Expansion of Treg cells by FV-infected DCs depends on naive CD4⁺CD25⁺ T cells. (A) Naive TCR transgenic CD4⁺ (purity, > 97%) or CD4⁺CD25⁻ T cells (purity, > 96%) from DO11.10 mice were incubated with uninfected (thin small gray line) or FV-infected DCs (fat black line) in a 3D collagen gel. After 72 hours, gels were digested and T cells were analyzed for the intracellular expression of Foxp3 by flow cytometry. The experiments were repeated 3 times with comparable results, and the results from representative cultures are shown. (B) 5 × 10³ uninfected (□) or FV-infected (■) DCs were loaded with the OVA peptide and cocultured with 5 × 10⁴ naive TCR transgenic total CD4⁺ or CD4⁺CD25⁻ T cells from DO11.10 mice in a 3D collagen gel. Cells were incubated for 3 days and then pulsed with [³H] thymidine overnight to determine T-cell proliferation. Assays were done in triplicates and mean counts per minute (cpm) and standard deviations are shown. Differences between cultures with CD4⁺ versus CD4⁺CD25⁻ were statistically significant (P < .001).