title: Single cell analysis of factor VIII-specific T cells in hemophilic mice after treatment with human factor VIII.
abstract: A multi-parameter flow-cytometry assay was established suitable for analyzing T-cell-specific cell surface markers (CD3, CD4) together with intracellular cytokines on a single cell level. This assay was used to identify the frequency and the kinetic of different populations of factor VIII (FVIII)-specific CD4+ T cells in hemophilic E-17 mice after treatment with human FVIII. A clear temporal correlation was found between the appearance of FVIII-specific CD4+ T cells in the spleen and the detection of anti-FVIII antibodies in plasma. These cells and antibodies were detectable in all experiments after two doses of FVIII and in a few even after a single dose. The IFN-gamma-producing T cells were the most prominent type of FVIII-specific T cells suggesting Th1-type T cells have an important role in regulating the anti-FVIII immune response in E-17 mice. IL-10-producing T cells were the second most dominant type. They were detectable after two doses of FVIII and increased in frequency after four. Cytokine co-expression studies analyzing IL-10 and IFN-gamma in the same cell indicated that there might be at least two types of IL-10 positive T cells, those cells that produce IL-10 only and in addition cells that produce IL-10 and IFN-gamma. Furthermore, FVIII-specific T cells producing IL-2 were found in all experiments after two doses of FVIII. In a few experiments IL-4-producing T cells were seen but in most experiments they were not detectable. In contrast, IL-4 could be found in supernatants of in vitro restimulated CD8- spleen cells.
authors.affiliation: Baxter BioScience, Vienna, Austria.
authors.forename: Maria
authors.initials: M
authors.lastname: Sasgary
authors.forename: Rafi U
authors.initials: RU
authors.lastname: Ahmad
authors.forename: Hans Peter
authors.initials: HP
authors.lastname: Schwarz
authors.forename: Peter L
authors.initials: PL
authors.lastname: Turecek
authors.forename: Birgit M
authors.initials: BM
authors.lastname: Reipert
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002469:Cell Separation; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000276:immunology* / Q000627:therapeutic use; D005434:Flow Cytometry; D006467:Hemophilia A / Q000188:drug therapy / Q000276:immunology*; D006801:Humans; D007114:Immunization; D007371:Interferon-gamma / Q000378:metabolism; D016753:Interleukin-10 / Q000378:metabolism; D007518:Isoantibodies / Q000097:blood / Q000276:immunology; D007519:Isoantigens / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D013045:Species Specificity; D013154:Spleen / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism
chemical_list: D007518:Isoantibodies; D007519:Isoantigens; D016753:Interleukin-10; D007371:Interferon-gamma; D005169:Factor VIII