Fig. 3.

CD4⁺ T cell-induced osteoclastogenesis. (A) T cells from XHIM patients induce robust osteoclastogenesis. Purified monocytes (5 × 10⁵) were cultured alone, with recombinant RANKL (100 ng/ml), or with positively selected autologous CD4⁺ T cells (1 × 10⁵) activated by plate-bound anti-CD3 and soluble anti-CD28. The total number of osteoclasts (as defined by TRAP-staining giant cells with more than three nuclei) was counted in each well after 7 days of culture. These values are expressed as the number of cells per mm² ± SEM, and they are represented in the top right of each image. One representative example of two independent experiments is shown. (B) CD4⁺ CD40L^−/− mouse T cells induce marked osteoclast differentiation of BMMs. Nonadherent mouse BMM cells were cocultured with CD4⁺ CD40^+/+ (Left) or CD4⁺ CD40L^−/− (Right) T cells. T cells were activated with anti-CD3ϵ antibody (10 μg/ml), and the number of cells added to the cocultures is specified on the left side of the figure. The total number of osteoclasts in each well was counted after 72 h of culture, and it is expressed as the number of cells per mm². These values are expressed in top right of each image. One representative example of three independent experiments is shown. ∗, P = statistically significant. (C) IFN-γ levels in mouse coculture supernatants were analyzed 36 h after the addition and activation of T cells (the columns represent the median ± SEM for three independent assays measured by specific ELISA). ∗, P = statistically significant.