FVIII-specific proliferating CD4 T cells have a TFH phenotype. Splenocytes from rhF8-immunized FVIII^null mice were labeled with CellTrace Violet and stimulated with rhF8 for 96 hours. Nonstimulated or unrelated protein rhF9-stimulated splenocytes were used as controls in parallel. Cells were stained for mouse CD3, CD4, CXCR5, BCL6, GATA3, and Tbet. Proliferating daughter cells were analyzed by flow cytometry. (A) CD4⁺ T-cell proliferation is depicted. Representative plots from 4 experiments are shown. (B) CXCR5⁺ and CXCR5⁻ T-cell proliferation is depicted. Representative plots from 4 experiments are shown. (C) BCL6 and CXCR5 expression levels on proliferating CD4 T cells (blue line) induced by ex vivo FVIII stimulation were compared with nonproliferating CD4 T cells (red line). (D) Transcription factor BCL6, GATA3, and Tbet expression on FVIII-induced proliferating CD4 T cells (top row). Corresponding isotype control antibody staining was used to define gating (bottom row). PI, proliferation index.