Ag-specific CD4 T-cell proliferation assay. (A) Schematic diagram of T-cell proliferation assay. CD4⁺ T cells isolated from rhF8-primed FVIII^null mouse were labeled with CellTrace Violet and cocultured with DMVECs that were pretreated with IFN-γ/TNF-α in the presence of CXCL13 and the absence and presence of rhF8 at 37°C in 5% CO₂ for 1 week. rhF9 was used as an unrelated Ag control. CD4 T cells plated in the absence of endothelium served as an additional control (supplemental Figure 6). (B) Flow cytometry analysis daughter (proliferated) cells. Cells were stained for CD4, TCRβ, and CXCR5, and analyzed by flow cytometry. Dead cells were excluded by the 7-AAD staining. CD4 T-cell divisions were quantified as the dilution of CellTrace Violet signal in daughter cells away from the initially labeled peak. Results for both CD4 Tfh (top row) and total CD4 (middle row) are shown. Bottom row shows expanded view of daughter cell in the red, boxed regions from the middle row. These data demonstrate that ECs can present FVIII to Tfh cells, promoting Ag-specific cell proliferation.