Chronic hepatitis B virus and hepatitis C virus infections and cancer: synergy between viral and host factors.

Hepatitis B virus (HBV) or hepatitis C virus (HCV) infections represent major causes of chronic liver disease and hepatocellular carcinoma. Despite inducing shared pathological events leading to oncogenic transformation, these two viruses present profound differences in their molecular features, life cycle and interplay with host factors, which significantly differentiate the prognostic and therapeutic approach to the related diseases. In the present review, we report the main mechanisms involved in the multistep process leading from HCV/HBV infection and cancer development, discussing side-by-side the analogies and differences between the two viruses. Such events can be broadly categorized into (a) direct oncogenic effects, involving integration in the host genome (in the case of HBV) and chromosomal instability, interference with oncosuppressor pathways, induction of oxidative stress, promotion of angiogenesis, epithelial-mesenchymal transition, alterations in the epigenetic asset and interaction with non-coding RNAs; and (b) indirect activities mostly mediated by host events, including chronic inflammation sustained by peculiar cytokine networks (such as interleukin-6 and lymphotoxins), metabolic dysfunctions promoted by steatohepatitis, interplay with gut microbiota and fibrotic events (mainly in HCV infection). This scenario suggests that the integrated study of viral and host factors may lead to the successful development of novel biomarkers and targets for therapy.

Non-covalent presentation of sulfamethoxazole to human CD4+ T cells is independent of distinct human leucocyte antigen-bound peptides.

BACKGROUND: It has been shown that drugs comprise a group of non-peptide antigens that can be recognized by human T cells in the context of HLA class II and that this recognition is involved in allergic reactions. Recent studies have demonstrated a MHC-restricted but processing- and metabolism-independent pathway for the presentation of allergenic drugs such as lidocaine and sulfamethoxazole (SMX) to drug-specific T cells. However, there is little information so far on the precise molecular mechanisms of this non-covalent drug presentation. OBJECTIVE: The aim of this study was to evaluate the requirements for a specific peptide occupying the groove of the MHC class II molecule for the efficient presentation of non-covalently bound drugs to CD4+ T cells. METHODS: We analysed the effect of coincubation or prepulse of antigen presenting cells (APC) with different peptides on the proliferative responses of SMX-specific CD4+ T cell clones. In a second series of experiments, we eluted HLA-bound peptides from the surface of antigen presenting cells by mild acid treatment. Successful removal of peptides was tested directly using labelled peptides and functionally by monitoring activation and proliferation of peptide-specific T cell clones. Finally, the presentation of SMX to SMX-specific T cell clones before and after elution of MHC class II bound peptides was tested. RESULTS: We found that neither peptide coincubation nor peptide prepulse of APC altered the proliferative response of SMX-specific T cells. APC treated with the acid for a short time retained cell viability, MHC class II expression and antigen presenting cell function. However, defined peptides could be eluted from surface MHC class II molecules nearly quantitatively. Nevertheless, the chemically non-reactive drug SMX could still be presented to specific T cells independent of the presence of distinct self-peptides. CONCLUSION: Our data suggest that small molecules like drugs can bind to a multitude of HLA-bound peptides or that, similar to superantigens, they might bind directly to HLA.

The immunology of HIV-infected long-term non-progressors--a current view.

Long-term non-progressors (LTNP) are human immunodeficiency virus (HIV)-infected individuals characterized by the absence of disease, low viral loads and stable or even increasing CD4(+) T cell counts for prolonged periods of time. In these subjects, an HIV-specific immune response which is either stronger or directed against a wider array of viral epitopes than that seen in progressors, can be often detected. Here, we summarize the characteristics of HIV-specific CD4(+) and CD8(+) T cell responses in LTNP, and discuss how a highly effective T cell-mediated immune response against HIV might contribute to the establishment of this particular condition.

Ex vivo host response to gastrointestinal cancer cells presented by autologous dendritic cells.

BACKGROUND: Dendritic cells (DCs) capture apoptotic tumors and cross-present their antigens in the MHC class I and class II pathways for recognition by CD4+ and CD8+ T lymphocytes. Here we have tested the ability of fresh surgically resected colon and gastric cancer tumors to specifically activate host T lymphocytes when presented by autologous DCs. METHODS: DCs derived from adherent blood mononuclear cells of five patients, after a 7-day culture with GM-CSF and IL-4, were exposed to apoptotic autologous tumor (AAT) or apoptotic autologous peritumor normal (AAN) cells and cultured 24 h with monocyte-conditioned medium to achieve full DC maturation. Tumor-specific response was evaluated as single-cell cytokine release in an enzyme-linked immunospot (ELISPOT) and as cytotoxicity in a cold target inhibition (51)Cr-release assay. RESULTS: AAT-DCs induced specific IFN-gamma by T lymphocytes of two patients (rectal and gastric cancer), whereas in another two patients (rectal and gastric cancer) this response was depressed with a similar tumor-specific pattern and in one patient (rectal cancer) there was no response. Activation of IFN-gamma release was accompanied by tumor cytotoxicity and both responses were enhanced by IL-12, indicating the functional integrity of patients' lymphocytes. CONCLUSION: These data show that T-cell memory against rectal/gastric carcinoma antigens can be triggered by tumor-loaded autologous DCs. However, escape mechanisms may exist among tumors of the same histological origin that can inhibit this host response. A DC-based antitumor immunological monitoring assay with autologous tumor biopsies may allow patients to be screened to determine those who are suitable candidates for immune-based immunotherapy.

Apoptotic cells overexpress vinculin and induce vinculin-specific cytotoxic T-cell cross-priming.

Here we show that apoptotic cells overexpress vinculin and are ingested by dendritic cells, which subsequently cross-prime vinculin-specific cytotoxic T lymphocytes (CTLs). Successful cross-priming requires that the apoptotic cells provide maturation signals to dendritic cells through CD40-CD40 ligand (CD40L) interactions. If apoptotic cells are CD40L-, the help of a third T cell is needed for priming, indicating a regulatory role for apoptotic cells in determining priming or tolerance. Vinculin-specific CTL priming is also related to apoptosis in vivo, given that in HIV-seropositive individuals, the frequency of specific CTLs depends on the proportion of peripheral CD40L+ apoptotic cells.

Spreading of HIV-specific CD8+ T-cell repertoire in long-term nonprogressors and its role in the control of viral load and disease activity.

Long-term non-progressors (LTNP) represent a minority of human immunodeficiency virus (HIV) infected individuals characterized by stable or even increasing CD4+ T-cell count and by stronger immune responses against HIV than progressors. In this study, HIV-specific effector CD8+ T cells, as detected by both a sensitive ex vivo enzyme-linked immunospot (ELISPOT) assay and specific major histocompatibility complex (MHC) peptide tetramers, were at a low frequency in the peripheral blood of LTNP, and recognized a lower number of HIV peptides than their memory resting cell counterparts. Both factors may account for the lack of complete HIV clearance by LTNP, who could control the viral spread, and displayed a higher magnitude of cytotoxic T lymphocyte (CTL) responses than progressors. By combining cell purification and ELISPOT assays this study demonstrates that both effector and memory resting cells were confined to a CD8+ population with memory CD45RO+ phenotype, with the former being CD28- and the latter CD28+. Longitudinal studies highlighted a relatively stable HIV-specific effector repertoire, viremia, and CD4+ T-cell counts, which were all correlated with maintenance of nonprogressor status. In conclusion, the analysis of HIV-specific cellular responses in these individuals may help define clear correlates of protective immunity in HIV infection.

Virus-specific CD8(+) T cells with type 1 or type 2 cytokine profile are related to different disease activity in chronic hepatitis C virus infection.

The present study demonstrates that the quality of the virus-specific CD8(+) T cell responses, as detected by both enzyme-linked immunospot assay and specific MHC-peptide tetramers, changed in relation to the different disease activity in chronically hepatitis C virus-infected patients. Indeed, both the serum alanine transaminase and the hepatic flogosis levels were related directly to the frequencies of peripheral memory effector CD8(+) T cells producing IFN-gamma (Tc1), but inversely to the frequencies of those producing both IL-4 and IL-10 (Tc2). Longitudinal studies highlighted that Tc1 or Tc2 responses fluctuate in relation to the different phases of the disease in the same individual. Furthermore, the Tc1 or Tc2 phenotype correlates with tetramer-positive cells expressing either CXCR3 or CCR3, promoting differential tissue localization of these cells and the maintenance of T cell homeostasis. Finally, studies at the level of liver-infiltrating lymphocytes indicated that they produced both IFN-gamma and IL-4 with an evident bias towards the Tc1-like phenotype. Our studies suggest that the progressive fluctuation of Tc1 and Tc2 responses may play a fundamental role in maintaining a long-lasting low-level liver inflammation, and may constitute the basis for new therapeutic strategies of immune regulation.

Persisting viruses and autoimmunity.

Viral infections can be responsible for the onset and sustaining of autoimmune processes. We discuss how chronic inflammation associated with viral persistence is the prerequisite for initiation of a multi-step process leading to autoimmunity. Firstly, chronic inflammation may favor the priming of autoreactive T cells that have escaped thymic selection and are specific for self-mimicking viral peptides in the periphery. In addition, viral persistence and inflammation can act synergistically to induce and sustain autoimmunity either unveiling cryptic self-epitopes, or favoring determinant spreading, or activating dendritic cells, or promoting constant priming of new autoreactive T cells, or contributing to the efficient generation of effector cells, or, finally, restimulating memory T lymphocytes.

Short-lived immunization site inflammation in self-limited active experimental allergic encephalomyelitis.

To understand the mechanisms underlying spontaneous remission of proteolipid protein (PLP) 139-151 peptide-induced experimental allergic encephalomyelitis (EAE), an acute autoimmune disease of SJL mice resembling human multiple sclerosis, we examined both the effector response site in the central nervous system (CNS) and the immunization site at different phases of the disease. In the CNS, the frequency of PLP 139-151 peptide-specific IFN-gamma-producing T cells as well as the amount of infiltrating CD4(+) and CD11b(+) cells decreased with recovery. However, IL-4-producing cells were always rare and cyclooxygenase-2(+) cells were numerous only at disease peak in the CNS, suggesting that T(h)2 cytokines and prostaglandins did not determine remission of EAE. By looking at the s.c. site of PLP 139-151 peptide plus adjuvant injection, we found that, although the inflammatory infiltrate was abundant, CD11b(+) cells started to decrease already during disease acute phase and DEC-205(+) cells were numerous only at early time points. We propose that immunization site inflammation is short-lived in PLP 139-151 peptide-induced EAE, and this leads to a temporary autoreactive T cell stimulation and to a self-limited disease.

Immunoglobulin A nephropathy complicating pulmonary tuberculosis.

A 31-year-old man who presented with smear- and culture-negative pulmonary tuberculosis had associated macroscopic hematuria, elevation of serum creatinine and immunoglobulin A (IgA) levels, overt proteinuria, and peripheral edema. Renal biopsy revealed focal mesangial proliferation with IgA deposits, and a diagnosis of IgA nephropathy was made. The patient received treatment with isoniazide and rifampin. After 4 months, pulmonary lesions were almost completely healed, and a significant improvement of creatinine clearance with normalization of serum creatinine and IgA levels and disappearance of proteinuria were observed. Treatment with isoniazide and rifampin was discontinued after 6 months, without reappearance of either pulmonary or renal symptoms. Two years after the diagnosis of IgA nephropathy, the patient is in good general condition. Serum creatinine and IgA levels are normal, proteinuria is absent, and there is neither macrohematuria nor microhematuria. These findings suggest that IgA nephropathy may be a consequence of tuberculosis, possibly due to an abnormal IgA-mediated immune response against Mycobacterium tuberculosis with formation of nephrotoxic immune complexes.

Presence of effector CD8+ T cells in hepatitis C virus-exposed healthy seronegative donors.

CTL responses against multiple hepatitis C virus (HCV) epitopes were detected in 7 of 29 (24.1%) healthy family members (HFM) persistently exposed to chronically HCV-infected patients (HCV-HFM). These precursor CTL were at very low or undetectable frequencies, as determined by limiting dilution analysis. However, when HCV-specific effector CD8+ T cells, freshly isolated from PBMC of HCV-HFM, were assessed by a sensitive enzyme-linked immunospot assay, their frequencies were severalfold higher than those of precursor CTL. These results indicate that the two assays detect two functionally distinct T cell populations and that the effector cells are not assayed by the 51Cr-release assay. Furthermore, the combination of cell depletion and enzyme-linked immunospot analyses showed that the effector cells were confined into a CD8+ CD45RO+ CD28- population. The persistence of effector CD8+ T cells specific for both the structural and nonstructural viral proteins in uninfected HCV-HFM, suggest that: 1) an immunological memory is established upon a subclinical infection without any evidence of hepatitis, in a large cohort of HCV-exposed individuals; 2) because these cells required neither restimulation nor the addition of particular cytokines in vitro for differentiating in effectors, they should be capable of prompt HCV-specific effector function in vivo, possibly providing antiviral protection; and 3) the maintenance of effector T cell responses may be sustained by persisting low-level stimulation induced by inapparent infections.

Dynamics of intra-hepatic lymphocytes in chronic hepatitis C: enrichment for Valpha24+ T cells and rapid elimination of effector cells by apoptosis.

Chronic viral hepatitis is characterized by a dramatic lymphocyte infiltrate in the liver. Although it is one of the most common chronic inflammatory diseases in humans, little information is available on the functional state of these intra-hepatic lymphocytes (IHL). To address this issue, we have optimized cytofluorimetric techniques to assess directly ex vivo the functions, dynamics and repertoires of IHL isolated from biopsies of patients with chronic hepatitis C. We estimate that 1% of the total body lymphocytes infiltrate the inflamed liver and find that, at variance with peripheral blood lymphocytes (PBL) isolated from the same patients, most IHL display an activated phenotype and produce Th1 type lymphokines when stimulated in vitro. Virtually all IHL are found in the G0/G1 state of the cell cycle, while a sizeable percentage of them is undergoing programmed cell death in vivo, as detected by the TUNEL assay performed on freshly isolated cells. In contrast again to PBL from the same patients, IHL show a preferential compartmentalization of NK and TCRgamma/delta+ cells, and a remarkable (up to 20-fold) enrichment for Valpha24+ T cells. Together our data suggest that in a liver injured by chronic hepatitis C, most IHL are pro-inflammatory activated cells which are highly enriched for effectors of innate resistance. These IHL do not undergo clonal expansion in the liver but rather display effector function and die in situ at a high rate, suggesting that maintenance of the IHL pool is dependent on continuous migration from extra-hepatic sites.

Defective Th1 and Th2 cytokine synthesis in the T-T cell presentation model for lack of CD40/CD40 ligand interaction.

In this study, T or NK cell clones used as antigen-presenting cells (T- or NK-APC) were shown to be significantly less efficient than professional APC in inducing Th1 and Th2 cytokines by antigen-specific T cell clones. This phenomenon was not related to a limited engagement of TCR by T-APC, since comparable thresholds of TCR down-regulation were shown when antigen was presented by either T-APC or professional APC. Rather, the stimulatory T-APC weakness was due to their inability, because they are CD40-, to provide the appropriate co-stimuli to responder T cells both indirectly via IL-12, and partially via direct CD40L triggering on T cells. Indeed, the simultaneous addition of IL-12 and reagents directly engaging CD40L on responder T cells restored T cell cytokine synthesis when antigen was presented by T-APC. In addition, either IL-12 production or blocking of T cell cytokine synthesis by anti-IL-12 p75 antibodies was evident only when professional APC were used in our antigen-specific system. The down-regulation of cytokine synthesis in the system of T-T cell presentation could represent a novel mechanism of immune regulation, which may intervene to switch off detrimental Th1- or Th2-mediated responses induced by antigen presentation among activated T cells infiltrating inflamed tissues.

Lack of Th2 cytokine increase during spontaneous remission of experimental allergic encephalomyelitis.

The mechanisms underlying spontaneous remission of autoimmune diseases are presently unknown, though regulatory T cells are believed to play a major role in this process. We tested the hypothesis that Th2 and/or other T cell regulatory cytokines cause the spontaneous remission of experimental allergic encephalomyelitis (EAE), a model of Th1-mediated autoimmunity. We analyzed the cytokine profile of lymph node and central nervous system-infiltrating cells in individual SJL mice at different stages of proteolipid protein (PLP) 139-151 peptide-induced EAE. We found that IFN-gamma slowly fades away after clinical recovery, whereas IL-4, IL-10 and transforming growth factor-beta remain low or undetectable. Our peptide-results therefore suggest that regulatory T cells producing anti-inflammatory cytokines are not involved in spontaneous remission of EAE and challenge the view that the Th1/Th2 balance has a key role in EAE regulation.

Persisting viruses and chronic inflammation: understanding their relation to autoimmunity.

Viral infections may induce and sustain autoimmune processes via several and overlapping mechanisms. We outline how chronic inflammation, sustained by persisting viruses, may be "the prerequisite" for initiation and maintenance of the multistep process leading to autoimmunity. Chronic inflammation may favour priming of autoreactive T cells which have escaped thymic tolerance and are able to mount a cross-reactive response to self-mimicking antigens carried by viruses in the periphery. Moreover, chronic inflammation and persisting viruses can synergistically support autoimmunity through other relevant mechanisms: unveiling of cryptic self-epitopes, determinant spreading, activation of dendritic cells, constant priming of new autoreactive T cells, and efficient generation and restimulation of memory cells. Therefore, viruses seem to play a key role among the many environmental factors which, together with the genetic background, have been implicated in the pathogenesis of autoimmune diseases. We will also discuss some hypotheses explaining why autoimmunity is a rare event.

Autoimmune T-cell response to the CD4 molecule in HIV-infected patients.

In a previous study, we demonstrated that by downregulating plasma membrane CD4 and increasing its processing, human immunodeficiency (HIV)-1-gp120 unveils hidden CD4 epitopes, inducing an in vitro anti-CD4-specific T-cell response. We report herein that this mechanism may potentially have important implications in HIV immunopathogenesis, because it could take part in the severe depletion of CD4+ cells that characterizes acquired immune deficiency syndrome (AIDS) and be related to disease progression. Freshly isolated peripheral blood lymphocytes (PBMC) from about 1/4 of a conspicuous cohort of HIV-infected patients responded to CD4 and this response was correlated with beta2-microglobulin levels, widely recognized as marker for progression of HIV infection. Moreover, we provide evidence that a CD4-specific T cell priming can occur in vivo, following a gp120 or anti-CD4 monoclonal antibody (mAb)-mediated CD4 molecule downregulation on antigen-presenting cells (APC). To our knowledge, this is the first study indicating that an autoimmune T-cell response is linked to HIV infection and that it could have an important impact on the immunopathogenesis of this disease.

Generation of an MHC class II-restricted T cell epitope by extracellular processing of hepatitis delta antigen.

Hepatitis delta virus is a human pathogen that is responsible for a severe form of hepatitis affecting hepatitis B envelope Ag carriers. We have previously identified a series of hepatitis delta Ag (HDAg) epitopes that are recognized by CD4+ T cell clones isolated from infected subjects. Herein, we show that the presentation of soluble HDAg to CD4+ T cell clones that are specific for the HDAg(106-121) epitope was exceptionally unaffected by the inhibition of the APC-processing machinery when APCs were fixed with glutaraldehyde before Ag pulsing or treated with chloroquine or brefeldin A. Interestingly, 5 h of pulsing was strictly required for the efficient presentation of the HDAg(106-21) epitope by fixed APCs, suggesting that some form of extracellular processing had occurred. Indeed, fixed APCs were able to present HDAg after only 1 h of pulsing when HDAg was preincubated with serum for 5 h. More important, presentation was completely abrogated when Ag was previously incubated in medium containing serum in the presence of a potent inhibitor of trypsin activity such as the secretory leukoprotease inhibitor. These results show that HDAg may undergo extracellular processing and suggest that the generation of immunogenic epitopes directly by serum proteases could play a role in the immune response against hepatitis delta virus during infection.

Human CD4+ T-cell response to hepatitis delta virus: identification of multiple epitopes and characterization of T-helper cytokine profiles.

The T-cell-mediated immune response plays a crucial role in defense against hepatotropic viruses as well as in the pathogenesis of viral chronic hepatitides. However, very little is known about the role of specific T cells during hepatitis delta virus (HDV) infection in humans. In this study, the T-cell response to HDV in chronic hepatitis B virus (HBV) carriers with HDV superinfection was investigated at different levels. Analysis of peripheral blood mononuclear cell (PBMC) proliferation in response to a recombinant form of large hepatitis delta antigen (HDAg) revealed that 8 of 30 patients studied (27%) specifically responded to HDAg. By employing synthetic peptides spanning the entire HDAg sequence, we found that T-cell recognition was directed against different antigenic determinants, with patient-to-patient variation in the pattern of response to peptides. Interestingly, all responders had signs of inactive HDV-induced disease, while none of the patients with active disease and none of the control subjects showed any significant proliferation. More accurate information about the specific T-cell response was obtained at the clonal level. A panel of HDAg-specific CD4+ T-cell clones from three HDV-infected individuals and fine-specificity analysis revealed that the clones tested individually recognized four epitopes corresponding to amino acids (aa) 26 to 41, 50 to 65, 66 to 81, or 106 to 121 of HDAg sequence. The study of human leukocyte antigen (HLA) restriction revealed that peptides 50 to 65 and 106 to 121 were presented to specific T cells in association with multiple class II molecules. In addition, peptide 26 to 41 was efficiently generated after processing of HDAg through the endogenous processing pathway. Cytokine secretion analysis showed that all the CD4+ T-cell clones assayed were able to produce high levels of gamma interferon (IFN-gamma), belonging either to T helper-1 (Th1) or Th0 subsets and that some of them were cytotoxic in a specific assay. This study provides the first evidence that detection of a specific T-cell response to HDAg in the peripheral blood of individuals with hepatitis delta is related to the decrease of HDV-induced disease activity. The HDAg epitopes identified here and particularly those recognized by CD4+ T cells in association with multiple major histocompatibility complex class II molecules may be potentially exploited for the preparation of a vaccine for prophylaxis and therapy of HDV infection.

Lymphoblastoid cells transfected with c-myc: downregulation of EBV-lytic antigens and impaired response of autologous CD4+ T cells in vitro.

Normal EBV-positive lymphoblastoid B-cell lines (LCL) were transfected with vectors containing the c-myc oncogene (pHEBO-E(mu)-myc) or control vectors (pHEBO-E(mu)) and analyzed for the expression of EBV-lytic and latent antigens. While EBV-latent antigens were normal in the c-myc transfectants, there was an almost complete downregulation of EBV-lytic antigens, including BZLF1, EA(D), gp340 and VCA. These observations were consistently repeated on 6 different LCLs transfected with c-myc. Unlike control LCLs, the c-myc transfectants did not release infectious EBV. PCR analysis demonstrated that BZLF1 mRNA was virtually absent in c-myc transfectants, possibly suggesting that the deregulated c-myc imposed a block in the EBV-lytic cycle at this particular level. c-myc transfectants failed to sustain the proliferative response of autologous CD4+ T-cell clones with specificity for EBV-lytic antigens. However, they regained this capacity after incubation with ultraviolet-inactivated EBV or gp340 antigen in vitro, also indicating that their antigen-presenting capacities were not impaired. c-myc transfectants failed to elicit a secondary proliferative response by autologous CD4+ T cells purified from the peripheral blood of EBV-seropositive donors. Exposure of c-myc transfectants to UV-inactivated EBV again resulted in a proliferative CD4+ T-cell response comparable to that elicited by the control LCLs. Collectively, our data provide evidence for the remarkable ability of an oncogene to influence the life cycle of a virus and to modify the antigenicity of the infected cells.