P14 transgenic CD8 T cells were adoptively transferred into naïve congenic recipients followed by infection with 1.5×10⁷ pfu PV i.p. A, CFSE profiles of donor P14 CD8 T cells at 0, 3, 6, 9, and 12 days post PV infection from individual mice representative of > 10 mice from 3 independent experiments. B, Frequency (left axis, closed diamonds) and number (right axis, open diamonds) of P14 CD8 T cells during PV infection, average of 3 mice/group, representative of 3 independent experiments. C, At day 0 (naïve) and day 5 post PV infection, splenocytes were harvested and stained for the surface markers shown. Representative examples of host CD8 T cells (shaded) and P14 CD8 T cells (thick black) are overlaid from the same host, representative of > 10 independent experiments.

P14, HY, or OT-I transgenic CD8 T cells were adoptively transferred into naive congenic recipients followed by inoculation with 5×10⁴ pfu LCMV, 1.5×10⁷ pfu PV, 1×10⁶ pfu VV, or 200 µg poly IC i.p. At days 0 (naive) and 5 post infection or day 1 post poly(I:C), splenocytes were stimulated with GP_33–41, Smcy, or SIINFEKL peptides ex vivo. Each transgenic population was gated and the ability of the transgenic CD8 T cells to produce IFN-γ in response to their cognate peptide was assessed. IFN-γ vs. CFSE is shown from representative mice from 8 independent experiments and the numbers depict the frequency of CFSE^hi IFN-γ⁺ events, except for OT-I + poly(I:C) which shows IFN-γ vs CD8.

A–B, P14 CD8 T cells were adoptively transferred into WT congenic recipients, followed by i.v. inoculation with A, anti-NK1.1 or IgG2a, or B, anti-GK1.5 or IgG2b to deplete NK cells or CD4 T cells respectively. One day post antibody treatment, mice were inoculated with 200 µg poly(I:C) i.p. At days 0 (untreated) and 1 post poly IC inoculation, splenocytes were stimulated with GP_33–41 peptide ex vivo and the intracellular accumulation of IFN-γ was assessed. Splenocytes were also stained with anti-NK1.1 clone PK136 (gated on CD3⁻ DX5⁺) and anti-CD4 clone RM4-4 (gated on CD3⁺) of different clones than the depletion antibodies to assess depletion. C–H, P14 CD8 T cells were adoptively transferred into WT, IFN-γ KO (C), IL-12 KO (D), IL-18 KO (E), or IL-15 KO (F–H) congenic recipients. One day post adoptive transfer, mice were inoculated with 200 µg poly(I:C) i.p. At days 0 (untreated) and 1 post poly(I:C) inoculation, splenocytes were stimulated with GP_33–41 peptide ex vivo and the intracellular accumulation of IFN-γ (C–F), GrzB (G), or degranulation (H) was assessed.

P14 transgenic CD8 T cells were adoptively transferred into naïve WT, K^bD^b KO, K^b KO, or D^b KO congenic recipients followed by inoculation with 200 µg poly(I:C) i.p. At days 0 (untreated) and 1 post poly(I:C), splenocytes were stimulated with GP_33–41 peptide + exogenous WT splenocytes ex vivo. The frequency of IFN-γ-producing (A), CD107⁺ (B), and GrzB⁺ (C) P14 CD8 T cells were graphed. D, Functional IFN production was graphed. E, TNF production by unstimulated (grey histograms) and GP_33–41-stimulated (black lined histograms) P14 CD8 T cells was assessed. Data depict 3–4 mice/group and is representative of 3 independent experiments.

IFN-αβ induced by viral infection or TLR agonist poly(I:C) induces expression of IL-15 and up-regulation of MHC I. Up-regulation of MHC I enhances presentation of self and virus-encoded peptides, which signal through the TCR of bystander CD8 T cells, inducing Eomes expression. Eomes induces expression of CD122, which confers IL-15 responsiveness and induces GrzB expression. In response to cognate antigen, the expression of Eomes allows for the rapid synthesis of IFN-γ. IL-15 expression is also required for the enhanced cognate antigen-driven degranulation.

P14 transgenic CD8 T cells were adoptively transferred into naïve congenic recipients followed by infection with 1.5×10⁷ pfu of PV or inoculation with 200 µg poly(I:C) i.p. A, At day 5 of PV infection or day 1 after poly(I:C) treatment, splenocytes were stimulated with GP_33–41 peptide ex vivo. P14 transgenic CD8 T cells were gated, and intracellular accumulation of IFN-γ was assessed. B, At days 0 (naïve), 5, 10, 15, and 20 post PV infection, splenocytes were stimulated with GP_33–41 peptide ex vivo and the frequency of P14 CD8 T cells producing IFN-γ was assessed. C, At days 0 (untreated), 1, 2, and 3 post poly(I:C) inoculation, splenocytes were stimulated with GP_33–41 peptide ex vivo and the frequency of P14 CD8 T cells producing IFN-γ was assessed. D, At day 0 (untreated), day 1 post poly(I:C), or day 5 post PV GrzB expression was assessed by intracellular staining directly ex vivo and E, degranulation (CD107 surface expression) was assessed after GP_33–41 peptide stimulation. The frequency of P14 CD8 T cells staining positive and MFI for both molecules were graphed. Representative experiments with 3–5 mice/group are shown, experiments were independently performed > 10 times. *p<0.05, **p<0.005, ***p<0.0005

P14 CD8 T cells were adoptively transferred into congenic recipients followed by inoculation with 0–100 µg poly(I:C) i.p. One day post poly(I:C) inoculation, serum was collected for bioassay and splenocytes were collected for stimulation. A, Functional IFN for each individual mouse was assessed via VSV-induced CPE bioassay, and the log₂ of the reciprocal of the serum dilution that provided 50% protection was graphed. Dashed line depicts the level of detection for the assay. B, The level of functional IFN was plotted against the ability of P14 T cells in each individual mouse to express IFN-γ in response to GP_33–41 peptide stimulation from 2 independent experiments. n= 34 mice, r²=0.560, and p<0.005. C, The level of functional IFN was plotted against the expression of granzyme B in P14 T cells in each individual mouse. n= 18 mice, r²=0.563, and p<0.005. D, The level of functional IFN was plotted against the ability of P14 T cells in each individual mouse to degranulate in response to GP_33–41 peptide stimulation. n=18 mice, r²=0.389, and p<0.05. E–F, WT or IFN-αβR KO P14 CD8 T cells were adoptively transferred into WT or IFN-αβR KO congenic recipients, followed by inoculation with 200 µg poly(I:C) i.p. At days 0 (untreated) and 1 post poly(I:C) inoculation, serum was collected for bioassay and splenocytes were stimulated with GP_33–41 peptide ex vivo. E, Functional IFN in all poly(I:C)-treated groups was graphed. F, The ability of P14 CD8 T cells to rapidly express IFN-γ was assessed. Cumulative data from 5 independent experiments is depicted. ***p<.0005

P14 transgenic CD8 T cells were adoptively transferred into naïve WT or β2m KO congenic recipients followed by inoculation with 200 µg poly(I:C) i.p. At days 0 (untreated) and 1 post poly(I:C), splenocytes were stimulated with GP_33–41 peptide + exogenous WT splenocytes ex vivo. A, P14 CD8 T cells were gated and intracellular accumulation of IFN-γ and TNF was assessed. B, The frequency of IFN-γ producing P14 cells was graphed (n=4/group). C, Functional IFN production was graphed. Data are representative of 4 independent experiments. **p<0.005

A, P14 transgenic CD8 T cells were adoptively transferred into naïve congenic recipients followed by inoculation with 5×10⁴ pfu LCMV, 1.5×10⁷ pfu PV, or 200 µg poly(I:C) i.p. P14 CD8 T cells were sorted from naïve P14 mice (Naïve), day 6 post LCMV (Effector), day 42 post LCMV (Memory), day 1 post poly(I:C), day 5 post PV, or day 20 post PV. RNA was extracted and converted to cDNA, followed by real-time PCR using primers for T-bet and Eomes. Standardized mRNA relative to 18S rRNA was calculated from an individual experiment that is representative of 4 independent experiments. B–D, P14 transgenic CD8 T cells were adoptively transferred into naïve congenic WT, H2D^b KO, or IFN-αβR KO recipients followed by inoculation with 1.5×10⁷ pfu PV or 200 µg poly(I:C) i.p. At day 5 of PV infection or day 1 after poly(I:C) treatment, splenocytes were stimulated with GP_33–41 peptide ex vivo. B, P14 transgenic CD8 T cells were gated, and IFN-γ production and Eomes expression was assessed from representative mice for each group. C, The frequency of Eomes⁺ P14 CD8 T cells in WT, H2D^b KO, and IFN-αβR KO mice was graphed. D, The frequency of Eomes⁺ CD44^lo naïve host CD8 T cells WT, H2D^b KO, and IFN-αβR KO mice was graphed. Data in (B–D) are representative of 3 independent experiments.