Tracking virus-specific CD4+ T cells during and after acute hepatitis C virus infection

Abstract

Background: CD4+ T cell help is critical in maintaining antiviral immune responses and such help has been shown to be sustained in acute resolving hepatitis C. In contrast, in evolving chronic hepatitis C CD4+ T cell helper responses appear to be absent or short-lived, using functional assays.

Methodology/principal findings: Here we used a novel HLA-DR1 tetramer containing a highly targeted CD4+ T cell epitope from the hepatitis C virus non-structural protein 4 to track number and phenotype of hepatitis C virus specific CD4+ T cells in a cohort of seven HLA-DR1 positive patients with acute hepatitis C in comparison to patients with chronic or resolved hepatitis C. We observed peptide-specific T cells in all seven patients with acute hepatitis C regardless of outcome at frequencies up to 0.65% of CD4+ T cells. Among patients who transiently controlled virus replication we observed loss of function, and/or physical deletion of tetramer+ CD4+ T cells before viral recrudescence. In some patients with chronic hepatitis C very low numbers of tetramer+ cells were detectable in peripheral blood, compared to robust responses detected in spontaneous resolvers. Importantly we did not observe escape mutations in this key CD4+ T cell epitope in patients with evolving chronic hepatitis C.

Conclusions/significance: During acute hepatitis C a CD4+ T cell response against this epitope is readily induced in most, if not all, HLA-DR1+ patients. This antiviral T cell population becomes functionally impaired or is deleted early in the course of disease in those where viremia persists.

Figure 1. Validation of HLA-DR1-HCV-1806-1818 tetramer staining and enrichment technology.

A CD4+ T cell clone specific for HCV 1808-1817 was stained with the HLA-DR1-HCV-1806-1818 tetramer and a control HLA-DR1 tetramer specific for the influenza epitope 307-319 and analysed by flow cytometry as described in the methods (A+B). The HCV clone was derived from patient AR1. Panel C shows staining of the clone diluted serially into PBMC negative for the tetramer at the concentrations shown. Panel D shows a strong correlation between observed pre-enrichment frequency and calculated post-enrichment frequency of HLA-DR1-HCV-tetramer positive CD4+ T cells in all PBMC samples with a frequency of HLA-DR1-HCV-tetramer positive CD4+ T cells of >0.03% (R² = 0.99, p<0.001). The dotted line is the bisecting line, the solid line represents the trend line of the measured frequencies. Panel E shows the staining of control groups; four healthy and four HIV-infected HLA-DR1 positive individuals, and two healthy individuals and five acute hepatitis C patients without HLA-DR1.

Figure 2. Examples of ex vivo HLA-DR1-HCV-1806-1818 tetramer staining from the acute hepatitis C cohort.

PBMC from one acute resolving patient (AR1) and one patient with acute hepatitis C and chronic evolution (AC1) were stained with the HLA-DR1-HCV-1806-1818 tetramer as described in the methods. For each patient the upper panels represent pre-enrichment stainings and the lower-panels the post-enrichment stainings. The numbers in the upper right corner of the post-enrichment panels are calculated according to the input CD4+ T cell number. The insert on top of the figure shows in detail the calculation method. These figures are plotted over time in both cases in Figure 2 (lower panels).

Figure 3. Clinical course of acute hepatitis C cohort in correlation to HCV specific CD4+ T cell response.

The clinical course of each individual is shown in the upper panel in each case. Note the time scale (days) is different in each case. The upper panel shows the ALT (IU/L; open boxes; left hand y axis) and the serum HCV RNA level (closed triangles; right hand y axis). For AR1, AR2 and AC3, at some time points only qualitative assays for HCV-RNA were available which have been marked with a +. Interferon alpha therapy is marked with a bar. In patients AC1 and AC2 time points where the sequence of epitope 1806-1818 was determined are indicated by arrows. The middle panel in each case corresponds to functional CD4+ T cell assays: proliferation in response to NS3/4-antigen (open boxes) and peptide-1806-1818 (open triangles) is shown as stimulation index (SI) on the left hand y axis and HCV-antigen induced interferon-gamma secretion as determined by Elispot (solid diamonds) is shown as spot forming units (SFU) on the right y-axis. The lower panel of each case shows the frequency of HLA-DR1-HCV-1806-1818 tetramer positive CD4+ T cells (grey circles). The cut-off for detection was 0.001%. The upper limit of normal for ALT is indicated in panel AR2 as dotted line.

Figure 4. Relative immunodominance of epitope HCV 1805-24 in the acute HCV cohort.

Ex vivo proliferation assays were performed on PBMC taken from the persons indicated over time. The responses to peptides including the sequence 1807-18 are marked with a dark bar. The ten peptides inducing the strongest proliferative response are shown in descending order with regard to the strength of the response. Asterisks indicate time points when the NS3-helicase/NS4 peptide library (covering aa 1207–2014) was used; all other assays were performed with a peptide library covering the entire HCV genome. A SI>3.0 was considered significant.

Figure 5. CD38 expression of HLA-DR1-HCV-1806-1818-tetramer positive CD4+ T cells.

Longitudinal phenotypic analysis of the DR1 tetramer positive cells was performed for the activation marker CD38. The dot plots shown are gated on the DR1 tetramer positive cell population. Representative examples of the staining for AR1, AC1, and AC3 at early and late time points are shown and the percentage of CD38 positive cells is given for each plot. The inserted histograms show CD38 staining on all CD4+ T cells pre-enrichment and have been used to set the quadrants for the DR1 tetramer staining.

Figure 6. Ex vivo HLA-DR1-HCV-1806-1818 tetramer staining in stable resolved and chronic HCV infection.

Tetramer staining was performed in five HLA-DR1-positive patients with chronic hepatitis C in comparison to three HLA-DR1-positive resolved patients. In all PBMC from control groups (five individuals with acute HCV who did not possess HLA-DR1, and four healthy and four HIV1+ individuals with HLA-DR1, respectively), tetramer staining was <0.001%. In all patients and healthy controls, proliferation assays following stimulation with recombinant NS4-antigen were performed. The SI for healthy controls was 1.2±0.48 (mean±SD, range 0.71 to 1.66), for chronic hepatitis C patients 1.7±0.9 (range 0.76 to 3.7), and for recovered patients 22.1±22.1 (range 1.74 to 52.9).