Functional threshold in CM and EM CD4 T cells. (A) Detection by FACS of intracellular production of IFN-γ and IL-2 in memory cells exposed for 1 night to graduated concentrations of immobilized anti-CD3 and 1 μg/mL soluble anti-CD28 antibodies. Shown is 1 representative response of 4 (left). Cumulated data of EM cells from 4 subjects in response to 5 μg/mL immobilized anti-CD3 antibody supplemented with 1 μg/mL soluble anti-CD28 or -CD4 antibodies are shown (right). (B) Proliferative potential of memory T cells measured by CFSE dilution in sorted CM and EM cells stimulated for 6 days as described for panel A (left). Shown are cumulated data of 4 subjects in response to 5 μg/mL of immobilized anti-CD3 antibody supplemented with 1 μg/mL soluble anti-CD28 antibody in the presence or absence of IL-2 (right). (C) Intracellular calcium mobilization in CM and EM cells in response to graduated concentrations of soluble mouse anti-CD3 and 1 μg/mL soluble anti-CD28 antibodies (left). The percentage of cell showing changes in intracellular calcium concentration was assessed by measuring variations of FL5/FL4 ratio (right).