The frequency of circulating antigen-specifi c CD4 T cells in healthy donors enrolled in the study was analyzed by fl ow cytometry, by using intracellular cytokine staining assay after the in vitro expansion of effector cells. To generate effector cells from their memory precursors, PBMC were challenged with antigen in vitro for 3 days and expanded for 6 additional days in the presence of IL-2 (18) .