Figure 3. Osteoclasts induce the transcription factors T-bet and Eomes in CD8 T-cells needed for IFN-γ production by Tc_REG.

(A) Flow cytometry plots showing stimulation of T-bet and Eomes expression in OT-I CD8 T-cells cultured under Tc_REG-inducing conditions (cocultured with osteoclasts for 48 h in the presence of the ovalbumin derived peptide antigen SIINFEKL). Cells were gated on CD45⁺CD3⁺CD8⁺ populations prior to determining T-bet and Eomes expression. Data are representative of five independent experiments. (B) Left panel: Inhibition of IKK, by IKK16, but not Notch signaling, by DAPT, blocks IFN-γ production by Tc_REG. OT-I CD8 T-cells were primed by osteoclasts for 48 h in the presence of 10 μM γ-secretase inhibitor DAPT or 10 μM IKK inhibitor IKK-16. GolgiStop was added to the cultures during last 3 h of incubation. T-cells were collected and stained for flow cytometry analysis. Right panel: Effect of the inhibitors on FoxP3 expression in CD8 T-cells. For both panels cells were gated on CD45⁺CD3⁺CD8⁺ population to determine IFN-γ or FoxP3 expression. (C) Reduction in IFN-γ production in Tc_REG by IKK-16 coincides with decrease in T-bet and Eomes expression levels. Flow cytometry plots showing cells from the experiment described in (B) and analyzed for T-bet and Eomes in CD45⁺CD3⁺CD8⁺ population. (D) Effect of IKK-16 on expression of IL-6 and IL-10 by Tc_REG. Flow cytometry plots showing that IKK inhibitor does not prevent induction of IL-6, whereas expression of IL-10 is blocked by IKK-16. Cells were gated on CD45⁺CD3⁺CD8⁺ population to determine expression of the cytokines.