Blockade of IFN-γ and NO production abolished NR286 T cell regulatory function.

<p>(<b>A</b>) CFSE-labeled CD4⁺ BDC2.5 T cells (2×10⁵/well) were cultured under seven different culture conditions. In control cell cultures, BDC2.5 T cells were cultured in conditions of either (1) alone or (2) with p79 plus CD4⁺ cell-depleted irradiated APCs for activation. BDC2.5 T cells were also cultured with (3) an equal number of NR286 T cells. In some experiments, antibodies against (4) IFN-γ or (5) IL-10 (24 µg/ml), and NO inhibitors, either (6) L-NMMA (active) or (7) D-NMMA (inactive) (0.2 µM), were added to the cultures to determine their effect on blocking the regulatory function of NR286 T cells. The number shown in each histogram represents the percentage of BDC2.5 T cells that underwent <2 rounds of cell division. Data are from at least 2 experiments. (<b>B</b>) CFSE-labeled CD4⁺ BDC2.5 T cells (2×10⁵/well) were cultured under three conditions. Similar to those in (A), in control cell cultures, BDC2.5 T cells were cultured in conditions of either (1) alone or (2) with p79 plus CD4⁺ cell-depleted irradiated APCs for activation. BDC2.5 T cells were also cultured with (3) antibody against IFN-γ (24 µg/ml), in the absence of NR286 T cells. <b>(C)</b> Cell culture supernatants from (A) were harvested for ELISA to detect the presence of IFN-γ, IL-10, and NO.