PD-1, PD-L1, and PD-L2 expression in WM BM cells. BM biopsies from WM patients were processed to isolate cells (CD19⁺/138⁺ and CD19⁻/138⁺) and plasma fractions. Cells were used for RNA extraction, cDNA synthesis, and RT-PCR analysis. (A) Bar graphs represent relative gene expression of PD-1 and the ligands normalized to housekeeping gene; CD19⁻/138⁻ (NBM, n = 9; WM, n = 18) and CD19⁺/138⁺ (NBM, n = 8; WM, n = 15). Significant differences and related P values are shown on each graph. (B) IHC staining shows the sections from WM and normal BM (NBM) stained for PD-1, PD-L1, and PD-L2 (brown stain; original magnification ×400). (C) CD19⁺/138⁺ and CD19⁻/138⁻ cell populations were stained with PD-L1 and analyzed using flow cytometry. Histograms show PD-L1 surface expression on CD19⁺/138⁺, CD3⁺, CD56⁺, CD14⁺ and CD15⁺ cell fractions (isotype control [red] and PD-L1 [blue]) in WM and NBM. Bar graphs compare mean fluorescence intensity of 19⁺/138⁺ cells between NBM and WM BM (NBM, n = 5; WM, n = 9). Data are presented as mean ± SEM and significant differences and related P values are shown on each graph. MFI, mean fluorescence intensity.