Higher TLR9 expression is seen in LNs and migrated CLL cells and is associated with preferential engraftment in a CLL xenograft model. (A) Primary PBMCs from 10 different patients with CLL were pumped through the circulating system for 48 hours; those that had migrated out of the circulating compartment were then harvested. CLL cells that remained circulating were harvested at the same time and both stained for expression of sTLR9 along with CD5 and CD19 for CLL cell identification. CD5⁺CD19⁺ CLL cells were gated on and the sTLR9 MFI positivity determined by fluorescence minus one. Due to the variation in MFIs, the circulating compartment results were normalized to 1 and the migrated results presented as fold difference. Migrated CLL cells from 9 of the 10 cases had higher sTLR9 MFI compared with those that remained circulating. (B) Fine needle aspirate and matching PB samples were taken from 7 CLL patients with palpable LNs. PBMCs were stained as above and CD5⁺CD19⁺ CLL cells gated on. Due to the variation in MFIs, the PB results were normalized to 1, and the LN results presented as fold difference. For all 7 cases, sTLR9 MFI was higher in the LN-derived CLL cells compared with those from the PB. (C) The scatter plots are representative examples showing matched PB and LN-derived gated CLL cells from the same patient. (D) CLL cells from 4 patients with raised TLR9 and 3 patients with low TLR9 (relative transcription normalized to β-actin) were xenotransplanted into NOD/Shi-scid/IL-2Rγ^null mice. Mice were euthanized according to the criteria described in the supplemental Materials and methods, and the percentage of CLL cells in the spleen were determined by using flow cytometry (supplemental Table 1). Mice engrafted with TLR9^hi CLL cells had considerably higher numbers in their spleen compared with those engrafted with TLR9^lo CLL cells. Q, quartile.