Human leukocyte antigen B27 selects for rare escape mutations that significantly impair hepatitis C virus replication and require compensatory mutations.

UNLABELLED: Human leukocyte antigen B27 is associated with spontaneous viral clearance in hepatitis C virus (HCV) infection. Viral escape within the immunodominant, HLA-B27-restricted, HCV-specific, cluster of differentiation (CD)8(+) T-cell epitope, nonstructural protein (NS)5B(2841-2849) (ARMILMTHF), has been shown to be limited by viral fitness costs as well as broad T-cell cross-recognition, suggesting a potential mechanism of protection by HLA-B27. Here, we studied the subdominant HLA-B27-restricted epitope, NS5B(2936-2944) (GRAAICGKY), to further define the mechanisms of protection by HLA-B27. We identified a unique pattern of escape mutations within this epitope in a large cohort of HCV genotype 1a-infected patients. The predominant escape mutations represented conservative substitutions at the main HLA-B27 anchor residue or a T-cell receptor contact site, neither of which impaired viral replication capacity, as assessed in a subgenomic HCV replicon system. In contrast, however, in a subset of HLA-B27(+) subjects, rare escape mutations arose at the HLA-B27 anchor residue, R(2937) , which nearly abolished viral replication. Notably, these rare mutations only occurred in conjunction with the selection of two equally rare, and structurally proximal, upstream mutations. Coexpression of these upstream mutations with the rare escape mutations dramatically restored viral replication capacity from <5% to ≥ 70% of wild-type levels. CONCLUSION: The selection of rare CTL escape mutations in this HLA-B27-restricted epitope dramatically impairs viral replicative fitness, unless properly compensated. These data support a role for the targeting of highly constrained regions by HLA-B27 in its ability to assert immune control of HCV and other highly variable pathogens.

Lack of ex vivo peripheral and intrahepatic α-fetoprotein-specific CD4+ responses in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignancies with a poor prognosis and limited therapeutic options that is often characterized by the expression of the tumor-associated antigen α-fetoprotein (AFP). CD4+ helper T cells are important in generating potent anticancer immunity as they prime and expand CD8+ T-cell memory and may also have direct antitumor activity. However, very little information is currently available about the relative frequency, immunodominance and peripheral versus intratumoral distribution of AFP-specific CD4+ T-cell responses in patients with HCC. We, therefore, analyzed AFP-specific CD4+ responses in blood and tumor tissue of patients with HCC by using overlapping peptides spanning the entire AFP protein and novel sensitive approaches such as antigen-specific upregulation of CD154. We found that AFP-specific CD4+ T-cell responses were not detectable in the peripheral blood ex vivo. However, after in vitro stimulation, AFP-specific CD4+ T-cell responses were detectable in a large fraction of patients targeting different previously unreported epitopes with no clear immunodominance. These results indicate that AFP-specific CD4+ T-cell responses are not completely deleted but only present at very low frequencies. Importantly, AFP-specific CD4+ T-cell responses were also rarely detectable in tumor tissue, suggesting that the relative absence of these cells in the circulation ex vivo is not due to a rapid accumulation to the tumor side. Taken together, these results suggest that the lack of sufficient CD4+ T-cell help, especially within the tumor tissue, may be one central mechanism responsible for the failure of AFP-specific immune responses to control HCC progression.

Protective effect of human leukocyte antigen B27 in hepatitis C virus infection requires the presence of a genotype-specific immunodominant CD8+ T-cell epitope.

UNLABELLED: Human leukocyte antigen B27 (HLA-B27) is associated with protection in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. This protective role is linked to single immunodominant HLA-B27-restricted CD8+ T-cell epitopes in both infections. In order to define the relative contribution of a specific HLA-B27-restricted epitope to the natural course of HCV infection, we compared the biological impact of the highly conserved HCV genotype 1 epitope, for which the protective role has been described, with the corresponding region in genotype 3 that differs in its sequence by three amino acid residues. The genotype 3a peptide was not recognized by CD8+ T cells specific for the genotype 1 peptide. Furthermore, patients with acute or chronic infection with HCV genotype 3a did not mount T-cell responses to this epitope region, and their autologous viral sequences showed no evidence of T-cell pressure. Finally, we found a significantly higher frequency of HLA-B27 positivity in patients with chronic HCV genotype 3a infection compared to genotype 1 infection, indicating that there is no protection by HLA-B27 in HCV genotype 3 infection. CONCLUSION: Our data indicate that the protective effect of HLA-B27 is limited to HCV genotype 1 infection and does not expand to other genotypes such as genotype 3a. This can most likely be explained by intergenotype sequence diversity leading to the loss of the immunodominant HLA-B27 epitope in viral strains other than genotype 1. Our results underline the central role of a single HLA-B27-restricted epitope-specific CD8+ T-cell response in mediating protection in HCV genotype 1 infection.

Virus-specific CD4+ T cell responses in chronic HCV infection in blood and liver identified by antigen-specific upregulation of CD154.

BACKGROUND & AIMS: Virus-specific CD4+ T cells play a major role in hepatitis C virus (HCV) infection. Viral clearance is associated with vigorous and multispecific CD4+ T cell responses, while chronic infection has been shown to be associated with weak or absent T cell responses. Most of these studies, however, have used functional assays to analyse virus-specific CD4+ T cell responses. Therefore, the important question, of whether virus-specific CD4+ T cells are completely absent or primarily impaired in specific effector functions during chronic infection, has yet to be analysed in detail. METHODS: To address this issue, a novel assay, where CD4+ T cell frequencies can be determined by de novo CD154 (CD40 ligand) expression in response to HCV antigens, was used in a cohort of chronically infected HCV patients and patients who spontaneously resolved HCV infection. These responses were compared to functional assays, such as the IFN-gamma ELISpot and flow cytometry-based proliferative assays. RESULTS: Our results reveal that using the CD154 assay, virus-specific CD4+ T cells are readily detectable during chronic HCV infection albeit at a lower frequency when compared to patients who spontaneously resolved the infection. Importantly, no CD4+ T cell responses were detectable from these patients when using functional assays. Finally, these cell populations were enriched in the intrahepatic compartment. CONCLUSIONS: Our findings suggest that HCV-specific CD4+ T cell responses are readily detectable in chronic HCV infection and enriched in the infected liver.

Analysis of CD8+ T-cell-mediated inhibition of hepatitis C virus replication using a novel immunological model.

BACKGROUND & AIMS: Virus-specific CD8+ T cells are required for the control of hepatitis C virus (HCV) infection. We investigated the extent to which different effector functions of CD8+ T cells contribute to the inhibition of viral replication. METHODS: We developed a novel immunologic model by stably transducing the HLA-A2 gene into the replicon system, matching the epitope sequence of the replicon to the sequence targeted by an HCV-specific CD8+ T-cell clone. Luciferase activity was then measured to quantitate HCV RNA replication. RESULTS: HCV-specific CD8+ T cells strongly inhibited viral replication, through cytolytic and noncytolytic mechanisms, in a dose-dependent manner. HCV replication was almost completely inhibited at an effector-to-target ratio of 1:1 with significant cytotoxicity; however, >95% viral inhibition occurred at ratios as low as 1:100. Importantly, no cytotoxicity was observed at low effector-to-target ratios, indicating a dominant effect of noncytolytic effector functions that was confirmed by Transwell experiments. Neutralization experiments revealed that interferon gamma mediates the noncytolytic inhibition. CONCLUSIONS: Only a very few HCV-specific CD8+ T cells are required to inhibit HCV replication; inhibition occurs primarily by noncytolytic effector functions.

CD161 expression on hepatitis C virus-specific CD8+ T cells suggests a distinct pathway of T cell differentiation.

UNLABELLED: Hepatitis C virus (HCV) causes chronic infection accompanied by a high risk of liver failure and hepatocellular carcinoma. CD8+ T cell responses are important in the control of viremia. However, the T cell response in chronic infection is weak both in absolute numbers and in the range of epitopes targeted. In order to explore the biology of this response further, we analyzed expression of a panel of natural killer cell markers in HCV compared with other virus-specific T cell populations as defined by major histocompatibility complex class I tetramers. We found that CD161 was significantly expressed on HCV-specific cells (median 16.8%) but not on CD8+ T cells specific for human immunodeficiency virus (3.3%), cytomegalovirus (3.4%), or influenza (3.4%). Expression was seen in acute, chronic, and resolved disease and was greatest on intrahepatic HCV-specific T cells (median 57.6%; P < 0.05). Expression of CD161 was also found on hepatitis B virus-specific CD8+ T cells. In general, CD161+CD8+ T cells were found to be CCR7- "effector memory" T cells that could produce proinflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha) but contained scanty amounts of cytolytic molecules (granzyme B and perforin) and proliferated poorly in vitro. Expression of CD161 on CD8+ T cells was tightly linked to that of CXCR6, a chemokine with a major role in liver homing. CONCLUSION: We propose that expression of CD161 indicates a unique pattern of T cell differentiation that might help elucidate the mechanisms of HCV immunity and pathogenesis.

Comprehensive analysis of the alpha-fetoprotein-specific CD8+ T cell responses in patients with hepatocellular carcinoma.

UNLABELLED: Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide, with a poor prognosis and limited therapeutic options. Therefore, the development of novel therapeutic strategies is of high priority. alpha-Fetoprotein (AFP) is overexpressed in the majority of HCCs. Priming of immune responses against AFP results in significant protective antitumoral T cell responses in the mouse model. Little information is available about the hierarchy, breadth, frequency, and peripheral versus intrahepatic distribution of AFP-specific CD8(+) T cell responses in patients with HCC. To address these important issues we comprehensively analyzed CD8(+) T cell responses against full-length AFP in peripheral blood, tumor liver tissue, and nontumor liver tissue from patients with HCC using overlapping AFP peptides. The AFP-specific CD8(+) T cell response was also tested in peripheral blood and liver from patients chronically infected with hepatitis C virus (HCV) and compared to the HCV-specific CD8(+) T cell response. The majority of patients with HCC showed AFP-specific responses, with many responses directed against previously unreported epitopes. These responses were primarily detectable in the HCC tissue and mainly targeted the C-terminus of AFP. Interestingly, AFP-specific T cells were not only found in patients with HCC but also in patients with chronic HCV infection, other liver diseases, and less frequently in healthy subjects. CONCLUSION: In patients with HCC, a high frequency of AFP-specific CD8(+) T cells directed against different epitopes suggest that AFP has a strong and broad immunogenicity. Further, CD8(+) T cells specific for the self-antigen AFP are present in the normal T cell repertoire and are not centrally or peripherally deleted. Our results provide support for strategies to boost AFP-specific CD8(+) T cell responses in patients with HCC but also demonstrate a diversity of immune responses that may be needed for protection.

Virological and immunological determinants of intrahepatic virus-specific CD8+ T-cell failure in chronic hepatitis C virus infection.

UNLABELLED: Virus-specific CD8+ T-cells play an important role in the outcome of acute hepatitis C virus (HCV) infection. In the chronic phase, however, HCV can persist despite the presence of virus-specific T-cell responses. Therefore, we set out to perform a full-breadth analysis of the intrahepatic virus-specific CD8+ T-cell response, its relation to the peripheral T-cell response, and the overall influence of viral escape and the genetic restriction on intrahepatic CD8+ T-cell failure. Intrahepatic and peripheral CD8+ T-cells from 20 chronically HCV infected patients (genotype 1) were comprehensively analyzed using overlapping peptides spanning the entire HCV polyprotein in concert with autologous viral sequences that were obtained for all targeted regions. HCV-specific CD8+ T-cell responses were detectable in most (90%) chronically HCV-infected patients, and two thirds of these responses targeted novel previously undescribed epitopes. Most of the responses were detectable only in the liver but not in the peripheral blood, indicating accumulation and enrichment at the site of disease. Of note, only approximately half of the responses were associated with viral sequence variations supported by functional analysis as viral escape mutations. Escape mutations were more often associated with HLA-B alleles. CONCLUSION: Our results show an unexpected high frequency of intrahepatic virus-specific CD8+ T-cells, a large part of which continue to target the present viral antigens. Thus, our results suggest that factors other than mutational escape contribute to the failure of intrahepatic virus-specific CD8+ T-cells.

Identification of naturally processed hepatitis C virus-derived major histocompatibility complex class I ligands.

Fine mapping of human cytotoxic T lymphocyte (CTL) responses against hepatitis C virus (HCV) is based on external loading of target cells with synthetic peptides which are either derived from prediction algorithms or from overlapping peptide libraries. These strategies do not address putative host and viral mechanisms which may alter processing as well as presentation of CTL epitopes. Therefore, the aim of this proof-of-concept study was to identify naturally processed HCV-derived major histocompatibility complex (MHC) class I ligands. To this end, continuous human cell lines were engineered to inducibly express HCV proteins and to constitutively express high levels of functional HLA-A2. These cell lines were recognized in an HLA-A2-restricted manner by HCV-specific CTLs. Ligands eluted from HLA-A2 molecules isolated from large-scale cultures of these cell lines were separated by high performance liquid chromatography and further analyzed by electrospray ionization quadrupole time of flight mass spectrometry (MS)/tandem MS. These analyses allowed the identification of two HLA-A2-restricted epitopes derived from HCV nonstructural proteins (NS) 3 and 5B (NS31406₋1415 and NS5B2594₋2602). In conclusion, we describe a general strategy that may be useful to investigate HCV pathogenesis and may contribute to the development of preventive and therapeutic vaccines in the future.

Analysis of CD127 and KLRG1 expression on hepatitis C virus-specific CD8+ T cells reveals the existence of different memory T-cell subsets in the peripheral blood and liver.

The differentiation and functional status of virus-specific CD8+ T cells is significantly influenced by specific and ongoing antigen recognition. Importantly, the expression profiles of the interleukin-7 receptor alpha chain (CD127) and the killer cell lectin-like receptor G1 (KLRG1) have been shown to be differentially influenced by repetitive T-cell receptor interactions. Indeed, antigen-specific CD8+ T cells targeting persistent viruses (e.g., human immunodeficiency virus and Epstein-Barr virus) have been shown to have low CD127 and high KLRG1 expressions, while CD8+ T cells targeting resolved viral antigens (e.g., FLU) typically display high CD127 and low KLRG1 expressions. Here, we analyzed the surface phenotype and function of hepatitis C virus (HCV)-specific CD8+ T cells. Surprisingly, despite viral persistence, we found that a large fraction of peripheral HCV-specific CD8+ T cells were CD127+ and KLRG1- and had good proliferative capacities, thus resembling memory cells that usually develop following acute resolving infection. Intrahepatic virus-specific CD8+ T cells displayed significantly reduced levels of CD127 expression but similar levels of KLRG1 expression compared to the peripheral blood. These results extend previous studies that demonstrated central memory (CCR7+) and early-differentiated phenotypes of HCV-specific CD8+ T cells and suggest that insufficient stimulation of virus-specific CD8+ T cells by viral antigen may be responsible for this alteration in HCV-specific CD8+ T-cell differentiation during chronic HCV infection.

Intrahepatic CD8+ T-cell failure during chronic hepatitis C virus infection.

The precise mechanisms responsible for the failure of intrahepatic hepatitis C virus (HCV)-specific CD8+ T cells to control the virus during persistent infection have not been fully defined. We therefore studied the CD8+ T-cell response in 27 HLA-A2-positive patients using four previously well-defined HLA-A2-restricted HCV epitopes. The corresponding HCV sequences were determined in several patients and compared with the intrahepatic HCV-specific CD8+ T-cell response. The results of the study indicate: (1) intrahepatic HCV-specific CD8+ T cells are present in the majority of patients with chronic HCV infection and overlap significantly with the response present in the peripheral blood. (2) A large fraction of intrahepatic HCV-specific CD8+ T cells are impaired in their ability to secrete interferon gamma (IFN-gamma). This dysfunction is specific for HCV-specific CD8+ T cells, since intrahepatic Flu-specific CD8+ T cells readily secrete this cytokine. (3) T-cell selection of epitope variants may have occurred in some patients. However, it is not an inevitable consequence of a functional virus-specific CD8+ T-cell response, since several patients with IFN-gamma-producing CD8+ T-cell responses harbored HCV sequences identical or cross-reactive with the prototype sequence. (4) The failure of intrahepatic virus-specific CD8+ T cells to sufficiently control the virus occurs despite the presence of virus-specific CD4+ T cells at the site of disease. In conclusion, different mechanisms contribute to the failure of intrahepatic CD8+ T cells to eliminate HCV infection, despite their persistence and accumulation in the liver.