Natural killer cells phenotypic characterization as an outcome predictor of HCV-linked HCC after curative treatments.

NK-cell number and function have been associated with cancer progression. A detailed analysis of phenotypic and functional characteristics of NK-cells in HCC is still lacking. NK-cell function is regulated by activating and inhibitory receptors determined by genetic factors and engagement with cognate ligands on transformed or infected cells. We evaluated phenotypic and functional characteristic of NK-cells in HCC patients undergoing curative treatment in relation to clinical outcome. NK-cells from 70 HCC patients undergoing resection or ablative treatment, 18 healthy volunteers and 12 cirrhotic patients with HCV-infection (controls) were phenotypically characterized. Unsupervised clustering based on the frequency of cells expressing different phenotypic NK-cell markers segregated HCC patients into different cohorts that were compared for outcome. NK-cell cytokine production and cytotoxicity were compared between cohorts with different overall survival (OS) and time to disease recurrence (TTR). By multivariate analysis, age, Child-Pugh class and NK-cell phenotypic clustering could independently identify patients with significantly different OS. NK-cells from patients with better outcome expressed higher levels of cytotoxic granules and CD3ζ and lower levels of natural cytotoxic receptors (NCRs) that were co-expressed with the inhibitory receptor NKG2A known to negatively regulate NCR function. Cytotoxic function and IFNγ production were significantly lower in the cohort of patients with worse outcome compared to controls (p < 0.05). Our results show a role for NK-cells in the control of HCC progression and survival providing the basis for the development of immunotherapeutic strategies to potentiate NK-cell response.

HLA and killer immunoglobulin-like receptor genes as outcome predictors of hepatitis C virus-related hepatocellular carcinoma.

PURPOSE: We evaluated the impact of the killer immunoglobulin-like receptors (KIR) of natural killer (NK) cells and of their HLA ligands over the clinical outcome of hepatitis C virus (HCV)-related hepatocellular carcinoma after curative treatment by either surgical resection or radiofrequency thermal ablation (RTA). EXPERIMENTAL DESIGN: Sixty-one consecutive patients with HCV-related hepatocellular carcinoma underwent KIR genotyping and HLA typing. A phenotypic/functional characterization of NK cells was carried out in patients with different KIR/KIR-ligand genotype. RESULTS: Activating KIR2DS5 was associated with significantly longer time to recurrence (TTR) and overall survival (OS; P < 0.03 each). Homozygous HLA-C1 (P < 0.02) and HLA-Bw4I80 (P < 0.05) were expressed by patients with significantly better OS, whereas HLA-C2 (P < 0.02) and HLA-Bw4T80 (P < 0.01) were associated with a worse OS. Multivariate analysis identified as parameters independently related to TTR the type of treatment (surgical resection vs. RTA; P < 0.03) and HLA-C1 (P < 0.03), whereas only KIR2DS5 was an independent predictor of longer OS (P < 0.05). Compound KIR2DL2-C1 and KIR3DS1-Bw4T80 genotypes were associated with better TTR (P < 0.03) and worse OS (P = 0.02), respectively. A prevalent cytotoxic (CD56(dim)) NK phenotype was detected in patients with both longer TTR and OS. Cytotoxic capacity measured by upregulation of CD107a was significantly higher in subjects with HLA-C1 alone or combined with KIR2DL2/KIR2DL3. CONCLUSIONS: These results support a central role of NK cells in the immune response against hepatocellular carcinoma, providing a strong rationale for therapeutic strategies enhancing NK response and for individualized posttreatment monitoring schemes.

Impact of soluble CD26 on treatment outcome and hepatitis C virus-specific T cells in chronic hepatitis C virus genotype 1 infection.

BACKGROUND: Interferon and ribavirin therapy for chronic hepatitis C virus (HCV) infection yields sustained virological response (SVR) rates of 50-80%. Several factors such as non-1 genotype, beneficial IL28B genetic variants, low baseline IP-10, and the functionality of HCV-specific T cells predict SVR. With the pending introduction of new therapies for HCV entailing very rapid clearance of plasma HCV RNA, the importance of baseline biomarkers likely will increase in order to tailor therapy. CD26 (DPPIV) truncates the chemokine IP-10 into a shorter antagonistic form, and this truncation of IP-10 has been suggested to influence treatment outcome in patients with chronic HCV infection patients. In addition, previous reports have shown CD26 to be a co-stimulator for T cells. The aim of the present study was to assess the utility of CD26 as a biomarker for treatment outcome in chronic hepatitis C and to define its association with HCV-specific T cells. METHODS: Baseline plasma from 153 genotype 1 and 58 genotype 2/3 infected patients enrolled in an international multicenter phase III trial (DITTO-HCV) and 36 genotype 1 infected patients participating in a Swedish trial (TTG1) were evaluated regarding baseline soluble CD26 (sCD26) and the functionality of HCV-specific CD8(+) T cells. RESULTS: Genotype 1 infected patients achieving SVR in the DITTO (P = 0.002) and the TTG1 (P = 0.02) studies had lower pretreatment sCD26 concentrations compared with non-SVR patients. Sixty-five percent of patients with sCD26 concentrations below 600 ng/mL achieved SVR compared with 39% of the patients with sCD26 exceeding 600 ng/mL (P = 0.01). Patients with sCD26 concentrations below 600 ng/mL had significantly higher frequencies of HCV-specific CD8(+) T cells (P = 0.02). CONCLUSIONS: Low baseline systemic concentrations of sCD26 predict favorable treatment outcome in chronic HCV infection and may be associated with higher blood counts of HCV-specific CD8(+) T cells.

Immunological and molecular correlates of disease recurrence after liver resection for hepatocellular carcinoma.

The definition of the risk of hepatocellular carcinoma (HCC) recurrence after resection represents a central issue to improve the clinical management of patients. In this study we examined the prognostic relevance of infiltrating immune cell subsets in the tumor (TIL) and in nontumorous (NT) liver (LIL), and the expression of immune-related and lineage-specific mRNAs in HCC and NT liver derived from 42 patients. The phenotype of infiltrating cells was analyzed by flow cytometry, and mRNA expression in liver tissue was examined by real-time reverse transcription (RT)-PCR. The tumor immune microenvironment was enriched in inhibitory and dysfunctional cell subsets. Enrichment in CD4+ T-cells and in particular CD4 and CD8+ memory subsets within TIL was predictive of better overall survival (OS) and time to recurrence (TTR). Increased programmed death ligand 1 (PDL1) mRNA content and higher prevalence of invariant NKT (iNKT) cells were associated with shorter OS and TTR, respectively. By combined evaluation of infiltrating cell subsets along with mRNA profiling of immune and tumor related genes, we identified the intratumoral frequency of memory T-cells and iNKT-cells as well as PDL1 expression as the best predictors of clinical outcome. HCC infiltrate is characterized by the expression of molecules with negative regulatory function that may favor tumor recurrence and poor survival.

Lack of full CD8 functional restoration after antiviral treatment for acute and chronic hepatitis C virus infection.

BACKGROUND: Hepatitis C virus (HCV) persistence is associated with impaired CD8 functions. Whether functional restoration of CD8 T cells chronically exposed to antigen can be obtained once the antigen is removed remains to be clarified. OBJECTIVE: To determine whether clearance of HCV by antiviral treatment can fully restore the antiviral function of HCV-specific CD8 cells. DESIGN: Peripheral blood HCV-, Flu- and cytomegalovirus (CMV)-specific CD8 cells were quantified by tetramer staining in 28 patients whose HCV infection resolved after peginterferon or peginterferon/ribavirin treatment for either acute or chronic hepatitis and in eight subjects with acute HCV infection which resolved spontaneously for comparison. HCV-specific CD8 cells were evaluated for their phenotypic and functional characteristics by comparing different patient groups and CD8 cells with different viral specificities in the same patients. RESULTS: Sustained viral response (SVR) did not lead to full maturation of a functional memory CD8 cell response. In particular, SVR in chronic infection was associated with a greater level of T cell dysfunction than responders after acute infection, who showed HCV-specific CD8 responses comparable to those of spontaneous resolvers but weaker than those of Flu-specific CD8 cells. Higher programmed death (PD)-1 expression was detected on HCV than on Flu- and CMV-specific CD8 cells and the effect of PD-1/PD-L1 blockade was better in SVRs after chronic than after acute HCV infection. CONCLUSION: A better restoration of HCV-specific CD8 function was detectable after SVR in patients with acute hepatitis than in those with chronic disease. Thus, the difficulty in achieving a complete restoration of the antiviral T cell function should be considered in the design of immunomodulatory therapies.

Radiofrequency thermal ablation for hepatocellular carcinoma stimulates autologous NK-cell response.

BACKGROUND & AIMS: Radiofrequency thermal ablation (RFA) is a minimally invasive technique used as standard local therapy of hepatocellular carcinoma and second-line treatment for metastatic liver tumors. Studies in preclinical models and in patients have shown that thermal destruction of tumor tissue can enhance anti-tumor cellular responses, but our knowledge of its impact on natural killer (NK) cells is still very limited. METHODS: Thirty-seven patients undergoing RFA for hepatocellular carcinoma were studied for peripheral blood lymphocytes counts followed by phenotypic and functional characterization of NK-cell population. RESULTS: Peripheral blood lymphocytes kinetics revealed an increased frequency and absolute number of NK cells expressing higher levels of activatory along with reduced levels of inhibitory NK receptors, and increased functional NK-cell activity. A prevalent expansion of the CD3(-)CD56(dim) NK subset was observed compared to the CD3(-)CD56(bright) counterpart. Interferon-gamma production, anti-K562 cell cytotoxicity, and antibody-dependent cell cytotoxicity, appeared consistently increased in terms of both absolute activity and killing efficiency at 4 weeks after RFA, as compared to baseline. Interestingly, when recurrence-free survival was assessed in 2 groups of patients separated according to higher vs lower enhancement of cytotoxicity and/or interferon-gamma production, a significant difference was observed, thus suggesting a potential predictive role of NK functional assays on efficacy of RFA. CONCLUSIONS: RFA can lead to stimulation of NK cells with a more differentiated and proactivatory phenotypic profile with general increase of functional activities. This observation may be relevant for development of adjuvant immunotherapeutic strategies aimed at enhancing NK-cell responses against primary and metastatic liver tumors.

The characteristics of the cell-mediated immune response identify different profiles of occult hepatitis B virus infection.

BACKGROUND & AIMS: Hepatitis B virus (HBV) DNA detection in serum and/or in the liver of hepatitis B surface antigen (HBsAg)-negative patients with or without serologic markers of previous viral exposure is defined as occult HBV infection. Because the role of the adaptive response in keeping HBV replication under control in occult infection still is undefined, this study was performed to characterize the features of the HBV-specific T-cell response in this condition. METHODS: HBV-specific T-cell frequency and function were tested ex vivo and after in vitro expansion in 32 HBsAg-negative patients undergoing diagnostic liver biopsy for chronic hepatitis C: 18 with occult HBV infection (11 anti-HBc-negative and 7 anti-HBc-positive patients) defined by the detection of intrahepatic HBV DNA by polymerase chain reaction; 14 without detectable intrahepatic HBV DNA (5 anti-HBc-positive and 9 anti-HBc-negative patients). Six patients with chronic hepatitis B and 7 HBsAg-inactive carriers were studied for comparison. RESULTS: The presence or absence of serologic HBV markers defined 2 profiles of HBV-specific T-cell responses in occult infection. Anti-HBc-positive patients showed a T-cell response typical of protective memory, suggesting that this condition represents a resolved infection with immune-mediated virus control. In contrast, HBV-specific T cells in anti-HBc-negative patients did not readily expand and produce interferon-gamma in vitro, suggesting the possibility of a low-dose infection insufficient to allow maturation of protective memory. CONCLUSIONS: Our results suggest different mechanisms of control of viral replication in seropositive and seronegative occult infections. Additional studies aimed at understanding possible different clinical implications are needed.

Increased immunostimulatory activity conferred to antigen-presenting cells by exposure to antigen extract from hepatocellular carcinoma after radiofrequency thermal ablation.

Radiofrequency thermal ablation represents an effective treatment for hepatocellular carcinoma (HCC) and it can also exert an "adjuvant" effect on spontaneous antitumor T-cell responses, as suggested by human and animal studies. The adjuvant effect is thought to depend on the huge amount of necrotic tumor antigen made available to the immune system by HCC thermal ablation. In addition, radiofrequency thermal ablation may result in the release of local stimuli responsible for activation and maturation of antigen-presenting cells (APCs). To test this hypothesis, we studied APC maturation and function in 19 patients undergoing thermal ablation for HCC. Patients' monocytes induced to differentiate with granulocyte macrophage colony-stimulating factor (GM-CSF), or GM-CSF plus IL-4, were cocultured in vitro with tumor debris generated by radiofrequency thermal ablation. Expression of costimulatory molecules, lymphnode homing chemokine receptor, antigen presentation, and cytokine secretion were enhanced by incubation with HCC treated tissue as compared with untreated HCC and nontumor liver tissue. Moreover, HCC-specific T-cell responses could be induced by monocytes activated with GM-CSF and incubated with thermally ablated HCC tissue. HCC thermal ablation can create an antigenic source along with stimuli appropriate for maturation of APCs to induce HCC-specific T-cell responses. These results contribute to explain at least in part the adjuvant effect of HCC thermal ablation and suggest a novel strategy to induce maturation of APCs and their loading with HCC antigens for active immunotherapy protocols aimed at reducing HCC recurrence after thermal ablation.

HCV-specific T-cell response in relation to viral kinetics and treatment outcome (DITTO-HCV project).

BACKGROUND & AIMS: The second slope of viral decline induced by interferon treatment has been suggested to be influenced mainly by the hepatitis C virus (HCV)-specific T-cell response; however, this hypothesis needs to be validated by results derived from experimental studies. METHODS: To address this issue, the HCV-specific T-cell response of 32 genotype-1-infected patients of the 270 patients enrolled in the dynamically individualized treatment of hepatitis C infection and correlates of viral/host dynamics phase III, open-label, randomized, multicenter trial was studied in relation to viral kinetics and treatment outcome. RESULTS: Greater proliferative responses by HCV-specific CD8 cells were found before treatment in patients with a fast viral decline and with a sustained viral response. However, no significant improvement of HCV-specific CD8 responses was observed in the first weeks of therapy in both rapid viral responder and non-rapid viral responder patients. A mild enhancement of proliferative T-cell responses and a partial restoration of the cytotoxic T-cell potential was expressed only late during treatment, likely favored by HCV clearance. CONCLUSIONS: Early restoration of an efficient T-cell response does not seem to be an essential requirement for a rapid viral decline in the first weeks of treatment. However, patients presenting a better HCV-specific CD8 cell proliferative potential at baseline are more likely to present a rapid and sustained viral response. Therefore, future treatment protocols should consider the development of strategies aimed at improving HCV-specific T-cell responses.

Dysfunction and functional restoration of HCV-specific CD8 responses in chronic hepatitis C virus infection.

UNLABELLED: The functional impairment of HCV-specific T cell responses is believed to be an important determinant of HCV persistence, but the functional T cell defects of patients with chronic hepatitis C (CH-C) are only partially defined. CD8 responses to HLA-A2-restricted epitopes of HCV and other unrelated viruses were studied in 23 HLA-A2-positive patients both ex vivo and after in vitro culture. Degranulation capacity, intracellular perforin, and granzyme-A content and cytokine production (IFN-gamma, TNF-alpha) by HCV- and non-HCV-specific CD8 cells were tested both ex vivo and in vitro, whereas cytolytic activity was studied after 10 days' expansion in vitro. Memory maturation and role of exhaustion were assessed ex vivo by HCV-specific CD8 staining for CD127 and PD-1, and in vitro after peripheral blood mononuclear cells (PBMC) culture in the presence of anti-PD-L1 monoclonal antibodies. IFN-gamma production and cytolytic activity were expressed less efficiently by HCV-specific than by non-HCV specific CD8 cells derived from the same CH-C patients. The amount of stored granzyme-A within single cells was always lower in HCV-specific CD8 cells, which were less efficient also in the release of lytic granules and in the production of TNF-alpha. The CD8 dysfunction was associated with high PD-1 expression by most HCV-specific CD8 cells, and PD-1/PD-L1 blockade by anti-PD-L1 antibodies in vitro was able to improve the HCV-specific CD8 function. CONCLUSION: Our study characterizes CD8 defects that may be important in maintaining HCV persistence; identification of strategies to correct these defects may help to define novel approaches to treat HCV infection.

Radiofrequency thermal ablation of hepatocellular carcinoma liver nodules can activate and enhance tumor-specific T-cell responses.

Radiofrequency thermal ablation (RFA) destroys tumoral tissue generating a local necrosis followed by marked inflammatory response with a dense T-cell infiltrate. In this study, we tested whether hepatocellular carcinoma thermal ablation can induce or enhance T-cell responses specific for hepatocellular carcinoma-associated antigens. Peripheral blood mononuclear cells derived from 20 patients with hepatocellular carcinoma were stimulated before and a month after RFA treatment with autologous hepatocellular carcinoma-derived protein lysates obtained before and immediately after RFA treatment. The effect of thermal ablation on memory T-cell responses to recall antigens [tetanus toxoid, protein purified derivative (PPD), Escherichia coli] was also assessed. T-cell reactivity was analyzed in an IFN-gamma enzyme-linked immunospot assay and by intracellular IFN-gamma staining. Treatment was followed by a significant increase of patients responsive either to tumor antigens derived from both the untreated hepatocellular carcinoma tissue (P < 0.05) and the necrotic tumor (P < 0.01) and by a higher frequency of circulating tumor-specific T cells. T-cell responses to recall antigens were also significantly augmented. Phenotypic analysis of circulating T and natural killer cells showed an increased expression of activation and cytotoxic surface markers. However, tumor-specific T-cell responses were not associated with protection from hepatocellular carcinoma relapse. Evidence of tumor immune escape was provided in one patient by the evidence that a new nodule of hepatocellular carcinoma recurrence was not recognized by T cells obtained at the time of RFA. In conclusion, RFA treatment generates the local conditions for activating the tumor-specific T-cell response. Although this effect is not sufficient for controlling hepatocellular carcinoma, it may represent the basis for the development of an adjuvant immunotherapy in patients undergoing RFA for primary and secondary liver tumors.

Heterologous T cell immunity in severe hepatitis C virus infection.

Hepatitis C virus (HCV) can cause liver disease of variable severity. Expansion of preexisting memory CD8 T cells by cross-reactivity with a new heterologous virus infection has been shown in mice to shape the repertoire of the primary response and to influence virus-related immunopathology. To determine whether this mechanism can influence the course of HCV infection, we analyzed the features of the HCV-specific CD8 T cell response in eight patients with acute HCV infection, two of whom had a particularly severe illness. Patients with severe hepatitis, but not those with mild disease, showed an extremely vigorous CD8 T cell response narrowly focused on a single epitope (NS3 1073-1081), which cross-reacted with an influenza neuraminidase sequence. Our results suggest that CD8 T cell cross-reactivity influences the severity of the HCV-associated liver pathology and depicts a model of disease induction that may apply to different viral infections.

Parenteral exposure to high HIV viremia leads to virus-specific T cell priming without evidence of infection.

Previous studies on CTL responses in HIV-exposed uninfected individuals assumed that the patients were exposed to replicating HIV, but the possibility that the immune responses detected were primed by exposure to a defective virus or viral antigen could not be excluded. Epidemiological and laboratory analysis of a nosocomial outbreak of acute hepatitis B unequivocally allowed the identification of an HIV-1- and HBV-co-infected patient with high plasma levels of both viruses, as the source case of the epidemics. This clinical setting provided a natural model for testing the HIV-specific T cell response in patients exposed to blood from a patient with highly replicating HIV. Parenteral exposure to both viruses led to acute hepatitis B in five subjects without evidence of HIV-1 infection. Cryopreserved lymphocytes derived from three exposed patients were tested ex vivo in an ELISPOT assay for IFN-gamma release upon stimulation with peptides from structural and non-structural HIV proteins; one of the patients was also tested with four HLA/class I tetramers. Circulating HIV-specific CD8 cells were detected by tetramer staining and a high frequency of T cells were able to release IFN-gamma upon stimulation with HIV peptides, showing in vivo T cell priming by HIV. These results unequivocally demonstrate a HIV-specific cell-mediated immune response in the absence of infection after exposure to highly replicating HIV.

Ex vivo characterization of tumor-derived melanoma antigen encoding gene-specific CD8+cells in patients with hepatocellular carcinoma.

BACKGROUND/AIMS: Members of the melanoma antigen encoding gene family are expressed in tumors of different histological types but not in normal tissue. For this reason, they are attractive targets for cancer immunotherapy. METHODS: In the present study, we analyzed the expression of MAGE-1 and -3 genes in the hepatocellular carcinoma (HCC) tissue as well as frequency, phenotype and function of circulating and tumor infiltrating CD8+ cells specific for HLA-A1 and -A2 restricted epitopes of MAGE-1 and -3. RESULTS: Our study shows for the first time the presence of MAGE/tetramer+ CD8 cells in the tumor tissue of patients with HCC. These cells are able to recognize the MAGE-1 sequence 161-169 and the MAGE-3 sequence 271-279. In a patient with a particularly high frequency of MAGE-1 sequence 161-169-specific T cells, phenotypic and functional analysis was performed showing a phenotype of recently-primed CD8 cells (CD28+CD27+CD45RA-CCR7). CONCLUSIONS: The observation of a spontaneous in vivo priming of a MAGE-specific T cell response in patients with HCC and the high frequency of MAGE antigens expression in this tumor, makes this antigen a potential candidate for a MAGE-specific immunotherapy in hepatocellular carcinoma.

Transient restoration of anti-viral T cell responses induced by lamivudine therapy in chronic hepatitis B.

BACKGROUND/AIMS: Lamivudine therapy in patients with chronic hepatitis B can induce the recovery of antiviral T cell responses. It is unknown whether the recovery of T cell responsiveness is long-lasting and persists throughout the treatment and whether the elevation of viremia which follows therapy withdrawal can restore a condition of T cell unresponsiveness. METHODS: Frequency and function of circulating hepatitis B virus (HBV)-specific CD4 and CD8 cells from 12 hepatitis e surface antigen + patients with chronic hepatitis B were studied longitudinally before, during and after lamivudine therapy by intracellular cytokine staining, proliferation and cytotoxicity assays against HBV proteins and peptides. CD4-mediated responses were analyzed in all patients, whereas CD8 cells were studied in 6 HLA-A2+ patients. RESULTS: HBV-specific CD4 and CD8 reactivity showed a bi-phasic behavior under lamivudine therapy with an early enhancement of T cell frequency and intensity of responses followed by a persistent decline starting from the 5th to 6th month of treatment. CONCLUSIONS: Since restoration of HBV-specific T cell reactivity is only transient, our study indicates that therapeutic stimulation of HBV-specific T cell responses to complement lamivudine treatment should be done early after the initiation of lamivudine. Moreover, the transient nature of the immune reconstitution may represent a favorable condition for virus reactivation once lamivudine therapy is withdrawn.

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.

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.