Upregulation of PD-1 expression on circulating and intrahepatic hepatitis C virus-specific CD8+ T cells associated with reversible immune dysfunction

Abstract

Infection with hepatitis C virus (HCV) is associated with persistence in the majority of individuals. We demonstrate here that the inhibitory molecule programmed death-1 (PD-1) is significantly upregulated on total and HCV-specific CD8(+) cytotoxic T lymphocytes (CTLs) in the peripheral blood and livers of patients with chronic infection compared to subjects with spontaneous HCV resolution, patients with nonviral liver disease, and normal controls. PD-1 expression on cytomegalovirus-specific CTLs also varies according to HCV status and is highest in patients with chronic infection. HCV-specific CTLs that are PD-1(high) express higher levels of the senescence marker CD57 than PD-1(low) CTLs, and CD57 expression is greater in chronic than in resolved infection. In vitro blockade of PD-1 by monoclonal antibodies specific to its ligands (PD-L1 and PD-L2) results in restoration of functional competence (proliferation and gamma interferon and interleukin-2 secretion) of HCV-specific CTLs, including those residing in the liver. This reversal of CTL exhaustion is evident even in individuals who lack HCV-specific CD4(+) T-cell help. Our data indicate that the PD-1/PD-L pathway is critical in persistent HCV infection in humans and represents a potential novel target for restoring function of exhausted HCV-specific CTLs.

FIG. 1.

PD-1 expression increased in chronic HCV infection. Representative dot plots and histograms of PD-1 expression on total CD8⁺ T cells (A) and HCV-specific CTLs (B), including patients with chronic and resolved HCV infection. (C) PD-1 expression on peripheral CD8⁺ T cells from patients with chronic infection (n = 18), subjects with spontaneously resolved infection (n = 11), subjects with non-HCV liver disease (n = 12), and healthy control subjects (n = 10), as well as intrahepatic lymphocytes (n = 13) from patients with chronic infection, is shown. Horizontal bars represent the medians. The PD-1 MFI on CD8⁺ T cells is higher in chronic infection than in resolved infection, non-HCV liver disease, or in healthy controls, with no difference observed between the peripheral and intrahepatic compartments. PD-1 expression is also higher in resolved infection than in healthy controls. (D) Total HCV-specific PD-1 expressed as a percentage of pentamer⁺ CD8⁺ T cells positive for PD-1 and as the MFI. Chronic HCV infection is associated with a greater percentage of PD-1⁺ HCV-specific CTLs in the periphery (n = 17 patients, 45 pentamers responses), as well as in the liver (n = 9 patients, 29 pentamers). The data are shown for seven patients (21 pentamers) with resolved infection. The intensity of PD-1 staining is also higher on the cells of chronically infected patients, showing the most concentrated PD-1 expression in the liver. (E) Breakdown of HCV-specific PD-1 expression by epitope displaying higher PD-1 in chronic infection compared to resolved for all pentamers tested. n, Number of patients with relevant HLA tested for PD-1 expression in pentamer-specific CTLs. B7- and B8-restricted pentamers are not shown, since there were only one chronic and one resolved patient tested against B7 and three chronic patients tested against B8. The graph shows the mean and standard error of the mean. The beginning HCV amino acid number associated with the seven pentamers is given on the x axis, along with their HLA restriction in parentheses. Comparisons were made by using Wilcoxon ranked sums.

FIG. 2.

CMV-specific CTLs express different levels of PD-1 according to HCV status. (A) PD-1 staining representative dot plots and histograms in healthy control, chronic-HCV, resolved-HCV, and non-HCV liver disease patients. (B) PD-1 expression of CMV-specific CTLs for patients with chronic HCV infection is significantly upregulated relative to non-HCV liver disease and healthy controls.

FIG. 3.

The proliferation of HCV-specific CTLs is enhanced in response to PD-1 ligand blockade. PBMC from patients with chronic and spontaneously resolved HCV infection were cultured for 7 days in the presence of either viral peptide alone or peptide plus anti-PD-L1, anti-PD-L2, both anti-PD-L1 and anti-PD-L2, or IL-2 (as a positive control). The HCV peptide concentration was 10 μg/ml. PD-L1 and PD-L2 were added at 10 μl/ml only initially; IL-2 was added at 5 ng/ml on days 0, 2, 4, and 6 of culture. Cells were gated on total CD8⁺ lymphocytes and analyzed for expression of CFSE and peptide-matched pentamer. The peptides and/or pentamers used were NS3 1436, core 132, NS3 1073, NS3 1406, NS5 1987, and NS5 2594 (see also Table 2). Experiments included seven patients with chronic HCV (12 pentamer responses) and three subjects with resolved HCV (5 pentamer responses). (A) The upper left quadrant of representative dot plots illustrate HCV-specific, proliferating CTLs in chronically infected patients (top row) and in patients who spontaneously resolved infection (bottom row). (B) In chronic infection, PBMC cultures with peptide plus IL-2, anti-PD-L1 and/or anti-PD-L2 exhibited significantly greater expansion of HCV-specific CTLs compared to stimulation with peptide alone. As expected, resolved patient cultures showed greater proliferation with peptide alone than cultures derived from chronic patients; although there were some increases in HCV-specific CD8 expansion with PD-1 ligand blockade and IL-2, they were not statistically significant. Comparisons were made by using the Wilcoxon signed-rank (matched pairs) test. (C) Intrahepatic lymphocytes stimulated in the presence of anti-PD-L1 and anti-PD-L2 demonstrated greater proliferation than intrahepatic lymphocytes culture with peptide NS5 1987 alone; CFSE comparisons are shown for three patients studied (see also Fig. S2 in the supplemental material).

FIG. 4.

Cytokine secretion in chronic HCV cultures with peptide and PD-1 ligand-blocking antibodies. Cell supernatants were collected after 7 day cultures of PBMC from patients with chronic (n = 8) HCV infection and assayed for cytokine quantity in pg/ml by Luminex. Cultures were performed in the presence of either HCV peptide alone or peptide plus anti-PD-L1, anti-PD-L2, both anti-PD-L1 and anti-PD-L2, or IL-2 as described in Fig. 2. IFN-γ, IL-2, and IL-13 were released in significantly higher quantities in cultures with PD-L1 and PD-L2 than in cultures with peptide alone, whereas IL-10 release did not differ between culture conditions. Comparisons were made by using the Wilcoxon signed-rank (matched-pairs) test.

FIG. 5.

PD-L1 levels in peripheral pDC and mDC. PBMC from patients with chronic HCV (n = 12) or resolved HCV (n = 8) and from normal patients (n = 8) and patients with non-HCV liver disease (n = 12) were stained and analyzed by flow cytometry to separate mDC (BDCA1 positive) and pDC (BDCA2 positive). PD-L1 was measured on each group and reported as a percentage (A) and as the MFI (B). The horizontal bar represents the median of the group. There was no appreciable difference in PD-L1 expression between patients with viral persistence and subjects with spontaneous recovery, but PD-L1 MFI was higher for pDC derived from patients with chronic infection compared to non-HCV patients (P = 0.01) and normal healthy controls (P = 0.07). PD-L1 MFI was also higher for mDC derived from patients with viral persistence compared to healthy controls (P = 0.04). In addition, in the chronic group the percentage of mDC expressing PD-L1 was higher than that for the pDCs (P = 0.007).