FIG. 5.

HCV specificity of expanded FoxP3⁺ Tregs demonstrated by MHC-II peptide tetramers. (A) Antigen specificities for HLA DRB1*04-restricted class II tetramers bound to influenza virus hemagglutinin (Flu HA), HCV NS3 1248, and HCV NS4 1770 epitopes in short-term peptide-stimulated T-cell lines are shown. (B) Specific binding of class II tetramers is shown by staining an HCV NS3/4-stimulated T-cell line with isotype antibody, HCV NS4 1770 tetramer, and influenza virus HA-specific tetramer. An HCV NS3/4-specific T-cell line binds HCV 1770 but not influenza virus HA tetramer. (C) Expansion of HCV-specific FoxP3⁺ CD4 T cells in vitro. CFSE-labeled PBMC from a DRB1*04⁺ HCV-seropositive subject (R19) were stimulated with recombinant HCV NS3/4 protein, with the expansion of FoxP3⁺ CD4 T cells (left panels) and HCV-specific (but not influenza virus HA-specific) DRB1*04-restricted class II tetramer⁺ CD4 T cells (middle three panels). Circled gates indicate class II tetramer⁺ CFSE^low CD4 T cells that divided following antigenic stimulation. FACS plots on the far right show FoxP3 expression in the gated HCV 1770-specific CFSE^low CD4 T cells. (D) FoxP3 expression by influenza virus HA tetramer⁺ CD4 T cells is shown in CFSE-labeled PBL following antigenic influenza virus HA peptide stimulation (gated on CD4 T cells on the upper graph) is shown. (Bottom) FoxP3 expression by 51% of gated CFSE^low influenza virus tetramer (Tet)⁺ CD4 T cells in an overlay with total CD4.