Figure 8.

T reg cell–mediated changes in AHR. (A–C) Data from a representative experiment using group sizes of n = 5 showing relative gene expression levels of Foxp3 (A), LAG-3 (B), and IL-10 (C) in sorted CD4⁺CD25⁺ cells from airway DLNs of sensitized animals exposed to 0, 1, or multiple OVA aerosols as indicated. Data are expressed as arbitrary RT-PCR units derived by normalization against the housekeeping gene Eef1α1. (D) Proliferative responses of OVA-primed LN cells obtained from OVA/CFA-sensitized animals in the presence of CD4⁺CD25⁺ cells isolated from airway DLN preparations of sensitized animals exposed to a single (dotted bars), or multiple (shaded bars) OVA aerosols. CD4⁺CD25⁺ cells were added at ratios (CD4⁺CD25⁺ to LN cells) of 1:20. Proliferation is shown as mean CPM from triplicate cultures. Addition of CD4⁺CD25⁺ cells from unexposed controls was without effect (not shown). Data are representative of three separate experiments. *, P < 0.05; **, P < 0.01. (E–M) Representative staining profiles for rat G2a isotype control (E) and Foxp3 (F) expression in airway DLN digest preparations from animals sensitized and exposed to multiple OVA aerosols. CD4 versus CD25 profiles of same DLN preparations (G and H) depict CD4⁺CD25⁺ and CD4⁺CD25⁻ populations of cells. Staining profiles of ratG2a isotype controls and Foxp3 for CD4⁺CD25⁺ cells (J and K, respectively) and CD4⁺CD25⁻ cells (L and M, respectively) are also shown. (N) EC200 was determined in OVA-sensitized animals after adoptive transfer of CD4⁺CD25⁺ or CD4⁺CD25^neg cells sorted from pooled airway DLN of groups of ≥5 animals sensitized to OVA and exposed to multiple OVA aerosols or control animals (no adoptive transfer) and naive animals, on four different occasions. Data are expressed relative to naive controls. Figures represent mean absolute reduction or increase in EC200 (in mg/ml MCh) relative to the mean control value (set at zero). OVA ×1 > OVA ×1 + mOVA CD4⁺CD25⁺; P = 0.005; OVA ×1 > CD4⁺CD25^neg, P = 0.873.