EmTh-1 supernatant inhibits nTreg immunosuppressive function. (A) CD4⁺CD25⁺ nTregs were isolated from BALB/c mouse lymphoid tissues and cultured for the indicated periods of time with plate-bound anti-CD3 (5 ng/mL), soluble anti-CD28 (5 ng/mL), and IL-2 (20 IU/mL) with or without emTh-1 supernatant. FoxP3 expression was then determined using flow cytometry. (B) CD4⁺CD25⁺ nTregs were cultured for 48 hours with plate-bound anti-CD3, soluble anti-CD28, and IL-2 with or without emTh-1 supernatant. Cells were then washed extensively with complete medium. Responder CD4⁺CD25⁻ T lymphocytes were stimulated with anti-CD3/anti-CD28 T-cell expander beads in the absence (CD25⁻) or presence of untreated nTregs (CD25⁻ + untreated nTreg) or in the presence of emTh-1 supernatant–treated nTregs (CD25⁻ + [nTreg]_{emTh-1 sup}). Responder CD4⁺CD25⁻ T-lymphocyte proliferation was determined after 48 hours using BrdU incorporation assays. NS, nonsignificant; *P < .001, a significant difference compared with responder CD25⁻ T cells cultured with untreated Tregs. (C) CD4⁺CD25⁻ T lymphocytes were first treated ([CD25⁻]_{emTh-1 sup}) or not (untreated CD25⁻) for 48 hours with emTh-1 supernatant, washed extensively with complete medium, and cocultured for 48 hours with freshly isolated CD4⁺CD25⁺ nTregs (+ nTreg). Proliferation of responder CD25⁻ T cells was then determined using BrdU incorporation assays. *P < .001. (D) IFN-γ concentration was assessed in the cocultures as described for panel C. *P < .001; **P < .0001.