Comprehensive Analysis of CD8⁺-T-Cell Responses against Hepatitis C Virus Reveals Multiple Unpredicted Specificities

Abstract

The hepatitis C virus (HCV)-specific CD8؉ -T-cell response is thought to play a critical role in HCV infection. Studies of these responses have largely relied on the analysis of a small number of previously described or predicted HCV epitopes, mostly restricted by HLA A2. In order to determine the actual breadth and magnitude of CD8؉ -T-cell responses in the context of diverse HLA class I alleles, we performed a comprehensive analysis of responses to all expressed HCV proteins. By using a panel of 301 overlapping peptides, we analyzed peripheral blood mononuclear cells (PBMC) from a cohort of 14 anti-HCV-positive, HLA A2-positive individuals in an enzyme-linked immunospot assay. Only four subjects had detectable HLA A2-restricted responses in PBMC, and only 3 of 19 predicted A2 epitopes were targeted, all of which were confirmed by tetramer analysis. In contrast, 9 of 14 persons showed responses with more comprehensive analyses, with many responses directed against previously unreported epitopes. These results indicate that circulating HCV-specific CD8؉ -T-cell responses can be detected in PBMC in the majority of infected persons and that these responses are heterogeneous with no immunodominant epitopes consistently recognized. Since responses to epitopes restricted by single HLA alleles such as HLA A2 do not predict the overall response in an individual, more comprehensive approaches, as shown here, should facilitate definition of the role of the CD8 ؉ -T-cell response in HCV infection. Moreover, the low level or absence of responses to many predicted epitopes provides a rationale for immunotherapeutic interventions to broaden cytotoxic-T-lymphocyte recognition. The CD8ϩ T-cell response is thought to play a crucial role in the course of hepatitis C virus (HCV) infection. In humans and in chimpanzees, a strong and broadly directed HCV-specific cytoxoxic-T-lymphocyte (CTL) response has been associated with viral clearance during acute HCV infection (7,29). In contrast, individuals with chronic infection are often found to have a relatively weak and narrowly directed CD8 ϩ -T-cell response against HCV (reviewed in reference 27). Whether these responses in chronic disease are still beneficial in containing viral replication or whether they are mediators of hepatic injury and disease is unclear. In several studies, the magnitude of the HCV-specific CD8 ϩ -T-cell response has been correlated to HCV viral load and to liver histology, but the results of these studies have been mixed (17,31,33,34,41). Overall, the levels of responses detected by most investigators, using a variety of methods, have been fairly low compared to those found in many other viral infections.Thus far, most studies of the HCV-specific CD8 ϩ -T-cell response have been undertaken in HLA A2-positive individuals, and the analysis usually has been restricted to a set of previously described HLA A2-restricted HCV epitopes or to HCV peptides shown to bind strongly to HLA A2 and therefore predicted to elicit responses (2,4,5,14,15,28,30,33,34,(37)(38)(3...

Subversion of effector CD8+ T cell differentiation in acute hepatitis C virus infection: exploring the immunological mechanisms

“…We addressed the issue of how the consistent expansion of activated virus-specific CTL contrasts with their poor capacity to perform immediate effector functions in acute HCV infection [21][22][23][24][25][26][27][28][29][30][31], at variance with the powerful CTL responses arising during resolving infections [32,34]. Nevertheless, our poorly competent tetramer + cells were inversely related to viral load, suggesting that they play a major role in the control of HCV, at least in the early phases of infection [35,36].…”

“…Recent studies in humans and in the chimpanzee model indicate that the promptness of both T helper cell and CTL responses after HCV exposure seem to be criti- cal factors in determining the outcome of acute infection, i.e. recovery versus viral persistence [21][22][23][24][25][26][27][28][29][30][31]. Moreover, these data demonstrate that CTL, despite being considerably expanded and expressing a memory effector membrane phenotype (CCR7 -) [30,32,33], are functionally inadequate to fight HCV in a multitude of acutely infected people undergoing a state of long-lasting liver immunopathology [21][22][23][24][25][26][27][28][29][30][31].…”

Ultra‐sensitive class I tetramer analysis reveals previously undetectable populations of antiviral CD8+ T cells

“…Nonetheless, it is clear that in some cases, these responses do exist as detection becomes possible following a single round of peptide stimulation in vitro [8], suggesting that memory populations of antigen-specific CD8 + T cells are present in some infected individuals but lie below the threshold of detection by use of conventional HLA class I tetramers. Furthermore, current analysis using conventional tetramer staining has been based on unusually large tetramer + populations that are detected in a very small minority of HCV-infected individuals and that may distort the true phenotype and function of HCV-specific cells typically arising during HCV infection.…”

“…However, once persistent infection develops, HCV-specific CD8 + T cell responses become undetectable ex vivo (using tetramers or IFN-+ ELISpot) in the majority of infected individuals [7]. In some persistently infected individuals with undetectable ex vivo responses, these responses are clearly detectable following a single round of peptide stimulation in vitro [8] or following the treatment of these patients with IFN- § and ribavirin [9], suggesting that memory populations of antigen-specific CD8 + T cells are present in some infected individuals but lie below the threshold of detection using conventional HLA class I tetramer staining techniques.…”

Immune responses during acute and chronic infection with hepatitis C virus

“…Several studies have shown an impaired capacity of HCV-specific T cells to proliferate and secrete IFNγ against HCV proteins [93; 133]. However, other studies have shown IFNγ production both in vitro and ex vivo [134; 135; 136] and complete exhaustion with deletion of HCV-specific CD8+ T cells does not occur because CD8+ T cell responses can be restored with stimulation by IL2 and HCV antigens [137].…”