Ex vivo T-cell proliferation assay assesses the immunogenicity of platelet-containing FVIII. The CFSE-labeled CD4⁺ T-cell proliferation assay was used to elucidate whether platelets that contain recombinant FVIII would stimulate rhF8-primed CD4⁺ T-cell proliferation. CD4⁺ T cells isolated from rhF8-primed FVIII^null splenocytes were labeled with CFSE and cocultured with dendritic cells from FVIII^null spleens in the presence of rhF8 or 2bF8^Tg platelets for 96 hours. The daughter CD4⁺ T cells (D-CFSE) were analyzed by flow cytometry. (A) The rhF8 dose-response curve in the CFSE-labeled CD4⁺ T-cell proliferation assay. (B) Representative histograms from flow cytometry analysis of daughter CD4⁺ T cells. (C) The graph of daughter CD4⁺ T cells after coculture with rhF8 or 2bF8^Tg platelets. Concanavalin A (ConA) was used as a positive control for T-cell proliferation. These results show that platelets that contain FVIII do not stimulate FVIII-primed CD4⁺ T-cell proliferation in vitro. plts, platelets.