Sustained Dysfunction of Antiviral CD8⁺ T Lymphocytes after Infection with Hepatitis C Virus

Dynamics of hepatitis and acute immune responses in two subjects. (a) Time course of disease and immune responses. (Upper panels) Time course of alanine aminotransferase (ALT [international units per milliliter]) in serum over time. Subject A was PCR positive (+) for HCV RNA at the first time point and subsequently became PCR negative (−) over time as indicated, with recrudescence at week 15. In subject B, virus did not recrudesce, even during longer follow-up periods of 1 year. (Middle panels) Dynamics of tetramer-positive responses. Frozen PBMCs were thawed and tested in parallel with tetramers for four HLA-A2-restricted peptides (NS3 1073, NS3 1406, NS4B 1807, and NS5B 2594). Thawed PBMCs were stained exactly as previously described (19, 21). Only positive stains are shown. The proportions were calculated after gating on live CD8⁺ lymphocytes. (Lower panels) Fresh PBMCs were tested in standard proliferation assays by incorporation of [³H]thymidine after stimulation with HCV antigens as previously described (11). (b) Phenotype of acute responses in subject A. Frozen PBMCs were thawed and stained in parallel with the tetramers for NS3 1073 and NS5 2094, shown to be positive, together with the antibodies for MHC class II CD38, CCR-5, or (after permeabilization) Ki-67 (see Materials and Methods). The proportions of tetramer-positive cells staining positive at each time point for each marker are shown. (c) PMA-ionomycin-stimulated cytokine synthesis over time in subject B. Frozen PBMCs were thawed, tetramer stained for 20 min, and then stimulated with PMA-ionomycin as previously described (21). Staining with PerCP-labeled anti-CD8 was followed by permeabilization as in panel b and intracellular staining with FITC–anti-IFN-γ (Becton Dickinson). After four-color flow cytometry, the proportion of tetramer-positive cells staining positive for intracellular IFN-γ was calculated. (d) Peptide-stimulated synthesis of IFN-γ in subject B: comparison of HCV and control response. Frozen PBMCs from subject B at weeks 2 and 14 were thawed, stained with tetramers for HCV NS3 1073 or CMV, stimulated with the appropriate peptide and costimulatory molecules, permeabilized, and stained for CD8 and intracellular IFN-γ (21). After flow cytometric analysis, the CD8⁺ population is displayed. The proportion of tetramer-positive cells staining positive for IFN-γ is shown. Staining of cells in the absence of peptide revealed stimulation of <2% in both cases. (e) Peptide-stimulated upregulation of CD69 in subject B: comparison of HCV and control responses. PBMCs from the same time points as in panel d above were stimulated in the same manner with peptide (21) and stained thereafter with PerCP–anti-CD8 and FITC–anti-CD69. The proportion of tetramer-positive cells expressing CD69 is illustrated. Expression in ex vivo samples or in the absence of peptide was <2%. (f) Example of CD69 upregulation in tetramer-positive populations by peptide stimulation. Examples from the first time point of CD69 surface staining in tetramer-positive cells. The tetramer-positive CD8⁺population was gated upon and CD69 expression was analyzed after peptide stimulation. No upregulation of CD69 on tetramer-negative cells was observed (data not shown).