FIG. 7.

HCV-specific CD8 T-cell suppression by in vitro-expanded HCV-specific CD4⁺CD25⁺ T cells. (A) HCV NS3 1406-specific CD8 T cells demonstrated directly ex vivo by class I tetramer staining in HCV-seropositive HLA-A2⁺ subject R23. Efficient antigen-specific expansion occurred following 7 days of in vitro stimulation with overlapping NS3-derived HCV peptides. (B) The top three FACS plots demonstrate preferential expansion of HCV-specific FoxP3⁺ CD4⁺ Tregs by day 14 following in vitro stimulation with recombinant NS3/4 protein, 1,000 U/ml rIL-2, and 10 μg/ml soluble anti-CD28 alone (NS3/4, 27.2% FoxP3⁺ CD4⁺) and with added TGF-β (NS3/4 plus TGF-β, 51.4% FoxP3⁺ CD4⁺), compared to control SOD (7.5% FoxP3⁺ CD4⁺). The bottom three graphs show FoxP3⁺ CD25⁺ T-cell content in autoMACS-sorted CD4⁺CD25⁺ (eTregs) and CD4⁺CD25⁻ (eCD25⁻) subsets from the expanded Treg cultures. (C) CD8⁺ T-cell expansion is shown by histograms representing CFSE dilution on day 7 in PBMC stimulated alone or with added eTregs isolated from the NS3/4-stimulated Treg cultures in panel B. The stimulating condition is indicated above each set of graphs. The ratios between eTreg and PBL are indicated below the graphs. (D) HCV-specific CD8⁺ T-cell expansion in PBL was directly monitored by a class I tetramer specific for HCV NS3 1406-specific CD8 T cells in the presence of NS3/4-expanded eTregs at various eTreg/PBL ratios (0:1, 0:25:1, and 1:1). Assays using eTregs expanded with TGF-β are indicated by 1:0.25T and 1:1T. The graphs at the extreme right represent assays in which CD4⁺CD25⁻ T cells are sorted from expanded Treg culture (eCD25⁻) and added at 1:1 ratio to PBL before antigenic stimulation with the NS3 peptide (top) or with anti-CD3/anti-CD28 stimulation (bottom).