Inflammatory CD4⁺ T cells from oc/oc mice are responsible for a high RANK-L expression and an increased osteoclastogenesis. (A) Flow cytometric analysis of splenic CD4⁺ T cells from oc/oc and +/+ mice using the activation marker CD62L. An isotype control (control) is presented. The results are representative of at least 5 oc/oc and 5 +/+ mice. (B) Intracytoplasmic analysis of IL-17 and IFN-γ production by flow cytometry on CD3⁺ T cells purified from the bone marrow. Nonactivated T cells served as control. Data are gated on pooled CD4⁺ cells from 4 or 5 oc/oc or +/+ mice. All results are representative of 3 independent experiments. (C) Real-time RT-PCR analysis of CD4⁺ T cells purified from the bone marrow of +/+ and oc/oc mice. The results are the mean plus or minus SD of 5 mice in each group. (D) Osteoblasts from +/+ mice were cultivated alone or in the presence of CD4⁺ T cells purified from the bone marrow of +/+ and oc/oc mice. Expression of Rank-l and M-csf was analyzed in the osteoblasts by real-time RT-PCR. The results are the mean plus or minus SD of 3 wells and are representative of 3 independent experiments. (E) Osteoclasts were generated by coculture of DCs with osteoblasts from +/+ mice and with our without CD4⁺ T cells purified from the bone marrow of +/+ and oc/oc mice. A representative result is presented, and multinucleated TRAP⁺ osteoclasts (arrow) were enumerated. The results are the mean of 8 wells and are representative of 3 independent experiments. Images were captured using an Axiovert 200 microscope (Carl Zeiss) with a 10×/0.25 Ph1 Plan objective (Carl Zeiss), a JVC KY-F50 camera, and AxioVision 4.5 software (Zeiss).