Figure 1.

Nine-colour flow cytometry for evaluation of Epstein–Barr virus (EBV)-specific T-cell responses. (a) Analysis of five functions of CD4⁺ and CD8⁺ T cells reactive to overlapping peptide pool of EBNA3B in a representative healthy long-term carrier. Gating scheme used to identify EBV-specific T cells was shown. Doublets were excluded from the single cells based on the Forward Scatter-Area (FSC-A) versus Forward Scatter-Height (FSC-H) profile. Live CD3-positive cells were selected by gating the CD3-positive and Aqua Blue-negative population. As CD3⁺ population form a ‘tail’ into the Aqua Blue channel, a back-gating procedure was performed, confirming that these cells were living CD4⁺ or CD8⁺ T cells. Lymphocytes were gated based on the FSC versus Side Scatter (SSC) profile. CD4⁺ and CD8⁺ T cells were analysed separately. (b) Analysis of five functions of EBNA3B-specific CD4⁺ and CD8⁺ T in a patient with post-transplant lymphoproliferative disorder (PTLD). The same gating strategies as those of long-term carriers were applied. The numbers in the flow diagrams represent the frequencies of cytokine-producing or degranulating T cells. IFN-γ, interferon-γ; MIP1-α, macrophage inflammatory protein 1-α; TNF-α, tumour necrosis factor-α; IL-2, interleukin-2.