Phenotype of EBNA1- and influenza-specific IFNγ-secreting and proliferating CD4⁺ T cells. EBNA1- and influenza-specific CD4⁺ T-cell responses of one representative volunteer are shown. (A) After gating on CD3⁺CD4⁺ T cells, EBNA1-specific cells were identified by intracellular IFNγ staining (y-axis), and the expression of the indicated surface markers was analyzed (x-axis). The frequencies of EBNA1-specific CD3⁺CD4⁺ cells are shown in the appropriate quadrants. Gates were determined following an analysis with isotype controls (data not shown). One of 3 experiments is shown. (B) As described for panel A but characterizing IFNγ-positive influenza-specific CD4⁺ T cells. One of 2 experiments is shown. (C) After gating on CD3⁺CD4⁺ T cells, EBNA1-specific proliferation was identified by CFSE dilution (x-axis), and the expression of the indicated surface markers was analyzed (y-axis). The frequencies of CFSE dilute or proliferating EBNA1-specific CD4⁺ cells are shown in the appropriate quadrants. One of 3 experiments is shown. (D) As described for panel C but following CD4⁺ T-cell proliferation after influenza stimulation. One of 2 experiments is shown.