Costimulation of TCR and TGF-β induces CD4⁺CD25⁻ T cell anergy, but fails to expand existing T_reg. (A) C57BL/6 CD25⁻ naive cells or T_reg (5 × 10⁴) were cultured (primary) with anti-CD3 and APCs (2 × 10⁶) in the absence (αCD3 + med) and presence (αCD3 + TGF-β) of 2 ng/ml TGF-β for 7 d. 3 × 10⁴ harvested viable CD4⁺ responder T cells or 5 × 10³ T_reg were restimulated with anti-CD3 and APCs for 72 h to monitor their proliferation. The data are representative of three separate experiments. (B–F) TGF-β induces OVA TCR transgenic CD4⁺ T cells (KJ1-26⁺) anergy. 2 × 10⁶ cells/ml spleen cells were cultured with OVA in the presence or absence of TGF-β for 7–10 d (primary). Viable CD4⁺ T cells were purified and restimulated with 100 μg/ml OVA, 100 μg/ml hen egg lysozyme (HEL), 1 μg/ml anti-CD3 mAb, or 10 U/ml IL-2 as indicated in the presence of BALB/c APCs or with PMA and ionomycin (secondary stimulation). The values are expressed as mean ± SD of triplicate wells for ³H incorporation (CPM, 5 × 10⁴ T cells) (B and C) or of duplicate wells of the ELISA (D–F, 2 × 10⁴ T cells). (B) TGF-β induces transgenic CD4⁺ TCR-specific anergy. (C) Inclusion of exogenous IL-2 in primary cultures blocks TGF-β–induced CD4⁺ T cell anergy. (D–F) TGF-β induces both Th1 and Th2 cell anergy. Cytokine levels of IL-2 (D), IFN-γ (E), and IL-4 (F) in secondary culture supernatants (after 24–48 h) were determined by ELISA. The data shown were repeated from two to six times with similar results.