(A) Rapid and enhanced production of IFN-γ in antigen-stimulated memory versus naive CD4 T cells. OVA-specific naive and memory CD4 T cells (see methods) were stimulated with OVA peptide and APC for 0–72hrs, and IFN-γ production was determined by intracellular cytokine staining. Results are expressed as percent of antigen-specific IFN-γ+ cells gated on CD4+KJ1-26+ cells at each timepoint, from one experiment representative of five independent experiments. (B) IFN-γ transcript expression in resting and activated naive and memory CD4 T cells. OVA-specific naive and memory CD4 T cells isolated as in (A) were activated with anti-CD3/anti-CD28 antibodies for 6–72hrs, and RNA was isolated from cell lysates of resting dn differentially activated, and IFN-γ-specific mRNA was amplified by quantitative realtime PCR (see methods). Results are expressed as expression of IFN-γ transcripts relative to control HPRT transcripts. Inset shows level of IFN-γ mRNA in naïve CD4 T cells with reduced scale, for direct comparison of peak mRNA production>