Reduced expansion of epitope-specific T cells in Lm-infected CD11c-Rac1(N17) mice. Spleens from Tg⁺ mice and WT controls were harvested 7 days after infection with 2000 Lm-OVA, and the frequencies of T cells specific for SIINFEKL (A), fMIGWII (B), and LLO_190-201 (C) were analyzed. A portion of cells was stained with PE-conjugated tetramers (H2-K^b/SIINFEKL or H2-M3/fMIGWII) in combination with anti-CD62L and anti-CD8α mAb and EMA to stain dead cells. Other cells were stimulated briefly (5 hours) in vitro in the presence of 10^-6 M SIINFEKL, fMIGWII, or LLO_190-201 peptide and brefeldin A. Intracellular staining (ICS) for IFN-γ followed staining with EMA and mAb specific for CD4, CD8α, and CD62L. Dot plots are gated on live CD8⁺ cells (SIINFEKL and fMIGWII) or CD4⁺ cells (LLO_190-201), with the frequency of activated (CD62L^low, x-axis) epitope-specific T cells as determined by tetramer or intracellular IFN-γ staining (y-axis, left and right dot plots, respectively) indicated. Total numbers of epitope-specific T cells are shown in the graphs to the right of each set of dot plots (n = 3 ± SD). Results are representative of 2 independent experiments. (D) CFSE-labeled OT-1 cells (2.5 × 10⁶) were transferred into Tg⁺ mice and WT controls, and mice were infected 1 day later with 1000 or 10 000 Lm-OVA. A group of control mice received OT-1 cells but was not infected. Splenocytes were harvested 3 days after Lm-OVA infection and stained with PI, anti-CD8α mAb, and K^b/SIINFEKL tetramers. The graph shows total numbers of OT-1 cells (positive for CFSE and tetramer) for 2 mice per group. Results are representative of 2 independent experiments.