S typhimurium–induced CD4⁺ Th1 cells are resistant against suppression by CD4⁺CD25⁺ Treg cells. (A) CD4⁺CD25⁻ T cells isolated from PBMCs of NC235 were cultured with APCs pulsed with a NY-ESO-1 peptide or infected with S typhimurium–NY-ESO-1 for 20 days. The presensitized T cells were labeled with CFSE as described in “CFSE labeling” and restimulated with T cell–depleted PBMCs pulsed with NY-ESO-1 or control peptides for 6 days. The cells with diluted CFSE staining were gated, and surface activation markers were analyzed as indicated. (B). CD4⁺CD25⁺ Treg cells were isolated from PBMCs and preactivated as described in “CFSE labeling.” S typhimurium–induced or peptide-induced CD4⁺ Th1 cells were cultured with T cell–depleted PBMCs pulsed with cognate NY-ESO-1 peptides in the absence or presence of these preactivated CD4⁺CD25⁺ Treg cells and/or 10 μg/mL blocking anti-GITRL antibody. Proliferation was assessed as described in “Proliferation assay.” These experiments were performed independently at least twice with similar results. Data in panel B are expressed as means plus or minus SD. *P ≤ .01.