LAPCs promote IL-4⁺ type-2 Tfh differentiation via ICOS–ICOSL–mediated interaction. (a) Sorted, in vivo Ag-primed 5 dpi TS-1 cells were incubated with either live or γ-irradiated (ir; 2,000 rads) 8 dpi LAPCs, which were isolated from the dLN of A/PR/8 virus–infected mice (n = 8). An aliquot of 5 dpi TS-1 cells were incubated with 8 dpi LAPCs, separated by a membrane (tw, transwell), according to the transwell protocol described in Materials and methods. Data are representative of at least two independent experiments are shown as means ± SEM. (b) ICOS and ICOSL expression were determined by FACS analysis on 5 dpi TS-1 T cells (Thy1.1⁺CD4⁺) and 8 dpi LAPCs (mPDCA1⁺CD11c⁻B220⁻TcRβ⁻), respectively. Data are representative of three independent experiments are shown. (c) To examine the molecular mechanisms involved in LAPC-mediated Tfh differentiation, 5 dpi TS-1 T cells were incubated with 8 dpi LAPCs, in the presence or absence of the indicated concentrations of ICOSL-blocking mAbs (HK5.3). At 24 h after incubation, the extent of Tfh differentiation (PD-1 and CXCR5) and related cytokine production (IL-4) were examined in the TS-1 (Thy1.1⁺CD4⁺) population. Data are representative of at least two independent experiments are shown as means ± SEM. *, P < 0.05.