FIG. 4.

FACS analysis with PBMC isolated from three M. bovis-infected cattle performed after stimulation with RPMI medium, each individual Rv3615c peptide (5 μg ml⁻¹), or the Rv3615c peptide pool (5 μg ml⁻¹). Secretion of IFN-γ was stopped by addition of brefeldin A (10 μg ml⁻¹). Lymphocytes were analyzed for intracellular IFN-γ production and the presence of CD4 and CD8 T-cell subset markers using anti-CD4-Alexa Fluor 647, anti-CD8-fluorescein isothiocyanate, and anti-IFN-γ-phycoerythrin conjugated antibodies. A live/dead differential stain analysis was performed using ViVid and a Dake Cyan ADP. The results are expressed as percentages of the lymphocyte population staining as CD4⁺ IFN-γ⁺ (bars above the x axis) or CD8⁺ IFN-γ⁺ (bars below the x axis) for each animal (animals 3420, 3450, and 3606).