Figure 3. IL-12 mediated bystander CD8 T cell responses require TCR signaling.

(A) OT-I CTLs were labeled with CFSE before treating with vehicle or PP2 followed by stimulation or not (NS) for 48 hours with IL-12 and assessed for CFSE dilution or CD25 expression or apoptosis (frequency of 7-AAD⁺ cells) by flow cytometry. Induction of CD25 expression is normalized to the NS control. (B) OT-1 CTLs were stimulated for 6 hours with IL-12 before assessing intracellular IFNγ or Granzyme B expression. (C) Day 4 OT-1CTLs were incubated with 20ng/mL IL-12 or not (dotted line) for 16 hours before treatment with SFK inhibitor. CD8 effectors were then mixed at indicated ratios with CFSE-labeled EL-4 target cells either pulsed or not with OVAp and cytotoxicity was measured. Data is representative of 3–5 independent experiments. (D) OT-I CTLs were pre-treated with vehicle or PP2 followed by IL-12 stimulation for 48h and assessed for surface NKG2D and ICAM-1. (E) by flow cytometry. Graphs show induction of the expression of the proteins indicated relative to NS controls or frequency of cells expressing CFSE^low, 7ADD, IFNγ, GranzymeB. All graphs show mean±SD. Data is representative of 3–4 independent experiments. *p < 0.05, **p=0.005 or *** p=0.0005.