Figure 4. Fyn is required for IL-12-dependent TCR signaling and bystander CD8 induction of IFNγ expression.
(A) OT-I or OT-IxLckind or OT-IxFynKO CTLs were generated upon OVA stimulation. On day 2, doxycycline was withdrawn from a cohort of the Lckind cultures (Lck-OFF). At day 3 CTLs were assessed for Kb-OVA, Lck and IL-12R expression before treating or not (NS) with 20 ng/mL IL-12 or congenic OVAp-pulsed APCs for 6 hours. Frequency of OT-1 cells expressing IFNγ expression is shown. (B) OT-1WT or FynKO CTLs were pre-treated with DMSO vehicle control, PP2 or anti-CD8β blocking antibody (5 µg/mL) and stimulated or not (NS) with 10 ng/mL IL-12 or Kb-OVA tetramer to determine levels of p21 CD3ζ by immunoblot (4G10 blot). Tubulin loading control was determined on the same membrane and Fyn control was obtained after stripping of the P-Y blot. Numbers and graph show densitometry values relative to NS control and corrected for loading control (α-tubulin). (C) Fyn (left) and Lck (right) immunoprecipitations were performed on lysates from OT-I CTLs stimulated with IL-12 after preclearing with beads and IgG controls. Immunoblots showing levels of pY416 SFK, p21ζ (4G10) or (D) Tyk2 pulled down with Fyn and Lck are shown. Extracts from an IgG precleared stimulated sample are shown as controls. Upon IL-12 stimulation (5ng/mL) in the absence (−) or presence of Tyk2 inhibitor (Tyk2 i), Fyn was immunoprecipitated and pY416 SFK and p21 CD3ζ were assessed by immunoblot. The percent inhibition of Fyn phosphorylation was measured relative to vehicle treated controls normalized to total Fyn. Fyn loading was determined after stripping on the same membrane. Data is representative n≥ 3 independent experiments with n=3 mice per group. All graphs show mean±SD. *** p=0.0005. N.S. (non significant).