1028 - Peripheral Blood CXCR4hiCD5hi Chronic Lymphocytic Leukaemia Cells are Primed for Returning to the Lymph Nodes for further Activation and Proliferation

Chronic Lymphocytic Leukaemia (CLL) cells proliferate in the lymph nodes, where the microenvironment promotes activation, proliferation, and drug resistance of malignant cells. Following activation and proliferation in lymph nodes, CLL cells may migrate into the periphery as CXCR4loCD5hi cells and thought to transform into CXCR4hiCD5lo cells that remain quiescent until they return to the lymph nodes for further proliferation and activation. However, we poorly understand which peripheral blood CLL cells are able to return to the lymph nodes for re-activation.
To identify which cells are most likely to return to the lymph nodes, we fractionated CD19+CD5+ cells of 22 CLL patients based on CXCR4 and CD5 densities and analysed their phenotype and functionality. Surprisingly, we found that the CXCR4hiCD5hi fraction of peripheral blood CLL cells is enriched for Ki67+ proliferating cells and high expression of the activation marker AID, demonstrating that CXCR4loCD5hi cells are not the only proliferating and activated fraction in the blood. Flow microscopy confirmed that the CXCR4hiCD5hi fraction contained proliferating cells. Analysis of blood and tissues of 10 Emu-Tcl1-transgenic mice revealed that murine CXCR4hiCD5hi CLL cells also expressed high levels of Ki67. Moreover, we found CXCR4hiCD5hi CLL cells were expanded in the spleens of Emu-Tcl1-transgenic mice and in the cervical lymph node of a CLL patient with mutated TP53 and de novo progressive disease.
The peripheral blood CXCR4hiCD5hi fraction displayed high surface expression levels of the chemokine receptors CCR7 and CXCR5, which ‒ together with CXCR4 ‒ facilitate homing to lymph nodes. To investigate if CXCR4hiCD5hi CLL cells are primed for migration to lymphoid tissue, we stimulated peripheral blood CLL cells with CCL21, the ligand of CCR7, and found that the CXCR4hiCD5hi fraction was most efficient at migrating through transwells. CXCR4hiCD5hi CLL cells expressed high levels of surface IgM and had low basal nuclear localisation of Nuclear Factor of Activated T cells (NFAT), implicating that these cells are B cell receptor (BCR) responsive. In contrast, quiescent CXCR4hiCD5lo CLL cells had a pre-dominantly nuclear localisation of NFAT. To mimic stimulation in lymph nodes, we cultured peripheral blood CLL cells on CD40L-expressing fibroblasts in the presence of interleukin (IL)-4 and IL-21, inducing extensive proliferation of CXCR4hiCD5hi and CXCR4loCD5hi CLL cells after six days, while CXCR4hiCD5lo CLL cells remained quiescent. Peripheral blood CXCR4hiCD5hi CLL cells persisted BTK inhibition in vitro, whereas the CXCR4loCD5hi fraction was effectively diminished by ibrutinib. Importantly, the CXCR4hiCD5hi cell fraction was expanded in patients that relapsed while on therapy, expressing higher levels of ki67, AID and chemokine receptors than before relapse.
In conclusion, we have identified CXCR4hiCD5hi CLL cells as a potentially dangerous fraction of CLL cells that is primed for migration to tissue, receipt of BCR signals and proliferation. We provide evidence that these cells may give rise to proliferating cell phenotypes in tissues and anticipate this cell fraction may represent critical targets for therapeutic intervention.