TGF-β–converted-CD4+ suppressor T cells prevent HDM-induced allergic pathogenesis in vivo. B6 CD4+CD25− T cells were cultured with anti-CD3 and APCs in the absence (Control cell) and presence of TGF-β (TGF-β cell) for 3–5 d, extensively washed and rested in DMEM complete medium containing 10 U/ml of IL-2 for an additional 3 d. Viable cells were harvested, washed, and resuspended in PBS for injection. For induction of allergic immune responses in lungs, B6 mice were injected with HDM allergen as depicted in schematic plan (A). 4 d after the last HDM intratracheal challenge, mice were killed, and lungs were immediately fixed with 10% PBS-Formalin for immunohistological staining (H&E). One representative lung from each group (three to five mice) is shown. The magnification of the images is 20. (B) Naive lung. (C) HDM-injected lung. (D) HDM-injected lung coinjected with Control cell (106 on day 1 and 5 × 105 on day 14, i.v.). (E) HDM-injected lungs coinjected with TGF-β cell (106 on day 1 and 5 × 105 on day 14, i.v.). (inset) Boxed images are mucin staining in airways by PAS (40×). The red staining represents mucin positive cells. (F–H) Spleen cells were harvested and pooled from three to five mice per group. 4 × 105 spleen cells were cultured with 100 μg/ml HDM (white bars) or 0.5 μg/ml anti-CD3 (black bars) for 24–96 h, the cell-free supernatants were collected to measure IFN-γ (F, 48 h), IL-4 (G, 24 h), and IL-13 (H, 96 h), respectively, by ELISA. IL-4 levels are shown as ΔOD (OD value of test supernatants − OD value of culture medium; OD = 0.107). n.d., not done.
