Evaluation of antigen specificity of CD4⁺CD25⁺Foxp3⁺ Tregs in the liver of PGK.142T-treated mice. (A) CD4⁺ T cells were isolated from the livers of mice treated 3 weeks prior with PGK.142T. Cell purity was determined by FACS analysis (> 90%). Purified hepatic CD4⁺ T cells from PGK.142T-treated mice were injected into naive recipient mice (10⁶ CD4⁺ T cells/mouse). Two days later, the mice received DNA vaccination to induce a cellular immune response to GFP or to hepatitis B small env subunit (HBs), an unrelated antigen. Twelve days after DNA vaccination, in vivo CTL assay was performed. Mice were infused with splenocytes labeled with different concentrations of CFSE and pulsed with either HBs_28-39 (CFSE^int) or GFP_200-208 (CFSE^hi), or left unpulsed (CFSE^low). Draining lymph nodes were collected, and FACS analysis was performed to quantitate the surviving cells. Mice receiving vaccination alone are show as filled histograms (n = 5), and mice receiving vaccination plus adoptive transfer of hepatic CD4⁺ T cells are shown as open histograms (n = 3). A representative histogram for each experimental group is shown (no vaccination, top panel; GFP vaccinated, midpanel; HBs vaccinated, bottom panel). The mean percentage ± SD of antigen-driven target cell lysis values for each experimental group of mice is reported (A right panel). (B) Liver-infiltrating CD4⁺ T cells isolated from mice treated with PGK or tolerized to GFP by PGK.142T vector were sorted by CD25 expression, and the 4 resulting populations were adoptively transferred (0.8 × 10⁵ cell/mouse) into naive mice that underwent GFP vaccination 2 days later. As described above, in vivo CTL assay was performed 12 days after vaccination. Mice receiving vaccination alone are shown as filled histograms (n = 10), and mice receiving vaccination plus adoptive transfer of hepatic CD4⁺CD25⁻ (n = 8) and CD4⁺CD25⁺ (n = 8) T cells derived either from PGK- or PGK.142T-treated mice are shown as open histograms. A representative histogram for each experimental group is shown. The mean percentage of antigen-driven target cell lysis ± SD values for each experimental group of mice is reported (B, right panel).