CD4⁺ T cells respond consistently to EBNA1 with IFNγ secretion and proliferation. EBNA1-specific CD4⁺ T cells were analyzed by intracellular IFNγ staining and CFSE proliferation assay. CD4⁺ T-cell responses in response to medium (no stimulus), Staphylococcal enterotoxin B (SEB), HCMV-derived CD8⁺ T-cell epitopes (CMV), all EBNA1 peptides (EBNA1), and subpools of EBNA1 peptides are shown. (A) Whole-blood assay after gating on lymphocytes based on size and on CD4⁺ T cells. The frequency of IFNγ-positive CD4⁺ T cells is indicated. Forward-scatter (FSC; x-axis) and intracellular cytokine staining for INFγ (y-axis) are depicted. The top row represents detected IFNγ responses from one of 20 EBV-positive carriers and the bottom row from one EBV-negative volunteer upon stimulation with the indicated stimuli. Gates for IFNγ-positive cells are based on isotype controls. (B) CFSE dilution assays characterize CD4⁺ T-cell proliferation in response to the indicated antigens and influenza virus infection (FLU). The frequencies of CD4⁺ T cells with diluted CFSE are indicated. The top row displays CD4⁺ T-cell responses from one representative of 20 healthy volunteers. The proliferation responses of an EBV-negative volunteer are depicted in the bottom row.