Production of IL-2, IL-9, and IFN-γ and the role of IL-2 and IL-9 in the proliferation of CD4⁺CD80^{acq hi} T cells. (A) Cytokine expression in CD4⁺CD80^{acq hi} T cells was examined using RPA when these T cells were incubated in the absence (i) or presence (ii) of 1 μg/mL PCC without APCs at various time points. Subpanels i and ii are originally from the same blot. (B-D) Secretion of IL-2, IL-9, and IFN-γ in the culture medium of CD4⁺CD80^{acq hi} T cells was examined using ELISA. □ indicates without PCC; ▪, with PCC. (E) CD4⁺CD80^{acq hi} T cells, cultured in the presence of exogenous PCC peptide (1 μg/mL) without APCs, were treated with neutralizing anti-IL-2 (5 to 40 μg/mL) Ab, neutralizing anti-IL-9 Ab (5 to 40 μg/mL), or IgG isotype Abs for 24 hours. The effects of anti-IL-2 and anti-IL-9 Abs on 3H uptake of CD4⁺CD80^{acq hi} T cells were assessed. CD4⁺CD80^{acq hi} T cells treated with anti-IL-2 Ab in the presence of PCC peptide (1 μg/mL) (♦); CD4⁺CD80^{acq hi} T cells treated with anti-IL-9 Ab in the presence of PCC peptide (1 μg/mL) (▪); CD4⁺CD80^{acq hi} T cells treated with anti-IL-2 IgG isotype Ab in the presence of PCC peptide (1 μg/mL) (⋄); CD4⁺CD80^{acq hi} T cells treated with anti-IL-9 IgG isotype Ab in the presence of PCC peptide (1 μg/mL) (□). Error bars represent mean ± SD.