The following anti-human monoclonal antibodies (clones) from eBiosciences, San Diego CA, were used for surface staining of PBMCs for total T cells, subsets and activation: CD3 (HIT3a), CD4 (SK3), CD8 (SK1) and CD69 (FN50) and Ki-67 (20Raj1) was used for intracellular staining to assess turnover. Intracellular expression of OPG and RANKL in T cells, was performed as previously described [10 (link)]. Briefly, cells were first stained for surface markers and then fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/permeabliziation kit (BD Biosciences). To quantify intracellular OPG, cells were incubated with 5 μg/ml of anti-human OPG-biotin (Leinco Technologies) for 30 minutes at 4°C,followed by incubation with Streptavidin-PE. RANKL was quantified by incubation with 5 μg/ml recombinant OPG-Fc (R&D systems) for 30 minutes at 4°C as previously described [12 (link)], followed by anti-human IgG Fc-PE (BD Biosciences) for another 30 minutes at 4°C. All cells were fixed in 4% paraformaldehyde, data acquired on an Accuri flow cytometer (BD Immunocytometry Systems, San Jose, CA) and analyzed using FlowJo software version 9.7 (Ashland, OR).