Determination of intracellular IFN-γ–producing antigen-specific T cells by 4-color FACS analysis.

Donor T cells were stimulated with autologous or allogeneic EBV BLCL overnight for 16 hours. Nonstimulated control tubes contained effector cells and stimulators mixed immediately before staining. Stimulated and nonstimulated tubes were then prepared simultaneously. (A) Lymphocytes were identified and gated. (B) A second gate including the CD3⁺CD8⁺ T lymphocytes was set, and the CD69⁺IFN-γ⁺ cells of the nonstimulated and stimulated samples were determined. (C) Nonstimulated control with background activity of 0.02%. (D) Stimulated cultures with 2.92% of CD8⁺CD69⁺IFN-γ⁺ reduced by background.