Ova a5503

Day 4 OC cultured in 20 ng/ml M-CSF and 50 ng/ml GST-RANKL, were seeded at 5×10⁵ cells/well in the presence of 5 μM OVA (A-5503; Sigma-Aldrich) in 24-well tissue culture-treated plates (Corning). After 14–16 h of incubation, medium was removed and (adherent) cells were washed with pre-warmed medium. 2.5×10⁵ freshly harvested splenic OT-I transgenic T cells purified by negative selection were added in 2 ml of complete T-cell media (RPMI, 10% ΔFBS, penicillin-streptomycin-glutamine, non-essential amino acids, sodium pyruvate, HEPES, and 55 μM β-mercaptoethanol). Following 48 h co-culture, T-cell aliquots were removed and intracellular staining was performed to assess FoxP3 expression. The Tc_REG were then further expanded, in the absence of OC, by splitting cells 1:2 and culturing in 100 U/ml IL-2 containing T-cell media for an additional 48 h. For polyclonal Tc_REG generation, T-cells were purified from spleens of C57BL/6 mice and incubated with day 4 OC in the presence of 1μg/ml anti-CD3. Control T-cells were activated with plate bound anti-CD3 (1 μg/ml) and anti-CD28 (2μg/ml; both from eBiosciences) for 48 hours; the activated T-cells were expanded further by splitting 1:2 and culturing for additional 48 hours in IL-2 (100 U/ml). 20×10⁶ Tc_REG (in 200 μl) were then injected by tail vein into 8-week-old OT-I mice.