Effect of PD-1 blockade on T-cell function. (a) PBMC from BLV+ cattle were cultured with Flag peptide as negative control and gp51 peptide mix. IFN-γ production was measured by ELISA (n = 13). (b, c, d) PBMC were cultured with rat IgG control or anti-PD-1 mAb (5D2; 20 μg/mL) in the presence of gp51 peptide mix. IFN-γ and IL-10 production was measured by ELISA (b; n = 22, d; n = 26). Positive correlation between increasing rate of IFN-γ production and percentages of PD-1+ cells in CD4+ T cells corresponding to Figure 4a (c; n = 22). Correlation statistics were analyzed using the Spearman correlation. (e) The proliferative responses were evaluated by detection of CFSElo cells in IgM- lymphocytes by flow cytometry (n = 12). Representative dot plots of CFSE-staining in lymphocytes other than B cells are shown. 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).