Figure 6. Viral epitopes are presented within hours of infection, and stimulate memory T cell effector functions.

Mice that contained approximately 3×10³ SMARTA/Ly5a CD4⁺ T cells were infected with LCMV and, 354 days later, were re-challenged intraperitoneally with 2×10⁶ PFU LCMV-Armstong. Six hours post-infection, the mice were given 0.25 mg Brefeldin A i.v., and 6 hours later the spleens were harvested and immediately surface stained for CD4, Ly5a, or CD8, then permeabilized and stained for intracellular IFNγ. The cells were not re-stimulated ex vivo with peptide antigen. A. ∼5% of all CD8⁺ T cells, and ∼1% of all CD4⁺ T cells, are actively producing IFNγ in response to infection. B. Using the SMARTA cells transferred ∼1 year previously as an indicator of the responsiveness of virus-specific CD4⁺ memory T cells, ∼14% of LCMV-specific CD4⁺ memory T cells actively produce IFNγ within 12 hours of virus infection. Data shown are from an individual mouse, and are representative of independent datasets. C. A separate set of naive mice were given CFSE-labeled pooled SMARTA cells (4×10⁵ naive SMARTA/Thy1.1 cells and 2×10⁴ memory SMARTA/Ly5a T cells). 4 days later, some of the recipient mice were given LCMV. Six hours later, BFA was administered to all mice, and after a further 6 hours splenocytes were harvested. The cells were immediately stained (without peptide re-stimulation) for CD4, Thy1.1, Ly5a and IFNγ, and were analyzed by flow cytometry. Approximately 2% memory SMARTA cells had begun to synthesize IFNγ in response to LCMV infection (top row) but none of those responding memory cells showed any dilution of CFSE signal. The naïve SMARTA cells (bottom row) failed to produce IFNγ at this early time point post-infection, and no sign of cell division was seen. Data are from one of two independent experiments.