation). There is a clear correlation between the percentage of CLL cells that migrate and the MFI of their eTLR9.