Reactivity to autologous LCLs in the PBMCs of EBV-seropositive healthy subjects. The mean reactivity to autologous LCLs was determined in 37 EBV-seropositive healthy subjects between the ages of 26 and 63 years (mean, 46 years) with flow cytometry and intracellular IFN-γ staining using a Gallios flow cytometer (Beckman Coulter) with 9-color acquisition. PBMCs were separated by density centrifugation and cryopreserved. LCLs were generated and grown for 3 months to ensure homogeneity as described previously.⁷  Each LCL was checked for CD3 and intracellular IFN-γ expression to ensure that there was no evidence of T-cell contamination. Cryopreserved PBMC samples were thawed and cultured for 24 hours before use to allow the cells to rest and reexpress cell-surface molecules. Cultures of 10⁶ PBMCs were stimulated with 5 × 10⁵ autologous LCLs for 6 hours in the presence of brefeldin A. Nonstimulated PBMC cultures were used to measure background IFN-γ expression. Cells were stained with Abs to cell-surface markers and then fixed and permeabilized before intracellular IFN-γ staining. Two Ab panels were used for each subject. Single labeled tubes for each Ab, isotype-matched control Abs, fluorescence-minus-one controls, dead-cell exclusion dyes, and doublet discrimination were used during panel development to ensure accurate positive cutoff values and compensation matrices and to validate cell phenotype detection sensitivity and resolution. The first Ab panel assessed the frequencies and memory phenotypes of LCL-reactive CD4⁺ and CD8⁺ T cells and consisted of the following fluorochrome-conjugated Abs: anti–IFN-γ-FITC (Beckman Coulter), anti–CCR7-peridinin chlorophyll protein (PerCP)–Cy5.5 (BD Biosciences), anti–CD45RA-PE-Cy7 (BD Biosciences), anti–CD3-allophycocyanin (APC; Beckman Coulter), anti–CD8-APC-A700 (Beckman Coulter), anti–CD62L-APC-Cy7 (BioLegend), anti–CD4-V450 (BD Biosciences), and Aqua Live/Dead cell exclusion dye (Invitrogen). For the first Ab panel tubes, 400 000 events were collected to enable accurate estimation of LCL-reactive T-cell frequencies. The numbers of background IFN-γ–expressing cells were subtracted from measures of LCL-reactive cells. The second Ab panel assessed the LCL reactivity of αβ T cells, γδ T cells, and NK cells and consisted of: anti–IFN-γ-FITC (Beckman Coulter), anti–TCR-γδ-PE (BioLegend), anti–TCRαβ-PE-Cy7 (BioLegend), anti–CD3-APC (Beckman Coulter), anti–CD19-APC-Cy7 (BD Biosciences), anti–CD16-V450 (BD Biosciences), anti–CD56-V450 (BD Biosciences), and Aqua Live/Dead cell exclusion dye. (A) LCL-reactive cells as percentages of PBMCs. (B) LCL-reactive cells as percentages of cell type. (C) Percentage contribution of cell types among the phenotypically identifiable LCL-reactive cells. (D) Percentage contribution of different memory phenotypes to CD8⁺ LCL reactivity; N indicates naive (CD45RA⁺CCR7⁺); CM, central memory (CD45RA⁻CCR7⁺); EM, effector memory (CD45RA⁻CCR7⁻); EMRA, CD45RA⁺EM (CD45RA⁺CCR7⁻). (E) Percentages of LCL-reactive cells within different CD8⁺ T-cell memory phenotypes.