Fig. 5.

Identification and characterization of IL-7–producing LECs localized in iBALT. (A–D) Effector Th2 cells from Ly5.1 OT-II Tg mice were transferred into IL-7:GFP knock-in mice (Il-7^gfp/+). Mice were challenged i.n. with OVA on days 1 and 3. The indicated assays were performed at day 42. (A) GFP⁺ cells or GFP⁻ cells among PECAM1⁺ cells (a mix of lymphatic and BECs) were sorted from the lung of mice. Quantitative RT-PCR analysis of expression of the indicated genes was performed. (B) Representative cell surface staining profiles of PECAM1⁺GFP⁻ (black lines) and PECAM1⁺GFP⁺ cells (red lines) are shown; gray-shaded curves show staining with isotype-matched control antibody. (C) Representative confocal micrograph of lung tissue from Il-7^gfp/+ mice stained with anti-GFP:IL-7 (green), anti-CD3ε (red), and anti–Lyve-1 (blue). Lyve-1 expression within iBALT can be observed on the vessel-like structures and reticular cells. (Scale bars: Left, 20 μm; Right, 10 μm.) (D) Representative GFP expression profiles of PECAM1⁻Podoplanin⁺ cells and PECAM1⁺Podoplanin⁺ cells from the lung of Il-7^+/+ (black line) or Il-7^gfp/+ (red line) mice, gated by the strategy as described in Fig. S5D. (E) Representative cell surface staining profiles of Thy1.2 expression on PECAM1⁺GFP⁻ cells (black line) and PECAM1⁺GFP⁺ cells (red line). (F) PECAM1⁺Thy1.2⁺ cells, PECAM1⁺Thy1.2⁻ cells, and VCAM1⁺ cells were sorted from the lungs of C57BL/6 mice in which iBALT had been induced as described in Fig. 1. Quantitative RT-PCR analysis of Il7 was performed. Three technical replicates were performed for quantitative RT-PCR (A and F). More than three independent experiments were performed with similar results (A–F).