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Pathogenesis and Immunity

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

Norbert H. Gruener, Franziska Lechner, Maria-Christina Jung, Helmut Diepolder, Tilman Gerlach, Georg Lauer, Bruce Walker, John Sullivan, Rodney Phillips, Gerd R. Pape, Paul Klenerman
Norbert H. Gruener
Institute for Immunology, D-80336 Munich, and
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Franziska Lechner
Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom;
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Maria-Christina Jung
Medical Department II, Klinikum Grosshadern, D-81366 Munich, Germany;
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Helmut Diepolder
Medical Department II, Klinikum Grosshadern, D-81366 Munich, Germany;
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Tilman Gerlach
Medical Department II, Klinikum Grosshadern, D-81366 Munich, Germany;
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Georg Lauer
Infectious Diseases Unit and AIDS Research Center, Massachussets General Hospital and Harvard Medical School, Boston, Massachusetts 02129; and
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Bruce Walker
Infectious Diseases Unit and AIDS Research Center, Massachussets General Hospital and Harvard Medical School, Boston, Massachusetts 02129; and
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John Sullivan
Australian Red Cross Blood Service, Sydney 2000, Australia
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Rodney Phillips
Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom;
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Gerd R. Pape
Institute for Immunology, D-80336 Munich, and
Medical Department II, Klinikum Grosshadern, D-81366 Munich, Germany;
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Paul Klenerman
Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom;
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DOI: 10.1128/JVI.75.12.5550-5558.2001
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    Fig. 1.

    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).

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    Fig. 2.

    Functional analysis of HCV-specific responses against three separate epitopes in three separate donors. (a) Cytokine release from CD8+ lymphocytes of different specificities. Frozen samples from three separate HCV antibody-positive individuals were thawed and tested for synthesis of antiviral cytokines after PMA-ionomycin stimulation exactly as in Fig. 1c. Both stains for TNF-α (right panels [marked FL4-H]) and IFN-γ (left panels [ifngFITC]) are illustrated. The numbers shown in each plot represent the percentage of tetramer-positive cells that stained positive for the particular cytokine. PCR-ve, PCR negative. (b) Control unstimulated cells and a reference for gating on the CD8 high population. Samples obtained from subject C (NS3 1073 specific) are illustrated. No synthesis of IFN-γ is seen. (c) Comparison of PMA-ionomycin and peptide stimulation. A CMV-specific response from a control HCV-negative patient is shown. The samples were tested in parallel for TNF-α synthesis according to the peptide stimulation and PMA stimulation protocols, and the CD8 high population is shown. Approximately similar proportions of CD8+ cytokine-positive cells were obtained, as indicated in the right upper quadrant of each FACS plot. (d) Analysis of Vβ usage of tetramer-positive cells in subjects A and B. PBMCs were anti-CD8 and tetramer stained as described above and costained with a panel of FITC-conjugated Vβ-specific MAbs (Immunotech, Marseille, France). Staining for Vβ3 only is shown, after gating on live CD8+ lymphocytes. A large population of Vβ3-positive, tetramer-positive lymphocytes is seen in subject B (30% of tetramer-positive cells), and a smaller population is seen in subject A (5%). No dominant Vβ usage was seen in subject A, C, or D. Tetramer-negative populations in these subjects did not reveal a major oligoclonal expansion when this restricted panel of antibodies was used (Vβ1, -2, -3, -5.1, -5.2, -5.3, -8, -9, -12, -13.1, -14, -16, -17, -20, -22, and -23).

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    Fig. 3.

    Functional analysis of a long-term antiviral CD8+ T-lymphocyte response. (a) Peptide-stimulated synthesis of cytokines in subject E: comparison of HCV and control responses. Experiments were performed exactly as in Fig. 1c. The CD8+ population is shown, and the proportions within the tetramer-positive populations that stain positive for intracellular cytokine are indicated. The upper panels represent stimulation with HCV peptide NS3 1073 (also indicated as “peptide 11” in the FACS plot title line), and the middle and lower panels represent HLA-A2-restricted peptides from CMV and EBV, respectively. (b) PMA-ionomycin-stimulated TNF-α synthesis in patient E. Experiments were performed exactly as in Fig. 2a. The CD8+ population is shown, and the proportions within the tetramer-positive and tetramer-negative populations positive for intracellular cytokine are indicated.

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    Fig. 4.

    Overall comparisons of HCV function and control responses. (Upper panel) Internal comparison. Five HCV antibody-positive subjects (all HCV PCR negative) for whom HCV and control responses were available were tested simultaneously for IFN-γ staining after PMA-ionomycin stimulation, and the proportion of tetramer-positive cells was calculated as in Fig. 2a, upper panels. Each HCV response was compared with the EBV and/or CMV response in the same individual (a total of eight comparisons). (Lower panel) Group comparison. Cytokine synthesis from HCV tetramer-positive populations in seven HCV antibody-positive subjects (five PCR negative) were tested exactly as described above (left-hand group, marked HCV) and compared with EBV and/or CMV responses within themselves and in seven normal controls (right-hand group). CMV and EBV responses from HCV antibody-positive subjects are shown by solid circles, and those from control subjects are shown by open circles.

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  • Table 1.

    Clinical details of subjects studied during or after acute infection

    SubjectSexAge (yr)Time of analysisHLA class IClinical outcome
    AFemale31During acute hepatitisA2, A26, B38, B50Relapse
    BFemale37During acute hepatitisA1, A2, B8, B40Sustained PCR negative in blood
    CMale42After acute hepatitisA2, A26, B37Sustained PCR negative in blood
    DFemale32After acute hepatitisA2, A3, B41, B44Sustained PCR negative in blood
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Sustained Dysfunction of Antiviral CD8+ T Lymphocytes after Infection with Hepatitis C Virus
Norbert H. Gruener, Franziska Lechner, Maria-Christina Jung, Helmut Diepolder, Tilman Gerlach, Georg Lauer, Bruce Walker, John Sullivan, Rodney Phillips, Gerd R. Pape, Paul Klenerman
Journal of Virology Jun 2001, 75 (12) 5550-5558; DOI: 10.1128/JVI.75.12.5550-5558.2001

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Sustained Dysfunction of Antiviral CD8+ T Lymphocytes after Infection with Hepatitis C Virus
Norbert H. Gruener, Franziska Lechner, Maria-Christina Jung, Helmut Diepolder, Tilman Gerlach, Georg Lauer, Bruce Walker, John Sullivan, Rodney Phillips, Gerd R. Pape, Paul Klenerman
Journal of Virology Jun 2001, 75 (12) 5550-5558; DOI: 10.1128/JVI.75.12.5550-5558.2001
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KEYWORDS

CD8-Positive T-Lymphocytes
cytokines
hepacivirus
Hepatitis C

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