(A) Splenocytes from short-boosted or long-boosted mice were stimulated at day 14 after 3° HPBB with or without SIINFEKL peptide and examined for TNFα and IFNγ production. Plots are gated on CD8+ T cells. (B) Kb-SIINFEKL tetramer+ CD8 T cells (black) and IFNγ -producing CD8 T cells (white) were enumerated in spleen at day 14 post 3°. Data are representative of 2 experiments N=1-5 mice per experiment. (C) Splenocytes were isolated 32 days post 3° from short- and long-boosted mice, enriched for CD8 T cells and equal numbers of Kb-SIINFEKL-specific CD8 T cells were transferred to naïve CD45.1 mice. New hosts were infected with VSV-OVA 1 day after transfer. 6 days after infection, the number of donor+, Kb-SIINFEKL+ CD8 T cells was enumerated in spleen. Data are representative of 1 experiment, N= 6-7. (D) Splenocytes were isolated at day 95 post 3° from short-boosted mice and ≥95 days post 3° from long-boosted mice, enriched for CD8 T cells and sorted for Kb-SIINFEKL-specific CD8 T cells. Either 2 × 103 or 2 × 105 Kb-SIINFEKL-specific CD8 T cells were transferred to CD45.1 mice. One day after transfer, recipients were infected with VSV-OVA and donor CD45.2 Kb-SIINFEKL-specific CD8 T cells from short-boosted (solid line) and long-boosted (dotted line) mice were measured in the blood at the indicated timepoints. Gray lines are low cell number transfers and black lines are high cell number transfers. At all timepoints, there is no significant difference in tetramer+ % between short- and long-boosted cells at low or high numbers of transferred cells. (E) Recipient mice from (D) were sacrificed at day 22 post infection and donor Kb-SIINFEKL-specific CD8 T cells from short-boosted (black) and long-boosted (white) mice were enumerated from spleen. Data in (D) and (E) are representative of 1 experiment N=at least 4.