Biomarker assessment of PD-1/PD-L1 expression and tumor-infiltrating T cells in treated patients. (A) The expression of PD-1, PD-L1, CD3, and CD20 detected by standard IHC in one representative RT- and one CLL-involved lymph node are shown in images taken using 40× visual field with magnification ×400. Scale bars = 50 μm. (B) The percentage of expression of PD-1, PD-L1, CD3, and CD8 in the baseline lymph nodes/tumors of 10 patients (6 RT and 4 CLL). PD-L1 expression is significantly increased (P = .03 by 2-tailed Student t test using 2-sample equal variance). PD-1 expression shows a trend for an increased level of expression (P = .10) in RT patients who have confirmed response in comparison with the CLL/RT patients who do not have confirmed responses. CD3 and CD8 expression are not different in these above 2 cohorts. Line represents median expression levels of individual antigens. Each open/filled square or triangle represents the % expression of individual antigen in 1 patient. (C) The representative images of PD-1 staining (green) in conjunction with CD3 (red) and PD-L1 expression (red) in conjunction with Pax 5 (green), tested in dual-color immunofluorescent staining using confocal microscopy. PD-L1 has minimal colocalization with Pax5-positive B cells (supplemental Table 2) and PD-L1–positive cells have the morphology of monocytic/histiocytic lineages. PD-1 partially colocalized with CD3, whereas the majority of PD-1 staining appears to be positive on tumor B cells (supplemental Table 2). Arrows point to the colocalization. Images were taken using 40× visual field with magnification ×400. H&E, hematoxylin and eosin.