Broadly directed virus-specific CD4+ T cell responses are primed during acute hepatitis C infection, but rapidly disappear from human blood with viral persistence

Abstract

Vigorous proliferative CD4(+) T cell responses are the hallmark of spontaneous clearance of acute hepatitis C virus (HCV) infection, whereas comparable responses are absent in chronically evolving infection. Here, we comprehensively characterized the breadth, specificity, and quality of the HCV-specific CD4(+) T cell response in 31 patients with acute HCV infection and varying clinical outcomes. We analyzed in vitro T cell expansion in the presence of interleukin-2, and ex vivo staining with HCV peptide-loaded MHC class II tetramers. Surprisingly, broadly directed HCV-specific CD4(+) T cell responses were universally detectable at early stages of infection, regardless of the clinical outcome. However, persistent viremia was associated with early proliferative defects of the HCV-specific CD4(+) T cells, followed by rapid deletion of the HCV-specific response. Only early initiation of antiviral therapy was able to preserve CD4(+) T cell responses in acute, chronically evolving infection. Our results challenge the paradigm that HCV persistence is the result of a failure to prime HCV-specific CD4(+) T cells. Instead, broadly directed HCV-specific CD4(+) T cell responses are usually generated, but rapid exhaustion and deletion of these cells occurs in the majority of patients. The data further suggest a short window of opportunity to prevent the loss of CD4(+) T cell responses through antiviral therapy.

Figure 1.

Responsiveness to HCV antigens by CD4⁺ T cells from patients with acute HCV infection and spontaneously resolving versus persistent viremia. (A) PBMCs obtained from patients within 6 mo of symptomatic HCV infection were stimulated for 6 d in vitro using 3 different recombinant HCV proteins (NS3, NS4, and NS5) in the absence of rIL-2. Proliferation was measured using thymidine uptake and expressed as SI over negative control. Each dot represents a single patient. The dotted line delineates an SI value of 5 that is considered the cut-off for a positive proliferative response. (B and C) PBMCs obtained as in A were depleted of CD8⁺ T cells, and short-term CD4⁺ T cell lines were generated using recombinant HCV antigens and overlapping HCV peptides together with rIL-2. After 10–14 d, cell lines were tested for IFN-γ production, initially with pools of 20 overlapping peptides, and then, if positive, by single 20-mer peptides. (B) For each subject, the number of single peptides that tested positive is shown, plotted separately for the AR versus AC cohort. (C) The percentage of IFN-γ–secreting cells for each reactive cell line as determined by ICS and ro compare the relative size of the expanded HCV-specific populations between AR and AC subjects. (D) Detailed results for subject AC9 with acute infection followed by chronic viremia. The bar graph on the left shows results (in SI) from the standard proliferation assay without rIL-2 as in (A), showing no response against any of the HCV proteins, but robust proliferative responses against cytomegalovirus lysate, tetanus toxoid (TT) and phytohemagglutinin (PHA) controls. On the right, six dot blots showing positive ICS assays from the experiments outlined in B and C, confirming expansion of HCV-specific CD4⁺ T cells targeting six different epitopes in the same individual using short-term in vitro culture under the addition of rIL-2.

Figure 2.

Frequency of detection of single HCV CD4⁺ epitopes using short-term in vitro culture with the addition of rIL-2. (A and B) The relative location of each epitope detected in the assays from Fig. 1 (B and C) within the HCV polyprotein and the frequency with which the respective peptide was recognized in the cohort of patients with self-limited (AR) and chronic (AC) infection. The six most frequently recognized peptides are marked with their exact location in the HCV protein sequence. More detailed information on all epitopes detected in each individual patient of the cohort can be found in Table S2.

Figure 3.

HCV-specific proliferation of virus-specific CD4⁺ T cells in the presence or absence of IL-2. (A) PBMCs of an early time point from patient AC7 with AC HCV infection were labeled with CFSE dye and stimulated with no peptide (−) or peptides p124 (aa 1241-1260) and p177 (aa 1771–1790) in the presence (left) or absence (right) of rIL-2. After 7 d, cells were stained with HCV-specific tetramers Tet124 and Tet177. (B) CFSE proliferation assay in a patient with acute HCV infection and self-limited disease. PBMCs were stained with CFSE dye and stimulated with HIV peptides (control peptides) or a pool of the six most frequently detected immunodominant HCV peptides (Fig. 2) for 7 d with or without addition of rIL-2. (C) Summary of the CFSE proliferation experiments performed as in A on six patients (filled circles) with AC infection and six patients (filled squares) with AR infection either in the absence or presence of IL-2. (SI, stimulation index with the frequency of proliferating cells without addition of peptide and rIL-2 as the denominator).

Figure 4.

Abortion of HCV-specific T cell proliferation by an anti–IL-2 blocking antibody. CFSE assay in a patient with resolution of the acute infection. PBMCs from a patient with resolved HCV infection were depleted of CD8⁺ T cells, stained with CFSE dye, and stimulated with recombinant c200 (NS3+NS4) protein in the presence of IL-2–blocking antibody or isotype control. At day 7, the number of CFSE^low cells was assessed by flow cytometry. Superoxide dismutase served as negative control and phytohemagglutinin and c200 plus IL-2 served as positive controls.

Figure 5.

Time dependency of detection of HCV-specific CD4⁺ T cell responses in patients with acute HCV infection. The number of HCV-specific CD4⁺ responses detected in HCV-antigen stimulated short-term T cell lines with rIL-2 (as shown in Fig. 1 B) was plotted against the time elapsed between symptom onset and sampling of blood for the experiment in AC infection (A) and patients with AR course (B). Each square represents the number of HCV peptide-specific CD4⁺ T cell responses of a single patient.

Figure 6.

Analysis of the frequency of HCV-specific CD4⁺ T cells by direct ex vivo class II tetramer staining in patients with acute HCV infection. (A) Sample dot blots for ex vivo tetramer staining with tetramer p177 in a patient with self-limited acute HCV infection (AR3) and a patient with chronically evolving HCV infection (AC1). (B) The ex vivo frequencies of all HCV MHC class II tetramers stainings performed on all samples of a total of 38 patients are plotted against the time elapsed between onset of symptoms and sampling of blood for each assay, depicted separately for AR infection and AC infection.

Figure 7.

Longitudinal assessment of HCV-specific CD4⁺ T cell responses by MHC class II tetramer staining. Detailed longitudinal results for three subjects with different clinical courses using standard proliferation assay and short-term cell lines with rIL-2, as in Fig. 1 D, as well as tetramer assays as shown in Fig. 6 (A and B). Frequencies of two class II tetramers (Tet124 and Tet177; triangles) over time for patient AR8 with AR infection blotted together with HCV viral load (gray; A) and as individual dot blots for each time point (B). (bottom) Results for the in vitro standard proliferation assay for comparison with the direct ex vivo tetramer results above. (C–F) Patients AC7 and AC10 were both diagnosed with acute HCV infection and persisting viremia. AC7 was treated at an early time point after developing symptomatic disease, whereas AC10 was treated only almost a year after the onset of symptoms. Both achieved a sustained virological response (SVR). Frequencies for detected tetramer responses (triangles) are plotted together with HCV viral load (gray) and the treatment period (red) in the top panel of C for AC7 and E for AC10. Asterisks in E mark time points at which tetramer assays were negative even with more sensitive tetramer staining protocols based on magnetic bead capture enrichment (Day et al., 2003). (C and E, bottom) Results from the standard proliferation assay over the same time periods as the tetramer results above. (D and F) ICS results detecting IFN-γ secretion are shown for short-term cultures at different time points of infection using rIL-2 and single HCV peptide that had tested positive in the first available blood sample.

Figure 8.

Analysis of HCV-specific CD4⁺ T cell responses in patients with AC infection and a sustained virological response (SVR) after early versus late induction of antiviral treatment. (A) PBMCs obtained after successful antiviral therapy of acute infection and either early (<6 mo after symptom onset; n = 5) or late (<6 mo after symptom onset; n = 3) initiation of treatment were depleted of CD8⁺ T cells, stimulated with recombinant antigens and overlapping peptides, and after 10–14 d the number of single peptides eliciting an IFN-γ response was measured (as in Fig. 1 B). Each dot represents the number of different responses in each patient. (B) Ex vivo HCV MHC class II tetramer frequencies before and after treatment. Frequencies of HCV-specific CD4⁺ T cells were assessed before and after treatment using HLA class II tetramer staining in subjects with chronically evolving infection who were treated early. Untreated patients with AC served as controls. Each dot or square represents one tetramer response in a single individual.