SLP-76 KI mice have intact CD8⁺ T-cell expansion but defective effector function in response to acute LCMV infection. (A) PBLs were isolated from serial bleeds from WT and KI mice at the indicated time points before and after LCMV infection and were analyzed by flow cytometry. CD8⁺ T cells (average ± SEM) are represented as a percent of the live lymphocyte gate (data are representative of 3 independent experiments each with 3-5 mice per group). Absolute numbers of (B) CD8⁺ (average ± SEM) and (C) tetramer-reactive (average ± SEM) splenocytes were calculated from day 8 infected WT and KI mice (representative of 2 independent experiments each with 5 mice per group). (D-F) Splenocytes from day 8 infected WT, Y145F, and Y112/128F KI mice were cultured in vitro with the indicated stimuli and analyzed by flow cytometry (representative of 2 independent experiments each with 5 mice per group). (D) Percent of CD8⁺ cells that produced IFNγ was calculated (average ± SEM). (E) The MFI of IFNγ staining in all CD8⁺ T cells producing IFNγ is shown (average ± SEM; left panel) and the percentage of total IFNγ producers that also produced IL-2 is shown (right panel). (F) Representative contour plots show IFNγ and TNFα expression in CD8⁺ splenocytes. Numbers indicate the percentage of CD8⁺, TNFα IFNγ double producers over the percentage of total CD8⁺ IFNγ producers (representative of 2 independent experiments each with 5 mice per group). Significant P values, when present, comparing KI to WT are indicated by asterisks: ***P < .001, **P = .001-.01, *P = .01-.05.