Figure 2.

Suppressive function of CD4⁺ T cells from tolerized mice. (A) Mice were exposed to PBS (airway inflammation) or 1% OVA (tolerance) and subsequently immunized with OVA and alum on days 21 and 27 after the initiation of OVA/PBS exposure. Splenic CD4⁺ T cells isolated from mice on day 34 were stimulated in vitro with different concentrations of OVA (0.01–100 ∝g/ml) and mitomycin C–treated, T cell–depleted APCs at equivalent cell numbers (105 cells each per well). After 72 hours of incubation, small samples of culture supernatants were removed for cytokine determination, and the remaining cells were pulsed for measurement of [3H]-thymidine incorporation. *P < 0.025 compared with proliferation of cells from mice immunized for inflammation. The proliferative response of CD4⁺ T cells from naive mice (open diamonds) is shown as a negative control. An additional control used was a mixture of cells from the inflammation group and from naive mice used in a 1:2 ratio. Each data point represents the mean plus or minus SEM of triplicate wells. Shown is a representative experiment of three. (B) As described above, CD4⁺ T cells isolated after day 34 were subjected to two rounds of stimulation with OVA and APCs in vitro, and nuclear extracts were prepared. Expression of GATA-3 and FOXP3 was assessed in the nuclear extracts (7–10 ∝g of total protein) by Western blotting techniques. CREB-1 expression is shown as a marker for protein loading. (C) Shown is an average densitometric reading of FOXP3 and GATA-3 expression relative to that of CREB-1 from two independent experiments.