EBNA 3c-specific T cells determined by tetramer staining and by intracellular IFN-γ production of an EBV-reactive T-cell line of an HLA-A*0201⁺ donor.

(A) Cells were triple stained with anti-CD3, anti-CD8, and HLA tetramer (EBNA3c [LLDFVRFMGV]). (B) Cells were additionally stained with anti-CD62L. (C, D) Cells were stained with an unrelated tetramer–peptide complex (EBNA 3a [QAKWRLQTL]) as a control. (E) Cells were stained with anti-CD3, anti-CD8, and anti–IFN-γ after secondary stimulation with EBNA3c [LLDFVRFMGV] peptide-pulsed autologous PBMCs. (F) Cells were additionally stained with anti-CD69. (G, H) Cells were stained after secondary stimulation with irrelevant EBNA 3a [QAKWRLQTL] peptide-pulsed autologous PBMCs as a control. Each plot is gated on live CD3⁺CD8⁺ T cells stained with the respective tetramer or anti-IFN-γ antibody. Percentage of peptide-specific CD8⁺ cells is indicated in the upper quadrants of each plot.