Selective binding of JML-1 IgG1 to primary B-CLL cells and B cells. (A) B-CLL PBMCs (5 × 105) were incubated with 1 μg/mL biotinylated JML-1 IgG1 or 1 μg/mL biotinylated TT11 IgG1 and subsequently stained with CD19-APC to allow gating of B cells. In a parallel experiment, MACS-separated CD19+ and CD19− subpopulations of PBMCs from a healthy volunteer were used. Shown are flow cytometry profiles of B-CLL PBMCs from patient α (used for library selection; top left), from patient A (the alloHSC transplant recipient from whom the library originated; bottom left), and CD19+ and CD19− subpopulations of PBMCs from a representative healthy volunteer (top right and bottom right, respectively). The histograms show the binding of biotinylated JML-1 IgG1 (green) and biotinylated isotype control TT11 IgG1 (red) detected by PE-coupled streptavidin. The background signal obtained for the detection reagent alone is shown in black. (B) PBMCs from 14 patients with B-CLL (including patient α and patient A), PBMCs subpopulations from 11 (CD19+) and 3 (CD19−) healthy volunteers, and 11 B-cell lines (including B-CLL cell lines EHEB and 232-B4) were analyzed for JML-1 IgG1 and TT11 IgG1 binding as described above. Each data point depicts the MFI of an individual sample minus the MFI obtained for the detection reagent alone. Horizontal lines indicate arithmetic mean values; P, probability based on the Mann-Whitney test.
