Effect of PD-1 blockade on gp51 expression and B-cell activation. (a , b, c) Percentages of gp51-expressing cells (a; n = 15) and mean fluorescence index (MFI) of WC4 (CD19 like molecule) (b; n = 12) and CD80 (c; n = 6) in IgM+ B cells were evaluated by flow cytometry in PBMC treated with rat IgG control or anti-PD-1 mAb (20 μg/mL). Representative contour plots showing gp51 expression (right panels) in PBMC treated with rat IgG control (upper panels) or anti-PD-1 mAb (lower panels) is shown in (a). No staining was observed in PBMC stained with isotype control for anti-gp51 mAb (left panels). (d) Expression of BAFF mRNA was evaluated by real-time PCR (n = 12). The results are indicated as relative change to control (no antibody treatment) when the amount of BAFF mRNA expression is divided by GAPDH mRNA expression. (e) Percentages of apoptotic cells in IgM+ B cells were measured by flow cytometry. Apoptotic B cells were identified as annexin-V+ 7-AAD- cells (n = 11). (f) Percentages of gp51-expressing cells were evaluated by flow cytometry in isolated B cells cultivated with rat IgG control or anti-PD-1 mAb (n = 11). Statistical comparisons between rat IgG control and anti-PD-1 mAb were made using the Wilcoxon matched-pairs test. Differences were considered statistically significant at P < 0.05 (*P < 0.05; ** P < 0.01).