FIG. 6.

Reevaluation of the total CD8⁺ T cell response to VACV. (A) C57BL/6 mice were administered OT-I CD8⁺ T cells by i.v. injection and then left uninfected (top plots) or were infected with 10⁶ PFU VACV WR (middle plots) or VACV-WR-SIIN (bottom plots). After another 7 days, spleens were taken and analyzed for surface CD8, CD62L, OVA₂₅₇-DimerX, and intracellular GzmB. GzmB against CD62L plots are shown for CD8⁺ (left plots) and for CD8⁺ and OVA₂₅₇-DimerX⁺ (right plots). Data are representative of the results obtained with 3 to 6 mice analyzed in 2 experiments. (B) Mice were infected with 10⁶ PFU VACV (Acute) or left uninfected (Naïve); after 7 days, spleens were taken and analyzed for surface CD8, CD62L, and intracellular GzmB. The percentages of CD8⁺ events representing the GzmB^hi-CD62L^lo population are shown. Data are representative of the results obtained with 5 mice in 2 experiments. (C) Total numbers of CD8⁺ cells in spleens were measured by flow cytometry for 5 naïve (filled bar) and 30 acutely infected (day 7; open bar) C57BL/6 mice over multiple experiments; data representing averages and SEMs of the results are shown. Using these values, the difference between the acutely infected and naïve (acute − naïve = excess in acute [striped bar]) mice was calculated. (D) The percentage of excess (acute − naïve) CD8⁺ cells in VACV-infected mice (shown as a percentage of all CD8⁺ cells in acutely infected mice) was calculated using the data shown in panel C. (E) A direct comparison between ICS for IFN-γ after restimulation of splenocytes with VACV-infected cells (left 2 columns) and staining of the same splenocytes (without restimulation) for CD8, CD62L, and GzmB (right column). For restimulations, uninfected cells (DC2.4) were used as a control for assays using VACV-infected cells (DC2.4+WR). Splenocytes were taken from uninfected (Naïve) or infected (WR 1 and WR 2) mice.