PD-1 expression in acute hepatitis C virus (HCV) infection is associated with HCV-specific CD8 exhaustion

Abstract

Hepatitis C virus (HCV)-specific CD8 cell exhaustion may represent a mechanism of HCV persistence. The inhibitory receptor PD-1 has been reported to be up-regulated in exhausted CD8 cells. Therefore, we studied PD-1 expression longitudinally during acute HCV infection. Most HCV-specific CD8 cells expressed PD-1 at the time of acute illness, irrespective of the final outcome. PD-1 expression declined with the acquisition of a memory phenotype and recovery of an efficient CD8 cell function in resolving HCV infections, whereas high levels were maintained when HCV persisted and HCV-specific CD8 cells remained dysfunctional. Blocking PD-1/PDL-1 interaction with an anti-PDL-1 antibody improved the capacity of expansion of virus-specific CD8 cells.

FIG. 1.

Analysis of PD-1 expression in HCV-specific CD8 cells. The dot plots show the percentages of tetramer-positive/PD-1-positive cells at two different time points corresponding to the acute phase of infection and to a later time point (24 to 30 weeks of follow-up after clinical presentation) in patients with self-limited (shaded area) or chronic evolution of (white area) disease. Flow cytometry analysis, gating on total CD8 cells, was performed with specific anti-PD1 or with isotype control immunoglobulin G1 antibodies (PD-1 fluorescein isothiocyanate; Becton Dickinson Biosciences, San Diego, CA) on HCV-specific CD8 cells detected by tetramer staining with two phycoerythrin-labeled tetrameric peptide-HLA class I complexes (Proimmune LTD, Oxford, United Kingdom), containing the HCV peptides NS3 1073-1081 (CINGVCWTV) and NS4B 1992-2000 (VLSDFKTWL). The study was approved by the Ethical Committee of the Azienda Ospedaliero-Universitaria di Parma, and all subjects gave written informed consent. N.D., Not determined; Pt, patient.

FIG. 2.

PD-1 expression in influenza virus-specific and total CD8 cells. (A) Representative dot plot analysis of PD-1 expression analyzed on FLU-specific tetramer-positive cells (influenza virus epitope matrix GILGFVFTL) for two representative patients with different evolution of hepatitis C virus infection at two different time points. Pt, patient. (B) Percentages of PD-1-positive cells among the total CD8⁺ subset detectable during the acute phase of HCV infection and 6 to 12 months of follow-up for patients with self-limited (black bars) or chronically evolving acute (white bars) hepatitis. Each rhomb in the left panel depicts the viremia levels detected for each patient at the time of PD-1 analysis. Statistical analysis was performed by the Mann-Whitney test. (C) Fluorescence intensities of PD-1 expressed by total CD8⁺ cells for patients with different outcomes of the disease.

FIG. 3.

Longitudinal evolution of CD127 expression on HCV tetramer-positive CD8 cells. (A) Percentage of tetramer-positive/CD127⁺ CD8 cells during a 50-week follow-up from the time of clinical presentation in patients with self-limited (white squares and upper panel) and chronically evolving (black triangle and lower panel) acute hepatitis C. The right panels illustrate CD127 expression for each individual patient at the different time points analyzed. (B) Dot plots of CD127/PD-1 expression analyzed at two different time points on HCV tetramer-positive cells (NS3 1073-1081) for two representative patients with different outcomes of acute HCV infection.

FIG. 4.

Effect of anti-PDL-1 antibody on peptide-induced proliferation of HCV-specific CD8 cells. Frequencies of tetramer-positive (tetramer-pos) CD8 cells are illustrated ex vivo (gray area) and upon specific peptide stimulation of T-cell lines (white area) in the absence or in the presence of different anti-PDL-1 concentrations. For these experiments, adherent mononuclear cells isolated from peripheral blood mononuclear cells of patients with chronically evolving infection were incubated for 45 min with different concentrations (2 or 5 μg/ml) of anti-PD-L1 (e-Bioscience, Boston, MA) or control antibody (negative control for mouse immunoglobulin G1 Ab-1; Fremont, CA). Cells were then washed and coincubated for 10 days with nonadherent cells and specific peptides (HCV NS3 1073-1081 or CMV pp65) at the final concentration of 1 μg/ml (interleukin 2 was added on day 3). White squares (□) indicate tetramer-positive cells specific for CMV pp65 (NLVPMVATV); black rhombs (⧫) indicate tetramer-positive cells specific for HCV (CINGVCWTV); gray triangles () indicate HCV NS3 1073 tetramer-positive CD8 cells stimulated with the specific peptide in the presence of a control isotype antibody.