<p>Mature osteoclasts (day 4) or osteoclast precursors were cultured with no Ag or OVA protein and then used to prime CD8 T-cells from an OT-I mouse. <b>A</b>. T-cells were collected at 48 h following initiation of co-culture. T-cells were stained for CD44, CD25 and FoxP3 and then analyzed by flow cytometry. T-cells co-cultured with osteoclasts in the absence of antigen do not express FoxP3<sup>+</sup> or CD25 (top); FoxP3 and CD25 were induced in the presence of antigen as shown in the representative flow plot. The expression of FoxP3 was confirmed by reverse-transcription of RNA isolated from the co-culture and subsequent PCR of cDNA. GFP sorted cells from FoxP3<sup>eGFP</sup> reporter mice were used as controls. Only mature (day 4) osteoclasts supported the generation of Tc<sub>REG</sub> (right panel). <b>B</b>. Anti-mouse TGFβ was added to co-cultures at the dose indicated (left). Addition of recombinant murine TGFβ1 to co-cultures of CD8 T-cells and osteoclasts at concentration indicated (right). The percent of input T-cells converted to FoxP3<sup>+</sup> are plotted in both panels. No statistically significant effect was observed on Tc<sub>REG</sub> induction with either the addition of neutralizing antibody or recombinant TGFβ. <b>C</b>. Media was collected and cytokine quantitated by multiplexed ELISA. After 48 h of co-culture, cells were treated with Golgi stop and PMA plus ionomycin for 6 h. The cells were permeabilized, stained and evaluated for cytokine production by flow cytometry. While the CD11b<sup>+</sup> osteoclasts were negative for all cytokines, the CD8<sup>+</sup> T-cells stained triple positive for IL-10, IL-6, and IFN-γ. Statistical significance was assessed by non-parametric paired T test: *: P<0.05, **: P<0.01, ***: P<0.001 and ****: P<0.0001.</p
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