SLP-76 KI memory cells expand in response to rechallenge in vivo. Equal numbers of CD8⁺ T cells from LCMV immune WT, Y145F, and Y112/128F mice were transferred into congenic hosts. Host mice were subsequently infected with LM:GP33 and analyzed 7-8 days later. (A) Fold increase of donor-derived H2D^b:GP33-reactive cells in spleens from host mice that received WT, Y145F, and Y112/128F donor cells after 7 days postinfection. Each point represents an individual mouse; bars indicate average ± SEM. Data are pooled from 3 independent experiments each with 2-7 recipients per group. (B) Percentage H2D^b:GP33-reactive cells of donor CD8⁺ T cells from spleens of day 7-8 postrechallenge mice (average ± SEM; left panel). Percentage of donor CD8⁺ T cells that produced IFNγ in response to GP33 peptide stimulation is graphed (average ± SEM; right panel). Data are pooled from 4 independent experiments, each with 2-7 recipients per group. (C) Splenocytes from recipient mice receiving WT, Y145F, and Y112/128F on day 7 following LM:GP33 infection were stimulated with GP33 or PMA plus ionomycin. Representative contour plots depict TNFα and IFNγ expression in CD45.2⁺CD8⁺ lymphocytes that were stimulated with GP33 peptide (left panel). MFI of IFNγ in response to GP33 is shown (left-most graph). Numbers indicate the percentage TNFα IFNγ double producers over the percentage of total IFNγ producers within the CD45.2⁺CD8⁺ gate. Peptide- or PMA/ionomycin-responsive splenocytes were quantified as of the frequency of CD45.2⁺CD8⁺ IFNγ, producers that coproduced TNFα (center graph), or IL-2 (right-most graph; representative of 4 independent experiments each with 2-7 recipients per group). Significant P values, when present, comparing KI to WT are indicated by asterisks: ***P < .001, **P = .001-.01, *P = .01-.05.