Optimized ex vivo expansion of 17195TCR-Tregs with FVIII-2191-2220 plus oligodeoxynucleotides. (A) FVIII-2191-2220–specific enrichment of transduced Foxp3⁺Helios⁺ Tregs via long-term expansion. After a second round of expansion (described in supplemental Figure 2), the CD4 T cells were stained using DR1 tetramers loaded with FVIII-2191-2220 (top contour plots). Expression of Foxp3 and Helios in the gated CD4⁺GFP⁺ population were also evaluated by intracellular staining (bottom contour plots). (B) Comparison of FVIII-2191-2220 specificity and Fox3⁺Helios⁺ phenotypes of short-term and long-term expanded 17195TCR-Tregs. First-round expanded (Exp) cells (1st Exp) were harvested 8 days after viral transduction, and second-round expanded cells (2nd Exp) were harvested 16 days after transduction. FVIII-2191-2220 specificity was determined by measuring the increase of tetramer⁺CD4⁺ cells as in A (right line graph). The Foxp3 and Helios expression levels were measured by intracellular staining, and the Foxp3⁺Helios⁺ cell count was obtained by gating of the GFP⁺CD4⁺ population (left line graph). Representative data from 1 of 2 experiments are shown. (C) Plasticity of long-term expanded 17195TCR-Tregs and Treg phenotype stability induced by antigen. Second-round expanded 17195TCR-transduced Tregs were rested for 3 days of culture without IL-2 and then restimulated for 4 hours with phorbol myristate acetate and ionomycin in the presence of Golgi-block reagent. Intracellular IFNγ (top contour plots) and IL-2 (bottom contour plots) levels were measured by FACs analysis. The bar graphs on the right indicate the relative expression levels of IFNγ (top graph) and IL-2 (bottom graph) in GFP⁺CD4⁺ cells from each treated group. (D) DNA methylation of TSDR in long-term expanded 17195TCR Tregs with FVIII-2191-2220 plus ODN. To analyze DNA methylation in the TSDR, mock-transduced and 17195TCR-transduced Tregs were expanded with anti-CD3ε antibody or FVIII-2191-2220 in the presence of ODN, as in A. TSDR is an unmethylated CpG-enriched region within the Foxp3 genome of natural Treg. Heat map analysis indicated the methylation status of 9 of the total 11 CpGs. The histogram summarizes the mean percent methylation of 9 TSDR CpGs in 17195TCR-transduced Tregs produced by FVIII 2191-2220/ODN stimulation vs Tregs produced by anti-CD3ε antibody stimulation.