Recognition of PD-1-expressing lymphocytes by anti-PD-1 mAb. (a) Gating strategies for CD4+ T cells, CD8+ T cells and IgM+ B cells in bovine lymphocytes. (b) Representative histograms obtained by flow cytometry of PD-1 expression in CD4+ T cells, CD8+ T cells and IgM+ B cells isolated from three BLV- cattle. Freshly isolated PBMC were stained with anti-PD-1 (5D2), CD4, CD8, and IgM Ab. PBMC were cultivated with PBS (No stimulation), PMA/ionomycin, and PWM for 24, 48, and 72 h, and stained in a similar way. (c) Proportions of PD-1 positive cells in CD4+ T cells, CD8+ T cells and IgM+ B cells. Statistical comparisons between percentages of PD-1 positive cells stimulated with PBS and PMA/ionomycin or PWM were made using two way ANOVA. Differences were considered statistically significant at P < 0.05 (** P < 0.01). (d) Representative dot plots of PD-1 and CD25 expression in CD4+ T cells, CD8+ T cells and IgM+ B cells 48 h after cultivation.