CD44 levels show a broad range in overt CLL and overexpression marks the developing malignant B-cell phenotype. (A) Surface expression of CD44 as detected by flow cytometry with the anti–pan-isoform antibody IM7 and recorded as isotype-corrected MFI in the CD19⁺ gate shows a markedly higher variability in 45 CLL (median, 223; range, 118-453) as compared with PB B cells from 14 healthy donors (median, 284; range, 250-334). (B) Intensity of B-cell–specific CD44 surface expression as per flow cytometry from tail vein blood increases in 3-, 6-, and 9-month-old TCL1-tg mice during evolution of CLL. Shown are medians (ranges) of isotype-corrected CD44 MFI of the CD5⁺CD19⁺ fraction at 3 months [60.0 (26.4-70.0)], 6 months [86.9 (60.9-99.1)], and 9 months [89.9 (57.3-150.3)]; data point 150.3 is not illustrated. Leukemic development is illustrated by the increasing percentage of CD5⁺CD19⁺ cells. CD44 levels do not significantly increase in the CD5-negative B-cell fraction of Eµ-TCL1 B cells. (C) Surface CD44 expression (MFI) of B cells differed significantly between splenocytes of Eµ-TCL1 mice (n = 10) at the leukemic stage (age ≥ 12 months) and their age-matched wt controls (n = 6), both within the population of CD5⁻CD19⁺ cells [median (range), 18.8 (13.5-21.6) vs 34.9 (20.9-93.6)] and of CD5⁺CD19⁺ cells (median (range), 44.9 [18.8-53.4] vs 74.7 [51.6-146.1]). (D) Immunoblots for pan-CD44 by IM7 from lysates of isolated CD19⁺ splenic B cells from age-matched wt (n = 2) and Eµ-TCL1 (n = 5) mice show the magnitude of differences in CD44 expression at the whole-cell level.