Ex vivo analysis of resident hepatic pro-inflammatory CD1d-reactive T cells and hepatocyte surface CD1d expression in hepatitis C.

Hepatic CD1d-restricted and natural killer T-cell populations are heterogeneous. Classical 'type 1' α-galactosylceramide-reactive CD1d-restricted T cells express 'invariant' TCRα ('iNKT'). iNKT dominating rodent liver are implicated in inflammation, including in hepatitis models. Low levels of iNKT are detected in human liver, decreased in subjects with chronic hepatitis C (CHC). However, high levels of human hepatic CD161(±) CD56(±) noninvariant pro-inflammatory CD1d-restricted 'type 2' T cells have been identified in vitro. Unlike rodents, healthy human hepatocytes only express trace and intracellular CD1d. Total hepatic CD1d appears to be increased in CHC and primary biliary cirrhosis. Direct ex vivo analysis of human intrahepatic lymphocytes (IHL), including matched ex vivo versus in vitro expanded IHL, demonstrated detectable noninvariant CD1d reactivity in substantial proportions of HCV-positive livers and significant fractions of HCV-negative livers. However, α-galactosylceramide-reactive iNKT were detected only relatively rarely. Liver CD1d-restricted IHL produced IFNγ, variable levels of IL-10 and modest levels of Th2 cytokines IL-4 and IL-13 ex vivo. In a novel FACS assay, a major fraction (10-20%) of hepatic T cells rapidly produced IFNγ and up-regulated activation marker CD69 in response to CD1d. As previously only shown with murine iNKT, noninvariant human CD1d-specific responses were also augmented by IL-12. Interestingly, CD1d was found selectively expressed on the surface of hepatocytes in CHC, but not those CHC subjects with history of alcohol usage or resolved CHC. In contrast to hepatic iNKT, noninvariant IFNγ-producing type 2 CD1d-reactive NKT cells are commonly detected in CHC, together with cognate ligand CD1d, implicating them in CHC liver damage.

Antigen-specific T lymphocyte proliferation decreases over time in advanced chronic hepatitis C.

To evaluate T cell immunity in advanced liver disease, antigen-specific lymphoproliferative (LP) responses were prospectively studied in the context of the Hepatitis C Antiviral Long-term Treatment against Cirrhosis trial. Peripheral blood responses to hepatitis C virus (HCV), tetanus and Candida protein antigens were measured at baseline, month 12 (M12), M24, M36 and M48 in 186 patients randomized to either low-dose peginterferon-alfa-2a (PEG-IFN) only or observation. Liver histology was evaluated at baseline, M24 and M48. Patients with cirrhosis (Ishak 5-6) were less likely to have positive LP responses to HCV at baseline than patients with fibrosis (15%vs 29%, P = 0.03) and had lower levels of HCV c100 responses at baseline, M24 and M48 (P = 0.11, P = 0.05, P = 0.02, respectively). For 97 patients with complete longitudinal data, the frequency of positive LP responses to HCV, tetanus and Candida antigens declined over time (P < 0.003), and the slope of this decline was greater in the PEG-IFN treatment group than the observation group (P < 0.02). Lower levels of tetanus LP responses were associated with fibrosis progression and clinical outcomes (P = 0.009). Poorer CD4+ T cell proliferative function was associated with more advanced liver disease in chronic hepatitis C and may be further affected by long-term PEG-IFN treatment.

Lower peripheral blood CD14+ monocyte frequency and higher CD34+ progenitor cell frequency are associated with HBV vaccine induced response in HIV infected individuals.

We evaluated immunologic predictors of response to HBV vaccine administered in the presence or absence of GM-CSF in HIV infected individuals. We measured peripheral blood hematopoietic progenitor, monocyte and myeloid-derived suppressor cell (MDSC) frequencies, and expression of GMCSF receptor on monocytes and MDSCs, at baseline and 4weeks after immunization in relation to antibody response. We observed higher baseline progenitor and lower monocyte frequencies among week 16 antibody responders. Week 4 decline in MDSC frequency was associated with week 16 antibody response, while administration of GM-CSF was associated with preservation of these cells. No significant differences in GM-CSF receptor expression were observed in the presence vs. absence of GM-CSF. These findings are consistent with a positive role of progenitor cells and a potential negative role of monocytes in vaccine response. Additionally, GM-CSF augmented the preservation of peripheral blood MDSC, which may contribute to the lack of improved vaccine responses.

Immune response to hepatitis B vaccine in HIV-infected subjects using granulocyte-macrophage colony-stimulating factor (GM-CSF) as a vaccine adjuvant: ACTG study 5220.

HIV-infected persons are at risk for HBV co-infection which is associated with increased morbidity and mortality. Unfortunately, protective immunity following HBV vaccination in HIV-infected persons is poor. This randomized, phase II, open-label study aimed to evaluate efficacy and safety of 40 mcg HBV vaccine with or without 250 mcg GM-CSF administered at day 0, weeks 4 and 12. HIV-infected individuals >or=18 years of age, CD4 count >or=200 cells/mm(3), seronegative for HBV and HCV, and naïve to HBV vaccination were eligible. Primary endpoints were quantitative HBsAb titers and adverse events. The study enrolled 48 subjects. Median age and baseline CD4 were 41 years and 446 cells/mm(3), 37 were on ART, and 26 subjects had undetectable VL. Vaccination was well tolerated. Seven subjects in the GM-CSF arm reported transient grade >or=2 signs/symptoms (six grade 2, one grade 3), mostly aches and nausea. GM-CSF had no significant effect on VL or CD4. Four weeks after vaccination, 26 subjects (59%) developed a protective antibody response (HBsAb >or=10 mIU/mL; 52% in the GM-CSF arm and 65% in the control arm) without improved Ab titer in the GM-CSF vs. control arm (median 11 mIU/mL vs. 92 mIU/mL, respectively). Response was more frequent in those with CD4 >or=350 cells/mm(3) (64%) than with CD4 <350 cells/mm(3) (50%), though not statistically significant. GM-CSF as an adjuvant did not improve the Ab titer or the development of protective immunity to HBV vaccination in those receiving an accelerated vaccine schedule. Given the common routes of transmission for HIV and HBV, additional HBV vaccine research is warranted.

Immunopathogenesis of hepatitis C virus in the immunosuppressed host.

The prevalence of chronic hepatitis C virus (HCV) infection among various groups of immunosuppressed patients is high. These groups include patients co-infected with human immunodeficiency virus (HIV), recipients of organ transplants, and those with hypogammaglobulinemia. The liver disease in the immunosuppressed host is typically severe with an unusually rapid progression to cirrhosis. This is somewhat paradoxical, as the classical model for HCV-induced liver disease assumes that cell-mediated immune responses induce liver injury. It is likely that a combination of viral-related factors and host-related factors plays a role in this accelerated natural history of HCV. Data are accumulating in immunocompromised hosts that address the immunopathogenesis of liver injury, although there are still fundamental gaps in our understanding of this process. In this review, we will focus on our current understanding of the mechanisms of liver injury and how it relates to the accelerated liver disease progression in immunocompromised hosts.

Influence of human immunodeficiency virus infection on the course of hepatitis C virus infection: a meta-analysis.

Studies have shown that rates of liver disease are higher in persons who are coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) than they are in persons with HCV alone, but estimates of risk vary widely and are based on data for dissimilar patient populations. We performed a meta-analysis to quantify the effect of HIV coinfection on progressive liver disease in persons with HCV. Eight studies were identified that included outcomes of histological cirrhosis or decompensated liver disease. These studies yielded a combined adjusted relative risk (RR) of 2.92 (95% confidence interval [CI], 1.70-5.01). Of note, studies that examined decompensated liver disease had a combined RR of 6.14 (95% CI, 2.86-13.20), whereas studies that examined histological cirrhosis had a pooled RR of 2.07 (95% CI, 1.40-3.07). There is a significantly elevated RR of severe liver disease in persons who are coinfected with HIV and HCV. This has important implications for timely diagnosis and consideration of treatment in coinfected persons.

Immunology of hepatitis C virus infection.

Hepatitis C virus (HCV) is known for its ability to establish persistent infection and cause chronic hepatitis in most infected individuals. The pathogenesis of hepatic injury and the precise mechanisms underlying viral persistence are unknown. Accumulating evidence indicates that successful elimination of HCV is associated with the induction and maintaining of strong helper T-cell and cytotoxic T-cell responses against multiple viral epitopes. In contrast, patients who develop chronic HCV infection are characterized by the lack of strong viral-specific helper T-cell responses. The failure to mount and maintain strong HCV-specific T-cell responses may be determined by the genetics, especially the major histocompatibility complex background, of the host. However, it is likely that other host and viral factors are also involved in determining the outcome of HCV infection. Available data suggest that HCV is not cytopathic to hepatocytes and that liver injury associated with chronic HCV infection is likely to be mediated by immune responses against HCV-infected hepatocytes. In addition to hepatitis, HCV infection may also cause breaching of immune tolerance, leading to autoimmune disorders. Although the lack of a small animal model and a tissue culture system has impeded research on hepatitis C virus (HCV) infection, recent studies in humans and chimpanzees have significantly enhanced our understanding of the interaction between HCV and the host's immune system. This review focuses on the most recent advances in our understanding of the immunology of HCV infection. In particular, the possible mechanisms of how HCV establishes chronic infection are discussed. The pathogenesis of liver injury, the immunogenetics of HCV infection, and the effect of HCV infection on host's immune function are also reviewed.

Association of multispecific CD4(+) response to hepatitis C and severity of recurrence after liver transplantation.

BACKGROUND & AIMS: After liver transplantation for hepatitis C virus (HCV), reinfection of the allograft invariably occurs. Indirect evidence suggests that the cellular immune response may play a central role. The purpose of this analysis was to determine the correlation between HCV-specific peripheral CD4(+) T-cell responses and the severity of recurrence after liver transplantation. METHODS: Fifty-eight HCV-seropositive patients, including 43 liver transplant recipients with at least 1 year of histological follow-up, were studied. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh heparinized blood and stimulated with either recombinant HCV antigens (core, E2, NS3, NS4, and NS5) or control antigens. RESULTS: Fourteen (40%) of 35 patients with mild or no evidence of histological recurrence within their allografts responded to at least 1 of the HCV antigens. Eleven responded to NS3, 5 to all the nonstructural antigens, and 3 to the HCV core polypeptide alone. In contrast, in the 8 patients with severe HCV recurrence, no proliferation in response to any of the HCV antigens was seen (P = 0. 03) despite responses to the control antigens. CONCLUSIONS: Despite immunosuppression, HCV-specific, major histocompatibility complex class II- restricted CD4(+) T-cell responses are detectable in patients with minimal histological recurrence after liver transplantation. In contrast, PBMCs from patients with severe HCV recurrence, despite being able to proliferate in response to non-HCV antigens, fail to respond to the HCV antigens. These findings suggest that the inability to generate virus-specific T-cell responses plays a contributory role in the pathogenesis of HCV-related graft injury after liver transplantation. It is hoped that further characterization of the immunoregulatory mechanisms related to recurrent HCV will provide the rationale for novel therapeutic strategies and diminish the incidence of inevitable graft loss.

Cytokines in viral hepatitis.

Cytokines play an important role in the defense against viral infections, both indirectly, through determination of the predominant pattern of host response, and directly, through inhibition of viral replication. However, in the context of an inflammatory response against a virus, cytokines may also lead to liver damage. The importance of this is best demonstrated in hepatitis B virus (HBV). In acute HBV infection, a vigorous polyclonal cellular immune response is critical; thus type 1 cytokine release is essential to initiating an effective immune response. The cytokines released by CD4+ and CD8+ cells also play an important role in downregulation of HBV replication, demonstrating that it is possible to control a viral infection without the death of infected cells. However, if there is a defect in the acute response, HBV becomes chronic; in that case, the presence of an ongoing suboptimal inflammatory response can activate the process of hepatic fibrosis. In hepatitis C infection, the role of cellular immune responses and cytokines is less clear. Hepatitis C may be resistant to inhibition by cytokines, so cytokines may have a more prominent role in liver damage than in controlling viral replication. Both hepatitis B and C may have specific mechanisms to inhibit cytokine production, highlighting the critical role of these molecules in recovery from infection.

Hepatitis C virus genotypes and viremia and hepatocellular carcinoma in the United States.

OBJECTIVE: Hepatitis C virus (HCV) is a well recognized cause of hepatocellular carcinoma (HCC). The pathogenic significance of HCV genotypes in hepatocarcinogenesis is undefined. The aim of this study was to investigate the genotypic distribution and viremic level of HCV in patients with HCV-associated cirrhosis with or without HCC. METHODS: A total of 28 HCV-infected patients with HCC (HCC+) and 38 patients with HCV-associated cirrhosis without HCC (HCC-) were studied. HCV genotype was assessed by the genotype-specific polymerase chain reaction (PCR) method of Okamoto and restriction fragment length polymorphism (RFLP) of the 5' untranslated region (5' UTR). Hepatitis C viremia was quantitated with the branched-chain DNA (bDNA) assay. RESULTS: Using the Okamoto method, we found genotype 1b in 64% of the HCC+ group and 74% of the HCC- group, 36% of the HCC+ group and 16% of the HCC- group were coinfected with a combination of genotype 1b and another genotype. Using the RFLP method, we found genotype 1b in 41% of the HCC+ group and in 24% of the HCC- group. Other genotypes accounted for 18% of the HCC+ group and 55% of the HCC- group; no combination genotypes were identified. Poor concordance occurred between the two genotyping methods. Mean bDNA levels were not significantly different between the two groups. CONCLUSIONS: Our study demonstrates that no particular HCV genotypes were associated with HCC and genotype did not appear to influence the development of HCV-associated HCC.

Characterization of HCV-Specific Cytotoxic T-Lymphocytes from Liver Tissue.

Cellular immune responses, especially those mediated by cytotoxic T-lymphocytes (CTLs), are an important component of the host immune response in many viral infections. For many years, it has been observed that CD8(+) cells were present in large numbers in the liver of patients with chronic HCV (1), but it was unknown whether these cells represented virus-specific immune responses. In order to understand the potential role of these CD8(+) lymphocytes in the disease course, it is first necessary to define the functional characteristics of the cells, including a precise definition of the epitopes, which are recognized by these CD8(+) lymphocytes.

Liver-derived CTL in hepatitis C virus infection: breadth and specificity of responses in a cohort of persons with chronic infection.

Hepatitis C virus (HCV)-specific CTL have been found within the inflammatory infiltrate of the liver of chronically infected individuals, but the breadth and specificity of the CTL response in relation to viral load are less well characterized. In this study, we analyzed the intrahepatic CTL response in liver biopsy specimens from 44 chronically infected subjects. Liver-infiltrating lymphocytes were expanded polyclonally in bulk cultures, and multiple clones were derived by limiting dilution. HCV-specific CTL responses directed at genotype 1a structural proteins were assessed in all subjects, and 22 subjects were tested more comprehensively using vectors expressing all structural and nonstructural HCV Ags. CTL responses were further characterized to determine the HLA restriction and optimal epitopes recognized. In those persons screened for recognition of all HCV Ags, HLA class I-restricted CTL were detected in 45%. Nineteen different CTL epitopes were identified, which were distributed throughout the genome; only one epitope was targeted by more than one person. In those persons with CTL responses, the breadth of response ranged from one to five epitopes. There was no correlation between the presence of a detectable CTL response and viral load. These results indicate considerable heterogeneity in detectable HCV-specific CTL responses in chronically infected persons. The mechanisms by which HCV persists during chronic infection remain to be clarified.

The immunopathogenesis of HBV infection.

Clinical manifestations of hepatitis B virus (HBV) infection are a balance between viral and host factors. The immune response against any virus consists of a coordinated defence of innate immunity and acquired, virus-specific immunity. In acute HBV, immune responses associated with recovery include vigorous, polyclonal CD4 T cells directed against multiple epitopes within HBV; antibodies directed against surface envelope proteins (anti-HBs), the development of which requires the presence of a CD4 response; and HBV-specific cytotoxic T lymphocytes (CTLs). HBV-specific CTLs can induce death of infected hepatocytes as well as produce cytokines. Most individuals with acute HBV recover without evidence of massive liver destruction; this, plus evidence from transgenic animal models, suggests that these cytokines produced by T cells play an important role in controlling HBV replication. Individuals who fail to mount a vigorous response in acute HBV develop chronic infection. In these cases, the persisting ineffective immune response appears to be responsible for liver damage and is likely to initiate the process of hepatic fibrosis. Based on our current understanding of the immune response in acute and chronic HBV, several groups are investigating the prospect of manipulating the immune response in chronic HBV.

Hepatitis C virus-specific cytolytic T lymphocyte and T helper cell responses in seronegative persons.

Hepatitis C virus (HCV) is a common infection worldwide, and in most persons, it leads to persistent viremia and liver damage. Efforts to identify the correlates of protective immunity are hampered by this high rate of persistent infection in both infected humans and the only animal model, the chimpanzee. Peripheral blood mononuclear cells from seronegative persons were stimulated with synthetic peptides that represent epitopes recognized by HCV-specific cytotoxic T lymphocytes (CTL) after natural infection. In addition, CD4+ proliferative responses to recombinant HCV proteins were examined in these same persons. CTL responses directed against a peptide epitope of HCV and proliferative responses in 2 HCV-seronegative persons with possible occupational exposure to HCV were found. These otherwise healthy persons were not viremic, suggesting that they may have recovered from acute HCV infection. Characterization of virus-specific immune responses in exposed but seronegative persons may provide important clues as to the nature of protective immunity in HCV.

Transgenic expression of hepatitis C virus structural proteins in the mouse.

Although hepatitis C virus (HCV) is a leading cause of morbidity and mortality worldwide, the role of viral cytopathic effects remains unclear. To study the biosynthesis of HCV structural proteins and their pathogenic role, we constructed transgenic mice, expressing type 1b HCV structural proteins (core, E1, and E2) in liver tissues. Two liver-specific promoters were used. The mouse major urinary protein (MUP) promoter has been shown to be developmentally regulated with little or no expression in utero but high-level expression after birth. The albumin (Alb) promoter provides constitutive, high levels of transgenes in live. Expression of both HCV transgenes was detected in several lines by Northern blots, HCV-specific reverse transcriptase-polymerase chain reactions (RT-PCR), and Western immunoblotting. Alb HCV lines showed higher levels of HCV expression than the MUP HCV lines. Immunohistochemical analysis revealed a predominantly cytoplasmic presence of core protein with occasional nuclear staining, and both cytoplasmic and membrane expression of the E2 protein in the transgenic livers. In both transgenes, the highest levels of both antigens were seen in perivenular hepatocytes, suggesting potential processing specificity in those cells. At six months of age, the livers of all transgenic lineages remained histologically normal. We concluded that HCV structural proteins are not directly cytopathic in this animal model.

Characteristics of the intrahepatic cytotoxic T lymphocyte response in chronic hepatitis C virus infection.

Based on our CTL studies of over 44 persons with chronic HCV infection, we are able to arrive at a number of conclusions. Clearly this cellular immune response is heterogeneous among infected persons. We have not identified any specific HCV protein which appears to be immunodominant for CTL responses, but rather we have detected diverse responses to both structural and non-structural proteins. Using an identical stimulation strategy for all persons studied, we have been able to detect responses in only approximately one third of persons with chronic infection. Among these persons, the responses among liver-infiltrating lymphocytes are greater than those detected in fresh peripheral blood, suggesting that the CTL are homing to the site of maximal viral burden in these persons. Some viral proteins contain overlapping epitopes presented by more than one HLA class I molecule, and we have also found cases where peptides in the same HLA superfamily, such as the HLA A3 superfamily which contains A11, for which the same peptide can be presented by both alleles (manuscript in preparation). Although sequence variation between the infecting strain and the vaccinia constructs used to test for responses may lead to non-recognition of some variants, even the highly conserved core protein appears to be an inconsistent and actually infrequent target for detectable CTL responses. The magnitude of the CTL response appears to vary greatly, from being undetectable to being so vigorous that it an be detected in stimulated peripheral blood. The breadth of the response also varies widely, ranging from the detection of a response to a single epitope in some persons, to the simultaneous recognition of up to five different epitopes in others. Even in persons of the same HLA type, we have not seen consistent targeting of the same epitopes except in rare cases. Despite the detection of over 20 epitopes and their restricting class I alleles using CTR derived from liver-infiltrating lymphocytes, we have identified only one epitope that has been shown to be targeted by more than one person of the same HLA type. These findings lead us to speculate that the CTL response may be submaximal in the majority of infected persons. The reasons for this are presently obscure, but could relate to a number of factors. The epitopes targeted are found within variable regions of the virus, such that immune escape from established CTL responses has to be considered a real possibility. Sequence variation may also lead to antagonism of CTL responses, as has been demonstrated for both HIV and HBV infections. Furthermore, sequence variation either within or adjacent to regions containing CTL epitopes can lead to altered antigen processing, either due to alteration of proteolytic processing of the viral peptides in the cytoplasm or to altered transport and altered association with class I molecules. A number of issues regarding the CTL response in HCV infection still require substantial attention. The apparent inability of CTL to clear this virus needs to be addressed, as does the potential role for viral immunomodulatory molecules in HCV persistence. Although we and others have shown CTL responses to be present in persons with chronic infection, the role of CTL in acute HCV infection needs to be determined. The best studied chronic human viral infection is HIV infection, in which expanding data indicate that the early events following primary infection predict the subsequent course of illness. Viral load in the first 1-2 years after infection is highly predictive of the subsequent disease course in HIV infection, and recent experimental data in humans suggest that early immune responses may be predictive of subsequent disease course. Such studies in HCV infection have been difficult to achieve, since primary HCV infection is often asymptomatic, and transfusion-related cases are now rare. (ABSTRACT TRUNCATED)

The role of immune responses in the pathogenesis of hepatitis C virus infection.

Hepatitis C virus (HCV) is notable for the high rate of chronic infection, which occurs in nearly all individuals who become infected. Liver biopsies from individuals with chronic HCV infection are notable for the presence of numerous mononuclear cells, at least some of which are CD4+ and CD8+ T lymphocytes. The immune response to HCV is polyclonal and multispecific, both in terms of antibody and cellular immune responses. Individuals who recover from acute HCV infection appear to have quantitatively more vigorous CD4+ proliferative responses against one or more HCV proteins compared with those individuals who develop chronic disease. CD8+ responses are less well characterized, in part because of the technical difficulties involved in isolating and characterizing these cells. HCV-specific CTL can be readily isolated from the liver and PBMC of chronically infected individuals, and recognize multiple epitopes. Even individuals with the same HLA type do not consistently recognize the same epitope. Thus, there does not appear to be an immunodominant response on the CD8+ level in this infection. CD8+ cells do appear to play some role in limiting viral replication. These responses are insufficient to eradicate virus completely, however, and may cause liver injury once chronic infection is established. Cytokines produced by both CD4+ and CD8+ cells may play an important role in both inhibiting viral replication and causing liver injury. A better understanding of the role of cellular immunity in the pathogenesis of HCV infection may aid in the development of vaccines and immunotherapeutic intervention strategies.