Figure 1. Tc_REG inhibit osteoclastogenesis via IFN-γ.

(A) Representative flow cytometry plots showing that OT-I CD8 T-cells primed by osteoclasts pulsed with SIINFEKL express Foxp3 and IFN-γ. T-cells were collected for staining after 48 h co-culture with osteoclasts. Protein transport inhibitor GolgiStop was added to the cultures during last 3 h of incubation. Cells were gated on CD45⁺CD3⁺CD8⁺ population to determine FoxP3 and IFN-γ induction. (B) IFN-γ^−/− Tc_REG have reduced anti-osteoclastogenic activity. Osteoclast-specific TRAP mean fluorescence intensity values are shown for osteoclast precursors after co-culturing them for 5 days with Tc_REG generated from WT or IFN-γ^−/− splenic CD8 T-cells (N=5–8 wells/group). (C) Tc_REG induced TRAF6 degradation in macrophages via IFN-γ. Bone marrow macrophages were cultured in the presence of WT or IFN-γ^−/− Tc_REG, or recombinant IFN-γ (20 or 100 U/ml) for 24 h. Adherent cells were removed and whole cell lysates of the macrophages were subjected to Western blotting for TRAF6 and β-actin. Band intensities were quantitated to show normalized TRAF6 levels. Data are representative of three independent experiments. MØ – macrophage, N – naïve CD8 T-cells. Statistical significance was assessed by non-parametric paired t-test: * p<0.05, ** p<0.01, *** p< 0.001.