Inhibition of the Tim-3/Gal-9 pathway increases cytokine expression in PBMC from BLV-infected cattle. (A and B) The expression of bovine Tim-3 on Cos7 cells. The detections were performed by (A) Western blotting analysis and (B) Flowcytometric analysis with anti-FLAG antibody as described elsewhere. Empty plasmid-transfected cells were used as mock for each assay. (C, D and E) Real-time PCR quantification of mRNA expression levels for cytokines in the treated-PBMC from BLV-infected cattle ( n = 3). Up-regulations of IL-2 (C) and IFN-γ (D) expressions in PBMC by the inhibition of the Tim-3/Gal-9 pathway using Tim-3 expressing cells (Tim-3-FLAG). Up-regulations of IFN-γ (E) expressions by blockade of the PD-1/PD-L1 pathway using anti-PD-L1 antibody (α-PD-L1) and enhancement of the expression by combination PD-L1 blockade with Tim-3 expressing cells. Each cytokine mRNA level was normalized by the bovine β-actin mRNA as above, and the relative index was determined in comparison to the cytokine mRNA level in the PBMC without Tim-3 expressing cells and antibodies. The results are means of three independent experiments using cells from three individual cattle. Asterisks donate significant differences between the types of the cells (* p < 0.05).