LAPCs promote IL-4⁺ type-2 Tfh differentiation of Ag-primed CD4⁺ T cells ex vivo. (a) Schematic diagram for ex vivo co-culture experiments. In vivo Ag-primed TS-1 cells were generated as described in Materials and methods. FACS-sorted 5 dpi TS-1 cells (5 × 10⁴ cells/well) were incubated with either total B cells (B; B220⁺CD19⁺CD11c⁻), activated B cells (acB; B220⁺CD19⁺CD69⁺CD11c⁻), DCs (CD11c⁺TcRβ⁻) or LAPCs (mPDCA1⁺CD11c⁻B220⁻TcRβ⁻; 2.5 × 10⁴ cells/well) isolated from the dLN of either X31(n = 8) or A/PR/8 virus-infected (n = 8) BALB/c mice on 8 dpi (b–d). 24 h after co-culture, Tfh differentiation (PD-1, CXCR5, and Bcl-6) and related cytokine production (IL-4 and IL-21) were examined in the TS-1 (Thy1.1⁺CD4⁺) population. Data are representative of at least two independent experiments are shown as means ± SEM Considered a significant difference at *, P < 0.05. (e) C57BL/6 mice (n = 6) were infected i.n. with a sublethal dose (0.05 LD50) of A/PR/8 virus. At 8 dpi, IL-4 production from Tfh cells (Thy1.2⁺CD4⁺PD-1⁺CXCR5⁺ or Thy1.2⁺CD4⁺CXCR5⁺Bcl-6⁺) was examined in the dLN by FACS analysis. The percentage of Tfh phenotypic cells, gated on IL-4⁺ CD4 T cells (Thy1.2⁺CD4⁺IL-4⁺), was also evaluated. Representative images from two independent experiments are shown.