10⁷ splenocytes from LCMV-gp33/H-2D^b–specific TCR-Tg 318 mice were injected i.v. into naive C57BL/6 mice. Mice were immunized with gp33 (1 mg in PBS) and CpG on days 0 and 4. A separate group of mice was additionally treated with poly(I:C) on day 7. The phenotype and function of CD8⁺ T cells were analyzed 24 hours (A–E; n = 3 per group) or 12 hours (F; n = 2–3 per group) later. (A) Splenocytes were analyzed by FACS for early activation marker CD25 and CD69. Histogram plots show cells gated on CD8 and Thy1.1 (marker for 318 T cells). Gray shading indicates staining with isotype control antibody. Values for FACS analysis give mean fluorecence intensity (MFI). For other plots in A, B, and C, the number of positive expressing cells (marked by bar or quadrant) are given. (B) Splenocytes were further analyzed for surface expression of CD44, CD62L, IL-7Rα, and VLA-4 (CD49d). Histogram plots show cells gated for CD8 expression and tet-gp33 expression (indicated by a black line) or tet-gp33–negative cells (control expression, indicated by gray shading). (C) Splenocytes were restimulated in vitro with or without gp33, then analyzed for intracellular expression of granzyme B and IFN-γ. Dot plots show cells gated for CD8 expression. (D and E) Splenocytes were analyzed for their ability to lyse peptide-loaded target cells in vitro (D) or in vivo (E). Naive splenocytes served as negative control; splenocytes from mice infected with 2 × 10⁶ pfu of LCMV-WE (WE high) served as positive control. (F) Eight hours after treatment with poly(I:C), splenocytes were analyzed for their expression of granzyme B (n = 2–3).