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cei12165-sup-0001-fs1.TIF815K

Fig. S1. The inhibitory effect of activated γδ T cells and CD4+ T cells on osteoclast formation is independent of cell–cell contact. Quantification of osteoclast formation following incubation of osteoclast precursor cells with anti-CD3/CD28-activated γδ or CD4+ T cells for 5 days, in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB-ligand (RANKL). Activated γδ T cells and activated CD4+ T cells were co-cultured with osteoclast precursor cells to permit cell contact, or cells were separated using a Transwell insert into which the activated T cells were placed. Osteoclast formation was assessed using immunohistochemical staining for vitronectin receptor (VNR) and counterstaining with 4′,6-diamidino-2-phenylindole (DAPI). Multi-nucleated (≥3 nuclei) VNR+ cells per well were quantified using fluorescence microscopy. Data shown are the mean ± standard deviation from two experiments from independent donors.

cei12165-sup-0002-fs2.TIF839K

Fig. S2. The blockade of granulocyte–macrophage colony-stimulating factor (GM-CSF) does not overcome the inhibitory effect of activated γδ T cells or CD4+ T cells on osteoclastogenesis. Osteoclast precursors were cultured in the presence of receptor activator of nuclear factor κB-ligand (RANKL) + macrophage colony-stimulating factor (M-CSF) and treated with 10% (v/v) conditioned medium (CM) from activated γδ T cells (a), or 10% (v/v) CM from activated CD4+ T cells (b), in the presence or absence of αGM-CSF or isotype control [immunoglobulin (Ig)G1], for 5–6 days. Following immunohistochemical staining for vitronectin receptor (VNR) and nuclear counterstaining with 4′,6-diamidino-2-phenylindole (DAPI), multi-nucleated (≥3 nuclei) VNR+ cells per well were quantified using fluorescence microscopy. Data shown are the mean ± standard error of the mean from three experiments from independent donors. *P < 0·05; ***P < 0·001.

cei12165-sup-0003-fs3.TIF885K

Fig. S3. Zoledronate-activated γδ T cells inhibit osteoclast differentiation via interferon (IFN)-γ. Peripheral blood mononuclear cells (PBMCs) were seeded in a 24-well plate at a density of 1 × 106 cells/ml (1 ml/well) in α-minimum essential medium (MEM) supplemented with 1 μM zoledronic acid (ZOL) plus 10 U/ml interleukin (IL)-2 and were cultured for 7 days. Cells were subjected to half-medium changes every 3 days. After 7 days γδ T cells were positively isolated using γδ T cell magnetic affinity cell sorter (MACS) beads. Macrophage colony-stimulating factor (M-CSF)-expanded osteoclast precursors were treated with M-CSF and receptor activator of nuclear factor κB-ligand (RANKL) for 5 days, in the presence or absence of autologous ZOL-activated γδ T cells. In some wells osteoclast precursors were incubated with 10% (v/v) conditioned medium from 72 h cultures of PBMCs treated with ZOL, as described above. To assess the contribution of interferon (IFN)-γ to the inhibitory effect of γδ T cells (or conditioned medium) on osteoclast formation, affinity-purified polyclonal goat anti-human IFN-γ antibody (10 μg/ml) was added to the cultures. Data shown are the mean ± standard deviation from two experiments from independent donors.

cei12165-sup-0004-fs4.TIF729K

Fig. S4. Activated γδ T cells and CD4+ T cells inhibit the resorptive activity of mature osteoclasts. Mature osteoclasts were seeded onto dentine discs and cultured with resting or activated γδ T cells (a), or resting or activated CD4+ T cells (b), for 6 days. The resorptive area was quantified using reflected light microscopy and in-house software to determine resorption pits. Data shown are the mean ± standard error of the mean from three experiments from independent donors. ***P < 0·001.

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