Outcome of acute hepatitis C is related to virus-specific CD4 function and maturation of antiviral memory CD8 responses^†

Patients and Virological Assessment

Sixteen patients with acute hepatitis C were enrolled at the Unit of Infectious Diseases and Hepatology of the University Hospital of Parma. The diagnosis of acute HCV infection was based on the following criteria: documented seroconversion to anti-HCV antibodies by RIBA assay, levels of serum alanine aminotransferase at least 10 times the upper limit of normal (50 U/L), detection of HCV RNA, and exclusion of other possible causes of acute hepatitis (i.e., viruses, toxins, alcohol, autoimmunity, metabolic factors). Anti-HCV antibodies were determined using commercial enzyme immunoassay kits (Ortho Diagnostic Systems, Raritan, NJ) and RIBA (RIBA II, Ortho Diagnostic Systems). Serum HCV RNA was quantified by branched DNA assay and is expressed as copies/mL of serum (Bayer System 340bDNA Analyzer, Bayer Corporation, Tarrytown, NY); it was also analyzed by qualitative polymerase chain reaction (Cobas Amplicor; Roche Molecular Systems, Inc., Branchburg, NJ). The lower limit of detection by the branched method and qualitative polymerase chain reaction is 2,500 copies/mL and 50 copies/mL, respectively. The study was approved by the ethical committee of the Azienda Ospedaliero-Universitaria di Parma, and all subjects gave written informed consent to participate in the study.

Synthetic Peptides, Recombinant Proteins, Peptide-HLA Class I Tetramers, and Antibodies

We used two distinct sets of peptides (Chiron Mimotopes, Victoria, Australia) in this study: (1) a panel of 601 15-mer peptides based on a genotype 1a sequence (HCV-1) covering all structural (core, E1, E2) and nonstructural (NS3, NS4, NS5) HCV regions and overlapping by 10 residues; and (2) a panel of 199 synthetic peptides (9, 10, or 11 amino acids in length) containing the HLA-A2–binding motif and selected among 962 HCV sequences (identified by scanning the entire HCV-1 genome for the presence of the HLA-A2–binding motif) based on the prediction of their high binding affinity to HLA-A2.20 The HCV antigens E1, E2, core, NS3, NS4, and NS5 were expressed as C-terminal fusion proteins with human superoxide dismutase in yeast and were kindly provided by Chiron Corporation (Victoria, Australia). Purity of the antigen preparations ranged from 85% to 95%. Phycoerythrin-labeled tetrameric peptide-HLA class I complexes were purchased from Proimmune LTD (Oxford, UK). HLA-A2 tetramers contained the HCV peptides NS3 1073-1081, NS3 1406-1415, NS4 1812-1820, NS4B 1992-2000, and NS5 2594-2602. Anti–IFN-γ FITC (conjugated fluorescein isothiocyanate) was purchased from Sigma Aldrich (St. Louis, MO). Anti-CD8 (APC), anti–CD-4 (phycoerythrin), anti-CD3 PerCP), anti–IL-4 (FITC), anti–IL-10 (APC), anti–IL-2 (APC), anti-CD127 (APC), and anti-CCR7 (FITC) were purchased from Becton Dickinson Biosciences (San Diego, CA).

Isolation of Peripheral Blood Mononuclear Cells and In Vitro Expansion of HCV-Specific T Cells

Peripheral blood mononuclear cells (PBMC) were isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation and resuspended in RPMI 1640 supplemented with 25 mmol/L HEPES, 2 mmol/L L-glutamine, 50 μg/mL gentamycin, and 8% human serum (complete medium). For T cell expansion, PBMC were resuspended in 96-well plates at a concentration of 2 × 106/mL in complete medium and stimulated with HCV peptides at a final concentration of 1 μmol/L. Recombinant IL-2 was added on day 4 of culture (50 UI/mL), and the immunological assays were performed on day 10.

Cell Surface and Intracellular Staining

ELISPOT Assay

The 601 15-mer peptides overlapping by 10 residues and covering the overall HCV-1 sequence were pooled in 60 mixtures, each containing 10 synthetic peptides. HCV-specific T cell responses were analyzed upon overnight stimulation with individual peptide mixtures (1 μmol/L of each peptide). Briefly, 96-well plates (Multiscreen-IP-Millipore S.A.S., Molsheim, France) were coated overnight at 4°C, as recommended by the manufacturer, with 5 μg/mL capture mouse anti-human IFN-γ monoclonal antibody (1 DIK, Mabtech, Nacka Strand, Sweden). Plates were then washed 7 times with phosphate-buffered saline/0.05% Tween 20 and blocked with RPMI/10% fetal calf serum for 2 hours at 37°C; 2 × 105 PBMC were seeded per well. Plates were incubated for 18 hours at 37°C in the presence or absence of peptides. After washing with phosphate-buffered saline/0.05%Tween 20, a biotinylated secondary mouse anti-human IFN-γ monoclonal antibody (1 μg/mL; 7B6-1, Mabtech) was added. After 3 hours of incubation at room temperature, plates were washed 4 times and 100 μL goat alkaline phosphatase anti-biotin antibody (Vector Laboratories Inc., Burlingame, CA) was added to wells, and the plates were incubated for a further 2 hours at room temperature. Plates were then washed 4 times, and 75 μL of alkaline phosphatase conjugate substrate (5-bromo-4-chloro-3-indolyl phosphate; Bio-Rad Laboratories, Hercules, CA) was added. After 4-7 minutes, the colorimetric reaction was stopped by washing with distilled water. Plates were air-dried and spots were counted using an automated ELISPOT reader (AID Elispot Reader System; Autoimmune Diagnostika Gmbh, Strassberg, Germany). IFN-γ–producing cells were expressed as spot-forming cells per 2 × 105 cells. The number of specific IFN-γ–secreting cells was calculated by subtracting the unstimulated control value from the stimulated sample. Unstimulated wells never exceeded 5-7 spots/well. Positive controls consisted of PBMC stimulated with PHA. Wells were considered positive if they were at least 2 times above background.

Antigen-Specific T Cell Responses Are Broader and Stronger in Self-Limited Than in Persistent HCV Infection.

Breadth and magnitude of the anti-HCV–specific T cell response was analyzed ex vivo in PBMC of 16 acutely infected subjects to define the role of adaptive T cell immunity in determining the course of HCV infection. PBMC were tested by ex vivo IFN-γ ELISPOT assay using a panel of 601 15-mer peptides overlapping by 10 residues and spanning the entire HCV sequence of genotype 1, pooled in 60 mixtures of 10 peptides each. All patients were enrolled at the time of clinical presentation and were infected by genotype 1 (Table 1); a self-limited outcome was observed in six of these patients, and ten developed a chronic disease as indicated by persistently elevated HCV RNA levels (Fig. 1). A sharp difference was observed in the global profile of the HCV-specific cellular immune response in the acute phase of infection, depending on the outcome of the disease. Patients with a self-limiting evolution displayed a multispecific T cell response, targeting structural and nonstructural proteins and recognizing most HCV antigens. In contrast, individuals with chronic evolution showed a narrower cellular immune response, targeting a limited number of peptides, predominantly located in nonstructural HCV regions (Fig. 2A).

The overall number of spots, calculated per 2 × 105 cells in each single patient at the time of the onset, was significantly greater among patients with a self-limited evolution of infection, showing that their T cell response was not only wider but also stronger compared to patients with persistent infection (Fig. 2B).

The breadth of the cellular immune response was analyzed longitudinally ex vivo from the onset of disease throughout a follow-up ranging from 20 to 24 weeks (Fig. 2C). Individuals with self-limited hepatitis showed a broader T cell response that persisted throughout follow-up.

In patients with chronic evolution of disease, the cellular immune response was already narrower during the acute phase of illness and declined further at later time points; in some patients the response was totally undetectable a few weeks from onset. Thus, the difference in the breadth of the T cell response between patients with self-limited or persistent infections was maintained throughout the follow-up, and this difference was statistically significant not only during the acute phase of infection but also at weeks 10-12 and weeks 20-24 from the onset of disease (Fig. 2D).

A Different Breadth and Strength of the CD4 But Not of the CD8 Response Distinguishes the Acute Phase of Self-Limited and Persistent HCV Infections.

Although virus elimination and control of HCV replication have been reported to be associated with a strong and multispecific cellular immune response, the real contribution of CD4 and CD8 T cell subsets is still controversial. To address this issue, the pools of peptides responsible for the detectable responses in ELISPOT were selected and used to stimulate PBMC of the earliest time point (clinical presentation). Through ex vivo intracellular cytokine staining performed with anti-CD4, anti-CD8, and anti–IFN-γ antibodies, the T cell subset responsible for IFN-γ production upon stimulation with each single pool was identified. As illustrated in Fig. 3, the overall number of peptides able to induce a T cell response detectable by ICS was higher in patients with self-limited outcome of infection (Fig. 3A) than in patients with chronic evolution (Fig. 3B). However, the cellular immune response was not similarly sustained by CD4 and CD8 T cell subsets in the two categories of patients. In self-limiting hepatitis, IFN-γ production was sustained predominantly by the CD4 T cell subset (Fig. 3A), whereas in patients with chronic evolution of disease (Fig. 3B), CD4-mediated responses were undetectable and IFN-γ production was induced by a small number of peptide pools and sustained exclusively by the CD8+ subset in all individuals, with the exception of a single pool in patient 1c (Fig. 3B). These results were confirmed with short-term T cell lines derived by 10-day stimulation of the same early PBMC samples with the 60 pools of peptides. T cell lines were first tested by IFN-γ ELISPOT analysis (data not shown) and T cell reactivity was confirmed byICS to define the contribution of the different T cell subsets. The results shown in Fig. 3A indicate that in patients with self-limited infection, a strong expansion of HCV-specific CD4 cells was detectable, whereas proliferation of CD8 cells was less vigorous and was induced by a more limited number of peptide pools. In patients with chronic evolution of the disease, neither CD4 nor CD8 cell subsets expanded efficiently upon peptide stimulation (Fig. 3B).

The Acute Phase Impairment of the CD8 Function Is Transient in Self-Limited But Not in Persistent HCV Infection.

To further investigate the behavior of the HCV-specific CD8 T cell response during the acute and the recovery phase of HCV infection, different complementary approaches were used simultaneously. In HLA-A2–positive patients, the analysis of the cytotoxic T lymphocyte response was also performed with a panel of 199 synthetic peptides containing the HLA-A2 binding motif pooled in 20 mixtures, assessing IFN-γ production by ex vivo ELISPOT analysis. These peptides were selected by scanning the entire HCV sequence (HCV-1 strain) for the presence of the HLA-A2–binding motif and selecting those sequences that were predicted to bind HLA-A2 more efficiently. Also through this approach, all patients tested during the acute phase of infection displayed a weak and narrowly focused CD8 response, irrespective of the outcome of disease (Fig. 4A). Defective IFN-γ production at this stage of infection was also confirmed by analyzing tetramer-positive CD8 cells specific for five epitopes widely recognized by HLA-A2–positive patients with acute HCV infection. Whereas a relevant frequency of tetramer-positive CD8 cells was present in the circulation, no IFN-γ tetramer-positive CD8 cells were detectable ex vivo upon stimulation with either specific peptide (Fig. 4A) or PMA and ionomycin (Fig. 4B).

Six months later (Fig. 4C), the CD8 response analyzed by ex vivo ELISPOT with HLA-A2 binding peptides became multispecific and directed toward different HCV proteins in patients who resolved the infection. In contrast, CD8 responses remained very weak or totally undetectable in patients with chronic evolution of infection. In this latter category of patients, frequencies of HCV-specific CD8 cells detectable by tetramer staining declined over time and, when detectable, CD8 cells remained unable to produce cytokines upon ex vivo peptide stimulation (Fig. 4C). In contrast, tetramer-positive CD8 cells were still detectable at this later time point in recovered patients and they acquired the capacity to produce IFN-γ upon peptide stimulation (Fig. 4C).

Memory CD8 Cells Are Generated in Self-Limited But Not in Persistent HCV Infection.

Acquisition of proliferative potential and capacity to secrete effector cytokines upon antigen exposure by CD8 cells of patients with a self-limited infection may reflect the generation of long-lived, IL-7 receptor–positive (CD127+) memory CD8 cells able to survive the contraction phase of the immune response that follows acute infection and clonal expansion. Lack of their generation in patients with chronic evolution of hepatitis may contribute to inefficient control of the infection, as proposed in different animal models.

To investigate the capacity of developing memory CD8 T cells in relation to the outcome of acute HCV infection, we examined in HLA-A2–positive patients the cell surface expression on antigen-specific CD8 cells of CD127 and CCR7, a lymph node homing receptor. Viremia and percentage of CD127/HCV-specific tetramer-positive cells were analyzed in parallel in patients with different evolution of the disease during a 15- to 34-week follow-up starting from clinical onset (Fig. 5). At the earliest time point analyzed, corresponding to the acute phase of infection, an elevated percentage of virus-specific CD8 cells were CD127−, irrespective of the outcome (Fig. 5A-B). With the resolution of the infection and in the absence of measurable HCV RNA, a diffferentiation to a CD127+/CCR7+ central memory phenotype occurred (Fig. 5A). In contrast, persistence of high levels of viremia were associated with a long term-maintenance of a CD127−/CCR7− effector phenotype by HCV-specific CD8 cells (Fig. 5B).

The results obtained by costaining with CD127 and CCR7 indicate that during the acute phase of infection, a progressive memory CD8 T cell differentiation occurs in patients who spontaneously recover, whereas an altered memory differentiation is associated with the persistence of infection.

A T Helper 1–Oriented CD4 Response Is Associated With Virus Control But Not Persistence of Infection.

Induction and maintenance of antiviral CD8+ responses generally depend on the presence of functionally efficient CD4 T cells. Moreover, the CD4 function may play a crucial role in virus control, irrespective of the effect on CD8 differentiation. To investigate the function of HCV-specific CD4 cells during the acute phase of infection and their possible role in HCV control, the cytokine secretion pattern of CD4+ cells in patients with different outcome of disease was analyzed by ex vivo ICS. PBMC were stimulated overnight with HCV recombinant proteins and then stained with anti–IFN-γ, IL-2, IL-10, and IL-4 antibodies. At the peak of alanine aminotransferase, the CD4 T cell response to HCV antigens was predominantly characterized by a T helper 1 (Th1) cytokine profile in all patients with self-limited evolution of the disease (Fig. 6A-B). In contrast, patients with chronically evolving disease showed impaired production of Th1 cytokines (Fig. 6C-D). No significant differences were detected between patients with different evolution of infection in IL-4 and IL-10 production.

Thus, CD4 T cells from patients with self-limited evolution of infection produced relevant amounts of effector Th1 cytokines, such as IL-2, that can help sustain cytotoxic T lymphocyte function and to generate stable and protective CD8 memory cells. In contrast, persistent viremia and persistent impairment of the CD8 function were associated with an almost complete lack of Th1 cytokine production by CD4 cells.