Fig. 5.

The absence of Spry1/2 is associated with reduced activity of the mTORC1–AKT–FoxO1/3a signaling axis. mTORC1 activity was evaluated in chimeric mice harboring both WT and DKO P14 T cells after infection with 2 × 10⁵ pfu LCMV_ARM. (A) p-S6 intensity was measured directly ex vivo as a marker of mTORC1 activity in WT and DKO P14 T cells isolated from Med LNs of chimeric mice at 3, 8, and 13 d.p.i. (B) Representative histograms and MFI of WT (solid black line) and DKO (red line) P14 effector T cells from day 13 after LCMV_ARM infection after ex vivo gp33 stimulation. Splenocytes were harvested and rested for 2–4 h, followed by restimulation with gp33 peptide for 60 min. p-S6 and p-4EBP1 were measured by phospho-flow. The shaded histogram and dotted histogram are unstimulated WT P14 and DKO P14 effector T cells, respectively. (C and D) Extracellular glucose uptake was measured in the spleen by ex vivo uptake of 2-NBDG by P14 T cells at 6 (C) and 13 (D) d.p.i. (E) Representative histograms and MFI of WT (solid black line) and DKO (red line) P14 effector T cells from day 13 after LCMV_ARM infection after ex vivo gp33 stimulation. Splenocytes were harvested and rested for 2–4 h, followed by restimulation with gp33 peptide for 60 min, and p-AKT(T308) and p-FoxO1/3a were measured by phospho-flow. The shaded histogram and dotted histogram show unstimulated WT P14 and DKO P14 effector T cells, respectively, from LCMV_ARM-infected mice. (F) The expression level of T-bet in WT and Spry1/2 DKO effector P14 T cells at the peak of the response. The y axes of histogram overlays were normalized to mode. Data are representative of two independent experiments with n = 4–5 mice per group. Each point represents one individual mouse. The P values represent the difference between WT and DKO P14 T cells (paired t test): *P < 0.05, **P < 0.009, and ***P < 0.0005.