NF-κB- and MAPK-induced CD44v6 expression is linked to human CLL cell proliferation. (A) CXCR4, CD5, and CD44v6 were costained on PBMCs from CD44v6⁺ CLL patients. (i) Cells were gated according to Calissano et al²⁴ ; representative dot plot is shown. (ii) Mean fluorescence intensity (MFI) of CD44v6 was compared between the CXCR4highCD5low and the CXCR4lowCD5high subpopulation of CD5⁺/CD19⁺ cells (n = 8). (Bi) PBMCs from CLL patients were cultured with M2 stromal cells with or without anti-CD3/CD28 activating beads (T act) or NIH3T3 fibroblasts transfected with or without CD40L (3T40L) for 24 hours. CD44v6 surface expression was determined by flow cytometry (n = 4). (Bii) PBMCs from CLL patients were cultured with M2 stromal cells with or without anti-IgM antibody or ibrutinib for 24 hours. CD44v6 surface expression was determined by flow cytometry (n = 4). (C) PBMCs from CLL patients were cultured with NIH3T3 fibroblasts or 3T40L and treated with 2 or 10 µM cobimetinib for 24 hours. CD44v6 surface expression was determined by flow cytometry (n = 5). (Di) Protein lysates from isolated CLL cells (patients 1-3, PAT 1-3) that were cultured with NIH3T3 fibroblasts transfected with or without CD40L for 24 hours were tested for their IkB α, phospho-IkB α, IKK α (upper band), and phospho-IKK α (upper band) β (lower band) content by western blot. (Dii-iii) PBMCs from CLL patients were treated for 0.5 hour with pan-caspase inhibitor (Q-VD-OPh) and an NF-κB inhibitor (BAY11-7082) for 1 hour and then cultured with NIH3T3 fibroblasts transfected with or without CD40L for 24 hours. (ii) CD44v6 and (iii) CD69 surface expression on viable CD5⁺/CD19⁺ CLL cells was determined by flow cytometry (n = 8). (iv) p65 ChIP analysis of the Cd44 promoter was conducted using an NF-κB activated, CLL patient-derived cell line Mec-1 (bars, mean ± standard deviation). One representative out of 3 independent experiments, performed in duplicates, is shown. (E) Unstimulated PBMCs from CLL patients were cultured for 24 hours and treated with HA for 10 minutes. Phosphorylation of ERK was measured in CD5⁺/CD19⁺ CLL cells via flow cytometry (n = 5). (F) PBMCs from CLL patients were cultured with NIH3T3 fibroblasts transfected with or without CD40L for 24 hours. HA binding and phosphorylation of p65 (i) and ERK (ii) were measured via flow cytometry (n = 5). (G) PBMCs from CLL patients were cultured with NIH3T3 fibroblasts transfected with or without CD40L for 72 hours with or without anti-CD44v6 or anti-panCD44 antibody. Intracellular Ki-67 expression was determined by flow cytometry (n = 6).