Th17 cells proliferate and differentiate in the lung. (A and B) WT B6 mice received BLM intratracheally (1.5 mg/kg). BLM-exposed mice were treated with PBS or FTY720 (2 mg/kg/day, i.p.) between days 3 and 7 (analyzed on day 7), between days 3 and 8 (analyzed on day 8), and between days 5 and 8 (analyzed on day 8). Mice were sacrificed and BALF and draining lymph nodes (DLN) harvested on the days noted above. The percentages of CD4⁺ cells producing IL-17 or IFN-γ in draining lymph nodes (DLN; A) and BALF (B) were determined by flow cytometric analysis. (C) BLM-exposed WT B6 mice were treated with PBS or FTY720 (2 mg/kg/day, i.p.) at day 5, 6, or 7 after BLM-exposure. Numbers and percentages of CD4⁺ cells producing IL-17 in BALF and DLN were determined by flow cytometric analysis 8 d after BLM exposure. (D and E) WT B6 (Thy1.2) mice received BLM intratracheally (1.5 mg/kg). On the next day, BLM-exposed mice were adoptively transferred with CFSE-labeled Thy1.1⁺ CD4⁺ T cells (5 × 10⁶ cells). To deplete endogenous T cells, anti-Thy1.2 antibody (30-H12; 300 µg/mouse) was administered i.p. to recipient mice on days −1 and 2 of BLM treatment. (D) Mice were sacrificed to harvest leukocytes from lungs on the days indicated. The percentages of donor Thy1.1⁺ CD4⁺ cells producing IL-17 were determined by flow cytometric analysis. (E) The percentages of donor Thy1.1⁺ CD4⁺ cells producing IL-17 in lungs and draining lymph nodes (DLN) of recipient mice were determined by flow cytometric analysis 8 d after PBS or BLM exposure. (F and G) WT B6 (Thy1.2) mice were adoptively transferred with CFSE-labeled naive phenotype Thy1.1⁺ CD4⁺ T cells (5 × 10⁶ cells) 1 d after BLM exposure. Some mice received FTY720 10 h before naive phenotype Thy1.1⁺ CD4⁺ T cell transfer. Percentages (F) and numbers (G) of donor Thy1.1⁺ CD4⁺ cells producing IL-17 in lungs and DLN of recipient mice were determined by flow cytometric analysis 8 d after PBS or BLM exposure. (C and G) Data represent means ± SD of three independent determinations with BALF or lung tissues from n = 5 mice/group.