Spleen- but not BM-derived CLL cells bind HA and express long CD44 isoforms containing the variant exon 6, which is important for leukemic cell proliferation. (A) Cytometric analysis of HA binding of SPL- or BM-derived CD5⁺/CD19⁺ cells from Tcl1-tg and Cd44ΔB Tcl1-tg that were cultured with or without M2 stromal cells for 48 hours was performed. (Bi) Schematic diagram of the Cd44 gene and the primer design used for RT-PCR. Binding regions of the CD44s forward (5 fw) primer used in combination with a constant exon-specific (16 rv) or variant reverse primer (v6 rv) are indicated with arrows. (Bii) RT-PCR analysis of isoforms containing CD44v6 (5 fw and v6 rv primer combination) and panCD44 (5 fw and 16 rv primer combination) in the Cd44 cDNA from SPL- or BM-derived CD5⁺/CD19⁺ cells from Tcl1-tg mice (n = 3, indicated as 1-3) was performed (M1 spleen: lower band CD44v6, upper band CD44v5-6; M2 spleen: lower band CD44v6, upper band CD44v4-6; M3 spleen: CD44v6; supplemental Table 5). Low-range DNA ladder (M) in the right panel. (Biii) RT-PCR analysis in the Cd44 cDNA from LNP-derived CD5⁺/CD19⁺ cells from 3 Tcl1-tg mice (indicated as 1-3) (M1: lower band CD44v6, upper band CD44v3v6; M2: main band CD44v6; M3: lowest band CD44v6, second lowest band CD44v5-6; supplemental Table 5) (C) Intracellular Ki-67 expression (i) and percentage of viability (ii) of splenic CD5⁺/CD19⁺ leukemic cells cultured on M2 stromal cells and with or without anti-CD44v6 or anti-panCD44 antibody treatment of 48 hours was determined by flow cytometry (n = 6).