T cell responses in hepatitis C virus infection: historical overview and goals for future research

Abstract

Hepatitis C virus (HCV)-specific T cells are key factors in the outcome of acute HCV infection and in protective immunity. This review recapitulates the steps that immunologists have taken in the past 25years to dissect the role of T cell responses in HCV infection. It describes technical as well as disease-specific challenges that were caused by the inapparent onset of acute HCV infection, the difficulty to identify subjects who spontaneously clear HCV infection, the low frequency of HCV-specific T cells in the blood of chronically infected patients, and the lack of small animal models with intact immune systems to study virus-host interaction. The review provides a historical perspective on techniques and key findings, and identifies areas for future research.

Keywords: Hepatitis C virus; History; Immunological techniques; Infection; T cell.

Fig. 1. Identification of T cell epitopes using pools of overlapping peptides arranged in a matrix format

A. A set of overlapping peptides (typically 15mer overlapping by 10 amino acids) are synthesized based on the HCV sequence. B. A matix of peptide pools is generated in which two pools share one peptide each. The figure shows how 30 peptides (1–30) are used to generate 11 peptide pools (A, B, C, D, E, F, AA, BB, CC, DD and EE). C. PBMC from an HCV-infected patient are tested against each peptide pool in standard immunological assay (typically IFN-γ ELISpot assay or intracellular cytokine staining/flow cytometry). In this case, pools B and DD yield the strongest response, suggesting that the shared peptide #20 is recognized. In subsequent assays, peptide #20 should be tested separately from the other peptides along with shorter, amino- and carboxyterminally truncated peptides to determine the minimal optimal epitope within the 15mer.

Fig. 2. Model systems to study immune responses to HCV infection

An NIH moratorium in 2013 restricted research with chimpanzees in 2013 (Wadman, 2013). Research performed prior to 2013 is described in this figure. PBMC, peripheral blood mononuclear cells.

Fig. 3. Effect of HCV mutations on CD8 T cell responses

A. In this simplified cartoon, a target cell (hepatocyte) is shown on the left and a CD8 T cell on the right. Within the HCV infected target cells, a peptide with the sequence of a CD8 T cell epitope is generated from the endogenously translated HCV NS4A polypeptide by the antigen processing machinery (proteasome depicted here as a representative antigen processing enzyme) and loaded onto MHC class I molecules. The two anchor moleculares of the peptide (e.g. amino acid in position 2 and carboxyterminal amino acid for HLA-A*0201-restricted peptides) bind correctly to the MHC molecule. The middle part of the peptide is recognized by the T cell receptor (TCR) of the CD8 T cells, resulting in activation and in effector functions such as cytokine production and lysis of the target cell. If CD8 T cells recognize epitopes that do not allow HCV escape mutations, they become functionally exhausted in persisting HCV infection and express high levels of inhibitory molecules such as PD-1 CTLA-4, CD160,, Tim-3, are CD127-negative and lack effector function. B. An HCV mutation associated with an amino acid change in the flanking sequence of an HCV CD8 T cell epitope results in proteasomal processing of the polypeptide sequence and cleavage within the epitope sequence. The correct epitope is not generated. C. The HCV CD8 T cell epitope is correctly processed but a mutation results in an altered amino acid in one of the two MHC binding sites. As a result the MHC binding affinity of the peptide is reduced and the epitope is not correctly presented to CD8 T cells. CD8 T cells that had been primed prior to the emergence of HCV mutations (B, C) typically display memorylike phenotype because they do not encounter their cognate antigen anymore. They express low levels of inhibitory molecules such as PD-1 CTLA-4, CD160,, Tim-3, express high levels of CD127 and display strong recall responses when stimulated with their cognate antigen in vitro. D. The HCV CD8 T cell epitope is correctly processed and bound to MHC class I molecules but an HCV mutation alters the amino acid sequence that is recognized by the T cell receptor. The result is reduced or absent TCR stimulation. The peptide may also compete with TCR stimulation by the wild-type peptide against which the CD8 T cells were originally primed, and thus act as an antagonist.