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

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.     

Differential CD4(+) and CD8(+) T-cell responsiveness in hepatitis C virus infection

This study was performed to compare the vigor and phenotype of virus-specific CD4(+) and CD8(+) T-cell responses in patients with different virologic and clinical outcomes after hepatitis C virus (HCV) infection. The results show that a vigorous and multispecific CD4(+) proliferative T-cell response is maintained indefinitely after recovery from HCV infection whereas it is weak and focused in persistently infected patients. In contrast, the HCV-specific CD8(+) T-cell response was quantitatively low in both groups despite the use of sensitive direct ex vivo intracellular interferon gamma (IFN-gamma) staining. Furthermore, although HCV-specific cytolytic CD8(+) memory T cells were undetectable ex vivo, they were readily expanded from the peripheral blood of chronically HCV-infected patients but not from recovered subjects after in vitro stimulation, suggesting that ongoing viremia is required to maintain the HCV-specific memory CD8(+) T-cell response. HCV-specific CD8(+) T cells displayed a type 1 cytokine profile characterized by production of IFN-gamma despite persistent HCV viremia. The paradoxical observation that HCV-specific CD4(+) T cells survive and CD8(+) T cells are lost after viral clearance while the opposite occurs when HCV persists suggests the existence of differential requirements for the maintenance of CD4(+) and CD8(+) T-cell memory during HCV infection. Furthermore, the relative rarity of circulating CD8(+) effector T cells in chronically infected patients may explain the chronic insidious nature of the liver inflammation and also why they fail to eliminate the virus.     

Discordant role of CD4 T-cell response relative to neutralizing antibody and CD8 T-cell responses in acute hepatitis C

BACKGROUND & AIMS: Acute hepatitis C virus (HCV) infection becomes chronic in the majority of patients. Although HCV-specific CD4 T-cell response is associated with HCV clearance, less is known about virus-specific CD8 T-cell or neutralizing antibody (nAb) responses and the role of CD4 help in their induction during acute infection. METHODS: HCV-specific CD4, CD8, and HCV pseudoparticle (HCVpp) nAb responses were monitored in acutely HCV-infected patients to define their relative contributions to viral clearance. RESULTS: Our results show that the outcome of acute hepatitis C is associated with a functional hierarchy in HCV-specific CD4 T-cell response and the scope of virus-specific, total T-cell interferon-gamma response. HCV-specific CD8 T-cell response was readily detectable in acutely HCV-infected patients regardless of virologic outcome or virus-specific CD4 T-cell response. In contrast, HCVpp-specific nAbs were readily detected in patients with chronic evolution and impaired virus-specific CD4 T-cell response but not in patients who cleared infection with robust virus-specific CD4 T-cell response. CONCLUSIONS: The outcome of acute hepatitis C is associated with efficient virus-specific CD4 T-cell response(s) without which HCV-specific CD8 T-cell and heterologous nAb responses may develop but fail to clear viremia. Furthermore, HCV-specific nAb responses may not be induced despite robust virus-specific CD4 T-cell response.     

Intrahepatic and circulating HLA class II-restricted, hepatitis C virus-specific T cells: functional characterization in patients with chronic hepatitis C

To compare the functional features of circulating and intrahepatic hepatitis C virus (HCV)-specific CD4+ T cells in chronic HCV infection, peripheral blood and liver-infiltrating lymphocytes from 29 patients with chronic hepatitis C were stimulated with structural and nonstructural HCV proteins to produce antigen-specific T-cell lines and clones. Antigen specificity, fine specificity, phenotype, cytokine production, and T-cell receptor (TCR)-vbeta chain expression were analyzed. The results indicate a hierarchy of stimulatory capacity by the different HCV proteins, core being the antigen most frequently recognized by CD4+ intrahepatic lymphocytes, followed by NS4 and NS5. The CD4 response was directed simultaneously against different HCV proteins in individual patients, but fine-specificity analysis indicated that the response was generally focused on a limited number of immunodominant epitopes. Although the narrowly focused nature of this response may favor the emergence of escape mutations, this event was not observed by following-up over time the sequence of 2 epitopes strongly immunodominant for intrahepatic CD4 cells of a patient with chronic HCV infection. In conclusion, simultaneous analysis of peripheral blood and intrahepatic CD4 cells in the same patients indicated a predominant Th1 profile of HCV-specific CD4 cells and suggests a specific compartmentalization of virus-specific T cells into the liver.     

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

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.     

Analysis of intrahepatic HBV-specific cytotoxic T-cells during and after acute HBV infection in humans

BACKGROUND/AIMS: Characteristics of the intrahepatic virus-specific T-cell response in patients with acute hepatitis B virus (HBV) infection have not been studied due to the risk of complications associated with standard liver biopsies. In this study we aimed to characterize the virus-specific CD8 + T-cell response in the liver of patients with acute HBV infection using fine-needle aspiration-biopsy (FNAB). METHODS: In HLA-A2 positive patients with acute HBV infection a FNAB was performed at first presentation, at the time of HBsAg-seroconversion and 3 months after HBsAg-seroconversion. HLA-A2 tetramers were used to identify HBV-specific CD8 + T-cells in FNAB-cytology and peripheral blood (PB). RESULTS: At first presentation there was a correlation between the frequency of intrahepatic CD8 + T-cells and the degree of liver damage. At all time points there was sequestering of HBV-specific CD8 + T-cells in the liver, and the percentage of intrahepatic HLA-DR expressing HBV-specific CD8 + T-cells was higher than in PB. Three months after HBsAg-seroconversion the frequency of intrahepatic HBV-specific CD8 + T-cells remained high. CONCLUSIONS: HBV-specific CD8 + T-cells are compartmentalized in the liver during acute HBV infection. Their presence in the liver may suggest a role in the resolution of the infection. Intrahepatic HBV-specific CD8 + T cells remain detectable at high frequencies after HBsAg-seroconversion.     

Success and failure of virus-specific T cell responses in hepatitis C virus infection

Hepatitis C virus (HCV) infection is only cleared in a minority of infected individuals, the majority of patients develop chronic infection. Chronic HCV infection potentially leads to liver fibrosis, cirrhosis and finally hepatocellular carcinoma. The host immune response is an important determinant in the outcome of HCV infection. Innate as well as adaptive cellular and humoral immune responses mediate important antiviral actions; however, virus-specific T cell responses appear to be most critical. Indeed, strong and multispecific CD4+ as well as CD8+ T cell responses are required for viral clearance. Interestingly, individuals who express certain HLA alleles (which are important for antigen presentation to CD4+ and CD8+ T cells) have a higher chance to clear the virus. The mechanisms of protection by HLA class I alleles such as HLA-B27 have been characterized recently. In most individuals, however, the HCV-specific immune response fails to clear the virus. Several mechanisms underlying this HCV-specific T cell failure have been identified. These include viral factors such as viral escape mutations and immunological factors such as the expression of inhibitory receptors, which lead to CD8+ T cell dysfunction. An in-depth understanding of the determinants of success or failure of the HCV-specific T cell response is critical for the development of prophylactic as well as therapeutic vaccination regimes against HCV. Here, we will discuss the virological and immunological determinants of HCV clearance and persistence.     

Recurrence of hepatitis C virus after loss of virus-specific CD4(+) T-cell response in acute hepatitis C.

BACKGROUND & AIMS: The prospective comparison of patients with acute hepatitis C virus (HCV) who spontaneously clear the virus with those who cannot achieve viral elimination and progress to chronic hepatitis offers the unique opportunity to analyze natural mechanisms of viral elimination. METHODS: We studied the HCV-specific CD4(+) T-cell response in 38 patients with acute HCV and correlated the clinical course with the antiviral immune response. The individual HCV-specific T-cell response was assessed in a proliferation assay ((3)H-thymidine uptake) and an enzyme-linked immunospot assay. RESULTS: Patients were classified according to their clinical course and pattern of CD4(+) T-cell responses in 3 categories: first, patients mounting a strong and sustained antiviral CD4(+)/Th1(+) T-cell response who cleared the virus (HCV RNA-negative; n = 20); second, patients who were unable to mount an HCV-specific CD4(+) T-cell response and developed chronic disease (n = 12); and third, patients who initially displayed a strong CD4(+) T-cell response and eliminated the virus (HCV PCR-negative) but subsequently lost this specific T-cell response (n = 6). The loss of the HCV-specific CD4(+) T-cell response was promptly followed by HCV recurrence. CONCLUSIONS: The results indicate that a virus-specific CD4(+)/Th1(+) T-cell response that eliminates the virus during the acute phase of disease has to be maintained permanently to achieve long-term control of the virus. The induction and/or maintenance of virus-specific CD4(+) T cells could represent a promising therapeutic approach in HCV infection.     

Differential antigen specificity of hepatitis C virus-specific interleukin 17- and interferon γ-producing CD8(+) T cells during chronic infection

A subset of CD8(+) T cells can secrete interleukin 17 (IL-17). However, very little information is currently available about their antigen specificity, tissue distribution, and biological relevance in chronic human viral infection. To address these issues, we comprehensively analyzed peripheral and intrahepatic CD8(+) T-cell responses in a cohort of patients with chronic hepatitis C virus (HCV) infection for the antigen-specific production of IL-17 and interferon (IFN) γ. We found that HCV-specific IL-17-producing and retinoic acid receptor related orphan receptorγt-expressing CD8(+) T cells are detectable in blood and liver and target different epitopes, compared with IFN-γ-producing CD8(+) T cells. Their highest frequency was found in patients with low inflammatory activity, suggesting a protective role in chronic HCV infection.     

Influence of ethnicity in the outcome of hepatitis C virus infection and cellular immune response

This study was performed to examine the immunologic basis for the apparent ethnic difference in clinical outcome of hepatitis C virus (HCV) infection between African Americans (AA) and Caucasian Americans (CA). To this end, we recruited 99 chronically HCV-infected and 31 spontaneously HCV-cleared subjects for clinical, virologic, and immunologic analysis. In particular, CD4-proliferative T-cell response to genotype 1-derived HCV antigens (core, NS3-NS5) was examined in 82 patients chronically infected with genotype 1 (54 AA, 28 CA) and in all HCV-cleared subjects (14 AA, 17 CA). HCV-specific Th1 response also was examined in 52 chronic and 13 recovered subjects. Our results showed that HCV clearance was associated with a vigorous HCV-specific Th1 response irrespective of ethnic origin. Although the HCV-specific CD4 T-cell response clearly was weaker during chronic infection, AA ethnicity in this setting was associated with a significantly greater CD4-proliferative T-cell response to HCV, particularly to the nonstructural antigens (22% AA vs. 0% CA, P =.007) as well as better clinical parameters of liver disease. Interestingly, most HCV-specific CD4 T-cell proliferative responses in AA patients were unaccompanied by concurrent interferon gamma (IFN-gamma) production, suggesting a dysregulated virus-specific, CD4 T-cell effector function during chronic HCV infection. In conclusion, our results suggest that host ethnicity does influence the clinical outcome and antiviral T-cell response during HCV infection. AA ethnicity is associated with a more robust antiviral CD4 T-cell response than CA ethnicity, although these T cells are limited in direct virus or disease control due to their dysfunctional nature.     

Liver-derived hepatitis C virus (HCV)-specific CD4(+) T cells recognize multiple HCV epitopes and produce interferon gamma

Virus-specific CD4(+) T-cell response at the site of inflammation is believed to play a decisive role for the course of viral disease. In hepatitis C virus (HCV) infection, the majority of studies focused on the peripheral blood T-cell response. In this study we analyzed intrahepatic virus-specific CD4(+) T-cell response and compared this with that in the peripheral blood. Liver and blood-derived T-cell lines were studied in 36 patients (18 with chronic hepatitis C and 18 with HCV-associated cirrhosis). Virus-specific interferon gamma (IFN-gamma) production at a single cell level to various HCV-proteins (core, nonstructural [NS] 3/4, NS5) were determined by enzyme-linked immunospot (ELIspot). Phenotyping was done by fluorescent-activated cell sorter analysis. In approximately half (16 of 36 [44%]) of intrahepatic T-cell lines a significant number of IFN-gamma spots were observed, whereas this was the case in only 19% (7 of 36 T-cell lines) in the blood. In relative terms, core and nonstructural proteins were recognized with the same frequency in both compartments, but HCV-specificity was significantly more often detected in liver tissue compared with the blood. Hepatitis activity index, viral load, and alanine transaminase levels did not correlate with the detection of HCV-specific CD4(+) T cells. All T-cell lines were dominated by CD4(+) T cells. In conclusion, HCV-specific CD4(+) T cells are multispecific, compartmentalize to the liver, and produce IFN-gamma. We speculate that our data would support the concept of compartmentalization of specific T cells at the site of inflammation and that a low frequency of specific T cells is associated with failure to clear the virus and a chronic course of disease.     

Intrahepatic virus-specific IL-10-producing CD8 T cells prevent liver damage during chronic hepatitis C virus infection

CD8 T cell killing of hepatitis C virus (HCV)-infected hepatocytes is thought to contribute to liver damage during chronic HCV infection, whereas the participation of HCV-nonspecific immune cells is unclear. To visualize the spatial relationship of HCV-specific CD8 T cells with parenchymal target cells, and to examine their local functional activity in relation to hepatocellular necrosis and fibrosis, we used HLA tetramers and confocal microscopy in biopsies from 23 HLA-A2 or HLA-B7 patients with chronic HCV infection. Intrahepatic tetramer+ (HCV-specific) CD8 T cells protected from hepatic necroinflammatory disease activity, independently of age, gender, viral load, and viral genotype. Indeed, tetramer+ cells were scattered in the liver within regions of weak fibrosis (low laminin expression) and low hepatocellular apoptosis (TUNEL method), and expressed IL-10 but not IFNgamma. By contrast, tetramer-negative CD8 T cells were associated with active necroinflammatory liver disease, colocalized with strong laminin expression and hepatocellular apoptosis, and expressed more frequently IFNgamma than IL-10. Overall, liver regions harboring HCV-specific CD8 T cells tended to be healthier than areas containing only inflammatory cells of undefined specificity. In conclusion, HCV-specific IL-10-producing CD8 T cells, although not cytotoxic and unable to control viral replication, can attenuate hepatocellular necrosis, liver fibrosis, and inflammation mediated by bystander T cells, and may thus represent antigen-induced regulatory CD8 T cells. Therapeutic modulation of the intrahepatic balance between specific and bystander CD8 T cells might be beneficial in patients with chronic hepatitis C.     

Costimulatory molecule programmed death-1 in the cytotoxic response during chronic hepatitis C

Hepatitis C virus (HCV)-specific CD8~+ T cells play an important role in the resolution of HCV infection. Nevertheless, during chronic hepatitis C these cells lack their effector functions and fail to control the virus.HCV has developed several mechanisms to escape immune control. One of these strategies is the upregulation of negative co-stimulatory molecules such us programmed death-1 (PD-1). This molecule is upregulated on intrahepatic and peripheral HCV-specific cytotoxic T cells during acute and chronic phases of the disease, whereas PD-1 expression is low in resolved infection. PD-1 expressing HCV-specific CD8~+ T cells are exhausted with impairment of several effector mechanisms, such as: type-1 cytokine production, expansion ability after antigen encounter and cytotoxic ability. However, PD-1 associated exhaustion can be restored by blocking the interaction between PD-1 and its ligand (PD-L1). After this blockade, HCV-specific CD8~+ T cells reacquire their functionality. Nevertheless,functional restoration depends on PD-1 expression level.High PD-1-expressing intrahepatic HCV-specific CD8~+ T cells do not restore their effector abilities after PD-1/ PD-L1 blockade. The mechanisms by which HCV is able to induce PD-1 up-regulation to escape immune control are unknown. Persistent TCR stimulation by a high level of HCV antigens could favour early PD-1 induction, but the interaction between HCV core protein and gC1q receptor could also participate in this process. The PD-1/PD-L1 pathway modulation could be a therapeutic strategy, in conjunction with the regulation of others co-stimulatory pathways, in order to restore immune response against HCV to succeed in clearing the infection.     

Longitudinal analysis of CD8 T-cell responses to HIV and hepatitis C virus in a cohort of co-infected haemophiliacs

OBJECTIVE: To investigate CD8 T-cell responses to HIV and hepatitis C virus (HCV) over time in a group of co-infected children with haemophilia to assess the influence of the virus infections on each other and on clinical outcome. DESIGN: The HIV and HCV CD8 T-cell response of HLA-A2 co-infected individuals in the cohort were analysed at two time points, looking at the frequency and phenotype of HIV-specific T cells and assessing overall responses to the two viruses. METHODS: Peripheral blood mononuclear cells (PBMC) from 72 HLA-A2 co-infected individuals were analysed using an HIV HLA-A2 tetramer and by IFN-gamma ELISpot using a panel of HIV and HCV antigens. PBMC from a group of 26 HLA-A2 HIV mono-infected adults were also analysed as a comparison. RESULTS: We identified two distinct patterns of response: some patients had a limited response to either virus whilst others made responses to a range of HIV epitopes. HCV responses were detected only in those who made multiple responses to HIV epitopes (P<0.0001). HCV infection had an influence on the phenotype of HIV-specific CD8 T cells, with a reduction in relative perforin and CD57 expression. Lack of functional or tetramer-positive HIV-specific T cells was associated with a decline in absolute CD4 T-cell counts between the time points (up to 7 years; P = 0.005). CONCLUSION: HCV infection has an impact on the phenotype of HIV-specific CD8 T cells. In this well-defined cohort, failure to maintain effective CD8 T-cell responses against HIV may contribute to disease progression.     

Hepatitis C virus-specific CD4+ T cell response after liver transplantation occurs early, is multispecific, compartmentalizes to the liver, and does not correlate with recurrent disease

The role of hepatitis C virus (HCV)-specific CD4+ T cells in recurrent HCV infection after orthotopic liver transplantation (OLTx) is unclear. In parallel, 73 intrahepatic and 73 blood-derived T cell lines were established from 34 patients. At a single cell level, virus-specific interferon (IFN)-gamma production to various HCV proteins was determined by ELISPOT assay: 45 (62%) of 73 liver- or blood-derived T cell lines produced IFN-gamma in response to one of the HCV antigens. HCV specificity was detected mainly in the liver (47% vs. 23% in the blood; P<.05, chi(2) test) and was detectable earlier (< or =6 months) significantly more often than later (>6 months) after OLTx (78% vs 49%; P<.05, chi(2) test). Histology, histologic activity index, liver enzymes, and virus load did not correlate with the occurrence of HCV-specific CD4+ T cells. Despite strong immunosuppressive treatment, OLTx recipients can develop an early, multispecific, preferentially intrahepatic CD4+ T cell response that decreases over time, making it a potential candidate target for novel therapeutic approaches in the transplant setting.     

Oral lichen planus pathogenesis: A role for the HCV-specific cellular immune response

Hepatitis C virus infection can be associated with different extrahepatic manifestations, including lichen planus; however, no clear role for HCV in their pathogenesis has been established. T cells were isolated from lichen biopsy specimens of 7 HCV positive patients with oral lichen planus. HCV-specific CD4(+) T-cell lines were obtained in 4 patients from lichen lesions but only in 2 of them from the peripheral blood. Different clonal populations were found in oral tissue and peripheral blood of individual patients, as shown by TCR-Vbeta analysis of antigen-specific T cells. Frequency of HCV-specific CD8(+) cells tested with 4 different HCV tetramers was significantly higher in the lichen tissue than in the circulation; moreover, lichen-derived HCV-specific CD8(+) T cells showed the phenotype of recently activated T cells because most of them were CD69(+) and produced interferon gamma (IFN-gamma) but expanded poorly in vitro upon antigen stimulation. The specificity of HCV-reactive T-cell recruitment into the lichen tissue was further confirmed by the absence of HBV-specific T cells within lichen lesions in 3 additional patients with lichen planus associated with HBV infection. Our study shows HCV-specific T-cell responses at the site of the lesions of an HCV-associated dermatologic disease, sustained by HCV-specific T cells with phenotypic and functional characteristics of terminally differentiated effector cells. In conclusion, this finding and the detection of HCV RNA strands in the lichen tissue strongly suggest a role for HCV-specific T-cell responses in the pathogenesis of oral lichen planus associated with HCV infection.     

Magnitude of CD8+ T-cell responses against hepatitis C virus and severity of hepatitis do not necessarily determine outcomes in acute hepatitis C virus infection

Aim:  We investigated the relationship between the magnitude of comprehensive hepatitis C virus (HCV)-specific CD8⁺ T-cell responses and the clinical course of acute HCV infection.
Methods:  Six consecutive patients with acute HCV infection were studied. Analysis of HCV-specific CD8⁺ T-cell responses was performed using an interferon-γ-based enzyme-linked immunospot assay using peripheral CD8⁺ T-cells, monocytes and 297 20-mer synthetic peptides overlapping by 10 residues and spanning the entire HCV sequence of genotype 1b.
Results:  Five patients presented detectable HCV-specific CD8⁺ T-cell responses against a single and different peptide, whereas 1 patient showed responses against three different peptides. Neither the magnitude of HCV-specific CD8⁺ T-cell responses nor the severity of hepatitis predicts the outcome of acute hepatitis. The maximum number of HCV-specific CD8⁺ T-cells correlated with maximum serum alanine aminotransferase level during the course ( r  = 0.841, P  = 0.036).
Conclusions:  HCV-specific CD8⁺ T-cell responses were detectable in all 6 patients with acute HCV infection, and 6 novel HCV-specific CTL epitopes were identified. Acute HCV infection can resolve with detectable HCV-specific CD8⁺ T-cell responses, but without development of antibody against HCV.     

CD8+ cell responses to hepatitis C virus (HCV) in the liver of persons with HCV-HIV coinfection versus HCV monoinfection

OBJECTIVE: Cellular immune responses are difficult to detect in the peripheral blood of persons with chronic hepatitis C virus (HCV) infection. We sought to determine whether T cell responses were present in the liver of patients with human immunodeficiency virus (HIV) and HCV coinfection. METHODS: T cells were expanded from liver-biopsy samples from 10 patients coinfected with HIV and HCV (median CD4(+) cell count, 456 cells/mm(3)) and 8 patients infected with HCV alone. CD8(+) cell responses were detected by use of a modified enzyme-linked immunospot (ELISpot) assay with recombinant vaccinia virus, and CD4(+) cell responses were detected by use of ELISpot with recombinant HCV proteins core, nonstructural (NS) 3, and NS5. RESULTS: Intrahepatic CD8(+) cell responses to HCV were detected in 7 of 10 patients coinfected with HCV and HIV (median frequency, 638 spot-forming cells [sfc]/1 x 10(6) cells) and were similar to those observed in patients singly infected with HCV (7/8; median, 647 sfc/1 x 10(6) cells). Intrahepatic HCV-specific CD4(+) cell responses were also comparable in both groups and correlated with the intrahepatic CD8(+) cell responses (r=0.59; P=.03). CONCLUSION: HCV-specific CD8(+) cell responses are present in the liver of persons with chronic HCV infection even when they are coinfected with HIV; these correlate with intrahepatic HCV-specific CD4(+) cell responses.     

Transient immunological control during acute hepatitis C virus infection: ex vivo analysis of helper T-cell responses

Summary.  Hepatitis C virus (HCV) readily sets up persistence after acute infection. Cellular immune responses are thought to play a major role in control of the virus. Failure of CD4+ T-cell responses in acute disease is associated with viral persistence but the dynamics of this are poorly understood. We aimed to assess such responses using a novel set of Class II tetrameric complexes (tetramers) to study helper T-cells ex vivo in acute disease. We analysed the HCV-specific CD4+ T-cell response in a patient with acute hepatitis c infection. We were able to track the virus-specific CD4+ T-cells directly ex vivo with HLA DR4 tetramers. Proliferative responses were absent initially, recovered as viral load dropped and were lost again during relapse. Longitudinal tetramer analyses showed expanded populations of antiviral CD4+ T-cells throughout acute infection despite lack of proliferation. A pattern of transient CD4+ T-cell proliferative responses as HCV is partially controlled is observed. Failure to control virus is associated with emergence of 'dysfunctional' CD4+ T-cell populations. Failure to control HCV in acute disease may relate to the capacity to sustain efficient immune responses as virus attempts to 'bounce back' after partial control.     

Selection of high-avidity CD8 T cells correlates with control of hepatitis C virus infection

Both strong antigenic avidity and acquisition of proper effector functions contribute to the efficacy of antiviral T cell responses. To correlate these parameters with the outcome of hepatitis C virus (HCV) infection, we characterized HCV-specific CD8 T cell lines isolated after immunomagnetic sorting of peripheral blood mononuclear cells from human leukocyte antigen A*02 (HLA-A*02) individuals with various HCV serological statuses, using recombinant HLA-A*0201 multimers loaded with three immunodominant HCV genotype 1-derived epitopes. CD8 T cells specific for these three epitopes were derived from most HLA-A*0201 individuals, regardless of their HCV serology or clinical outcome. Donors recovered from genotype 1 HCV infection were enriched for high-avidity T cells with enhanced interferon gamma (IFN-gamma), tumor necrosis factor alpha, and cytotoxic T lymphocyte responses, when compared with seronegative donors and seropositive patients infected with irrelevant HCV genotypes. Patients chronically infected with genotype 1 strain yielded almost exclusively low-avidity T cells, whose hyporesponsiveness was primarily attributable to low T cell receptor (TCR) avidity rather than intrinsic functional defects. CONCLUSION: This study suggests that strong IFN-gamma responses associated with efficient viral clearance primarily result from Ag-driven selection/survival of HCV-specific T cells expressing high-avidity TCR. It also suggests a link between the quality of the initial HCV-specific T cell repertoire and susceptibility to chronic infection.