EMMPRIN expression in CLL cells and S100-A9 binding in vitro. (A) Reverse-transcription polymerase chain reaction analysis of EMMPRIN mRNA isoforms in CLL. EMMPRIN mRNA isoforms 1 to 4 expression was evaluated in MACS-sorted B lymphocytes (CD19⁺) and non-B cells (CD19⁻) as described by Oppezzo et al.¹⁸ EMMPRIN isoform 2 is expressed in leukemic B cells at higher levels than in normal B cells, whereas isoforms 3 and 4 are also expressed but at basal levels. (B) Flow cytometry analysis of EMMPRIN protein levels in B lymphocytes. Median fluorescence intensity analysis of EMMPRIN staining in CLL and HD B cells reveals higher levels of EMMPRIN staining in CLL B cells (mean, 8.751; n = 15) compared with normal B cells (mean, 5.964; n = 5; Mann-Whitney test, P = .0037). MFI, mean fluorescence intensity. (C) EMMPRIN in situ immunostaining. Direct in situ immunolocalization of EMMPRIN (magenta) shows higher staining intensities in CLL B cells compared with their normal counterparts (CD19⁺ in yellow). EMMPRIN staining in normal T cells (CD3⁺ in cyan) was assessed as positive control. Single confocal planes are shown; deconvolution was performed with Huygens Essential 4.5 (Scientific Volume Imaging, Hilversum, The Netherlands; http://svi.nl). Scale bar, 5 µm. (D) rhS100-A9 B-cell CLL in vitro binding assay. The binding ability of rhS100-A9 (green) on B-cell CLL cells (IgM⁺ in yellow) expressing EMMPRIN (magenta) was evaluated on incubation of 10⁶ cells with 5 µg/mL for 1 hour at 37°C. An increase from basal 8% to 34% of S100-A9⁺ cells was found. Flow cytometry analysis was performed with R/Bioconductor. Confocal microscopy analysis showed an increase in S100-A9 immunostaining and suggested colocalization of rhS100-A9 with EMMPRIN receptor, shown in white in the overlaid images (arrows). Deconvolved single confocal planes are shown. Scale bar, 5 µm.