Stability of the central-memory phenotype and chemokine receptor expression on proliferating EBNA1-specific CD4⁺ T cells. (A) CFSE-labeled CD3⁺CD4⁺CD62L⁺ cells, purified by flow-cytometric cell sorting, were stimulated with the indicated antigens: medium (no stimulus), Staphylococcus enterotoxin B (SEB), influenza A infection (FLU), and the cognate EBNA1 peptide pool I. After 6 days, CD3⁺CD4⁺ T cells were analyzed for CD62L expression (y-axis) and CFSE dilution (x-axis). The frequencies of CD3⁺CD4⁺ T cells in each quadrant are indicated. One representative of 2 experiments is shown. (B) After gating on CD3⁺CD4⁺CD62L⁺ T cells, proliferation in response to 4 stimuli (medium, no stimulus; SEB, Staphylococcus enterotoxin B; influenza, influenza infection; and EBNA1, master mix of all 51 overlapping peptides of EBNA1) was identified by CFSE dilution (x-axis), and the expression of the indicated chemokine receptors (CXCR3, CCR4, and CXCRR5) was analyzed (y-axis). The frequency of CFSE dilute, and therefore proliferating, CD4⁺ cells is indicated in the appropriate quadrants. Gates and quadrants were determined after analysis with isotype controls (data not shown). One of 3 experiments is shown.