Osteoclast-induced Tc_REG inhibit osteoclast differentiation, but not survival.

<p><b>A</b>. Osteoclast precursor cells (day 0) were cultured alone, with pre-differentiated Tc_REG, iT_REG, anti-mouse CD3/CD28 re-stimulated iT_REG, or with naïve T-cells in the presence of GST-RANKL. After four days, non-adherent cells were removed by aspiration and remaining cells were stained with a fluorescent TRAP substrate ELF-97. Representative images from four experiments are shown in top panel, and quantitated cells counts are shown below. To test if suppression of osteoclastogenesis was mediated solely by IFN-γ, bone marrow cells from IFNγR1^−/− mice were used. The results of TRAP staining were confirmed by quantitative real-time PCR using primers for Acp5 (TRAP), Cathepsin K (CTSK) and matrix metalloprotease-9 (MMP-9). β-actin expression was used for normalization. <b>B</b>. Osteoclasts, cultured in the absence or presence of Tc_REG for five days were counted after TRAP staining with ELF-97. To test for increased apoptosis adherent cells were stained with Annexin V. Each data point is average of three wells per experiment. Statistical significance was assessed by non-parametric paired T test: *: P<0.05, **: P<0.01 and ***: P<0.001 by comparison to untreated (Untx) osteoclast wells.