LAPCs modulate anti-IAV Tfh differentiation via ICOS stimulation in ex vivo LN organ culture. (a) To examine LAPC migration from lungs into dLN, an in vivo migration assay was performed as described in Materials and methods. LAPC (mPDCA1⁺CD11c⁻B220⁻TcRβ⁻) accumulation in the dLN was examined at 5 and 6 dpi by FACS analysis. 24 h after FITC-dextran treatment (6 dpi), cells were harvested from the dLNs (n = 12) and were stained for IAV-NP protein (intra-cellular). The percentage of FITC⁺ NP⁺ cells in the LAPC population was examined by FACS analysis. Representative images of two independent experiments are shown. (b–d) Thy1.1⁺ TS-1 T cells were transferred into Thy1.2⁺ WT mice as described in Materials and methods. The TS-1 T cell recipient mice (n = 30) were infected with A/PR/8 influenza 24 h later. At 3 dpi, groups of IAV-infected mice (n = 12) were inoculated i.v. with day 8 LAPCs (5 × 10⁵ cells/mouse), isolated from the dLNs of A/PR/8 virus–infected BALB/c mice and pretreated with either rat IgG control Ab or αICOSL-blocking mAb (100 µg/ml). Intact dLNs (mediastinal LNs) from IAV-infected mice were excised on 5 dpi. The intact dLNs were cultured for 24 h in hyperoxy chambers, as described in Materials and methods, and then analyzed by FACS analysis for the presence of LAPCs (PDCA1⁺CD11c⁻B220⁻TcRβ⁻) and TS-1 Tfh cells (Thy1.1⁺CD4⁺PD-1⁺CXCR5⁺; c), and Ki-67⁺ cells (Ki-67⁺), B cells (B220⁺TcRβ⁻), and DCs (CD11c⁺TcRβ⁻; d). Data are representative of at least two independent experiments are shown as means ± SEM. *, P < 0.05.