Clinical relevance of increased serum preneoplastic antigen in hepatitis C-related hepatocellular carcinoma.

BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) patients remains poor despite advances in treatment modalities and diagnosis. It is important to identify useful markers for the early detection of HCC in patients. Preneoplastic antigen (PNA), originally reported in a rat carcinogenesis model, is increased in the tissues and serum of HCC patients. AIM: To determine the diagnostic value of PNA for discriminating HCC and to characterize PNA-positive HCC. METHODS: Patients with hepatitis C virus (HCV)-related hepatic disorders were prospectively enrolled in this study, which included patients with hepatitis, with cirrhosis, and with HCC. A novel enzyme-linked immunosorbent assay was developed to measure serum PNA concentrations in patients. RESULTS: Serum PNA concentrations were measured in 89 controls and 141 patients with HCV infections (50 hepatitis, 44 cirrhosis, and 47 HCC). Compared with control and non-HCC patients, PNA was increased in HCC. On receiver operating characteristic curve analysis, the sensitivity of PNA was similar to the HCC markers des-γ-carboxy-prothrombin (DCP) and α-fetoprotein (AFP), but the specificity of PNA was lower. There was no correlation between PNA and AFP and a significant but weak correlation between PNA and DCP in HCC patients. Importantly, the correlations with biochemical markers were completely different for PNA, AFP, and DCP; glutamyl transpeptidase was highly correlated with PNA, but not with AFP or DCP, and was significantly higher in PNA-high patients than in PNA-low patients with HCV-related HCC. CONCLUSION: PNA may have the potential to diagnose a novel type of HCC in which glutamyl transpeptidase is positively expressed but AFP or DCP is weakly or negatively expressed.

Targeting cryptic epitope with modified antigen coupled to the surface of liposomes induces strong antitumor CD8 T-cell immune responses in vivo.

Active cancer immunotherapy, such as cancer vaccine, is based on the fundamental knowledge that tumor­associated antigens (TAAs) are presented on MHC molecules for recognition by specific T cells. However, most TAAs are self-antigens and are also expressed on normal tissues, including the thymus. This fact raises the issue of the tolerance of the TAA­specific T­cell repertoire and consequently the inability to trigger a strong and efficient antitumor immune response. In the present study, we used antigens chemically coupled to the surface of liposomes to target telomerase reverse transcriptase (TERT), a widely expressed self/tumor antigen. Taking advantage of the high homology between mouse and human TERT, we investigated immunogenicity and antitumor efficiency of the liposomal TERT peptides in HLA-A*0201 transgenic HHD mice. Using the heteroclitical peptide-modifying approach with antigen­coupled liposomes, we identified a novel cryptic epitope with low affinity for HLA*0201 molecules derived from TERT. The heteroclitical variant derived from this novel low affinity peptide exhibited strong affinity for HLA*0201 molecules. However, it induced only weak CD8 T­cell immune responses in HHD mice when emulsified in IFA. By contrast, when coupled to the surface of the liposomes, it induced powerful CD8 T­cell immune responses which cross-reacted against the original cryptic epitope. The induced CD8 T cells also recognized endogenously TERT­expressing tumor cells and inhibited their growth in HHD mice. These data suggest that heteroclitical antigen derived from low affinity epitope of tumor antigens coupled to the surface of liposome may have a role as an effective cancer vaccine candidate.

Chimeric SV40 virus-like particles induce specific cytotoxicity and protective immunity against influenza A virus without the need of adjuvants.

Virus-like particles (VLPs) are a promising vaccine platform due to the safety and efficiency. However, it is still unclear whether polyomavirus-based VLPs are useful for this purpose. Here, we attempted to evaluate the potential of polyomavirus VLPs for the antiviral vaccine using simian virus 40 (SV40). We constructed chimeric SV40-VLPs carrying an HLA-A*02:01-restricted, cytotoxic T lymphocyte (CTL) epitope derived from influenza A virus. HLA-A*02:01-transgenic mice were then immunized with the chimeric SV40-VLPs. The chimeric SV40-VLPs effectively induced influenza-specific CTLs and heterosubtypic protection against influenza A viruses without the need of adjuvants. Because DNase I treatment of the chimeric SV40-VLPs did not disrupt CTL induction, the intrinsic adjuvant property may not result from DNA contaminants in the VLP preparation. In addition, immunization with the chimeric SV40-VLPs generated long-lasting memory CTLs. We here propose that the chimeric SV40-VLPs harboring an epitope may be a promising CTL-based vaccine platform with self-adjuvant properties.

Effect of the infectious dose and the presence of hepatitis C virus core gene on mouse intrahepatic CD8 T cells.

AIM: Chronic hepatitis C viral (HCV) infections often result in ineffective CD8 T-cell responses due to functional exhaustion of HCV-specific T cells. However, how persisting HCV impacts CD8 T-cell effector functions remains largely unknown. The aim of this study is to examine the effect of the infectious dose and the presence of HCV core gene. METHODS: We compared responses of intrahepatic CD8 T cells during infection of wild-type or HCV core transgenic (Tg) mice with various infectious doses of HCV-NS3-expressing recombinant adenovirus (Ad-HCV-NS3). RESULTS: Using major histocompatibility complex class I tetramer and intracellular interferon (IFN)-γ staining method to track HCV-NS3-specific CD8 T cells, we found that a significant expansion of HCV-NS3-specific CD8 T cells was restricted to a very narrow dosage range. IFN-γ production by intrahepatic CD8 T cells in HCV core Tg mice was suppressed as compared with wild-type mice. Higher levels of expression of regulatory molecules, Tim-3 and PD-1, by intrahepatic CD8 T cells and PD-L1 by intrahepatic antigen-presenting cells were observed in HCV core Tg mice following Ad-HCV-NS3 infection, and the expression increased dependent on infectious dose. Furthermore, we found a significant inverse correlation between the percentages of IFN-γ-producing cells and expression of regulatory molecules in antigen-specific intrahepatic CD8 T cells. CONCLUSION: High infectious dose and the presence of HCV core gene were strongly involved in ineffective CD8 T-cell responses. We consider that HCV core Tg mouse infected with high infectious dose of Ad-HCV-NS3 is useful as a chronic infection model in the development of immunotherapy for chronic hepatitis C.

Introduction of a point mutation into an HLA class I single-chain trimer induces enhancement of CTL priming and antitumor immunity.

We previously discovered one particular HLA-A*02:01 mutant that enhanced peptide-specific cytotoxic T lymphocyte (CTL) recognition in vitro compared to wild-type HLA-A*02:01. This mutant contains a single amino acid substitution from histidine to leucine at position 74 (H74L) that is located in the peptide-binding groove. To investigate the effect of the H74L mutation on the in vivo CTL priming, we took advantage of the technology of the HLA class I single-chain trimer (SCT) in which three components involving a peptide, ß2 microglobulin and the HLA class I heavy chain are joined together via flexible linkers. We generated recombinant adenovirus expressing SCT comprised influenza A matrix protein (FMP)-derived peptide, ß2 microglobulin and the H74L heavy chain. HLA-A*02:01 transgenic mice were immunized with the adenovirus, and the induction of peptide-specific CTLs and antitumor immunity was investigated. It was clearly shown that the H74L mutation enabled the HLA-A*02:01 SCT molecule to dramatically enhance both in vivo priming of FMP-specific CTLs and protection against a lethal challenge of tumor cells expressing FMP. These data present the first evidence that a simple point mutation in the HLA class I heavy chain of SCT is beneficial for improving CTL-based immunotherapy and prophylaxis to control tumors.

Coupling to the surface of liposomes alters the immunogenicity of hepatitis C virus-derived peptides and confers sterile immunity.

We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen presenting cells to cytotoxic T lymphocytes (CTLs). Liposomal form of immunodominant CTL epitope peptides derived from lymphocytic choriomeningitis virus exhibited highly efficient antiviral CTL responses in immunized mice. In this study, we coupled 15 highly conserved immunodominant CTL epitope peptides derived from hepatitis C virus (HCV) to the surface of liposomes. We also emulsified the peptides in incomplete Freund's adjuvant, and compared the immune responses of the two methods of presenting the peptides by cytotoxicity induction and interferon-gamma (IFN-γ) production by CD8(+) T cells of the immunized mice. We noticed significant variations of the immunogenicity of each peptide between the two antigen delivery systems. In addition, the immunogenicity profiles of the peptides were also different from those observed in the mice infected with recombinant adenoviruses expressing HCV proteins as previously reported. Induction of anti-viral immunity by liposomal peptides was tested by the challenge experiments using recombinant vaccinia viruses expressing corresponding HCV epitopes. One D(b)-restricted and three HLA-A(*)0201-restricted HCV CTL epitope peptides on the surface of liposomes were found to confer complete protection to immunized mice with establishment of long-term memory. Interestingly, their protective efficacy seemed to correlate with the induction of IFN-γ producing cells rather than the cytotoxicity induction suggesting that the immunized mice were protected through non-cytolytic mechanisms. Thus, these liposomal peptides might be useful as HCV vaccines not only for prevention but also for therapeutic use.

Monoclonal antibodies reveal multiple forms of expression of human microsomal epoxide hydrolase.

In a previous study, we developed five kinds of monoclonal antibodies against different portions of human mEH: three, anti-N-terminal; one, anti-C-terminal; one, anti-conformational epitope. Using them, we stained the intact and the permeabilized human cells of various kinds and performed flow cytometric analysis. Primary hepatocytes and peripheral blood mononuclear cells (PBMC) showed remarkable differences. On the surface, hepatocytes exhibited 4 out of 5 epitopes whereas PBMC did not show any of the epitopes. mEH was detected inside both cell types, but the most prominent expression was observed for the conformational epitope in the hepatocytes and the two N-terminal epitopes in PBMC. These differences were also observed between hepatocyte-derived cell lines and mononuclear cell-derived cell lines. In addition, among each group, there were several differences which may be related to the cultivation, the degree of differentiation, or the original cell subsets. We also noted that two glioblastoma cell lines reveal marked expression of the conformational epitope on the surface which seemed to correlate with the brain tumor-associated antigen reported elsewhere. Several cell lines also underwent selective permeabilization before flow cytometric analysis, and we noticed that the topological orientation of mEH on the ER membrane in those cells was in accordance with the previous report. However, the orientation on the cell surface was inconsistent with the report and had a great variation between the cells. These findings show the multiple mode of expression of mEH which may be possibly related to the multiple roles that mEH plays in different cells.

Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis of "preneoplastic antigen"-like molecules.

Microsomal epoxide hydrolase (mEH) is a drug metabolizing enzyme which resides on the endoplasmic reticulum (ER) membrane and catalyzes the hydration of reactive epoxide intermediates that are formed by cytochrome P450s. mEH is also thought to have a role in bile acid transport on the plasma membrane of hepatocytes. It is speculated that efficient execution of such multiple functions is secured by its orientation and association with cytochrome P450 enzymes on the ER membrane and formation of a multiple transport system on the plasma membrane. In certain disease status, mEH loses its association with the membrane and can be detected as distinct antigens in the cytosol of preneoplastic foci of liver (preneoplastic antigen), in the serum in association with hepatitis C virus infection (AN antigen), or in some brain tumors. To analyze the antigenic structures of mEH in physiological and pathological conditions, we developed monoclonal antibodies against different portions of mEH. Five different kinds of antibodies were obtained: three, anti-N-terminal portions; one anti-C-terminal; and one, anti-conformational epitope. By combining these antibodies, we developed antigen detection methods which are specific to either the membrane-bound form or the linearized form of mEH. These methods detected mEH in the culture medium released from a hepatocellular carcinoma cell line and a glioblastoma cell line, which was found to be a multimolecular complex with a unique antigenic structure different from that of the membrane-bound form of mEH. These antibodies and antigen detection methods may be useful to study pathological changes of mEH in various human diseases.

The route of immunization with adenoviral vaccine influences the recruitment of cytotoxic T lymphocytes in the lung that provide potent protection from influenza A virus.

Virus-specific cytotoxic T lymphocytes (CTLs) in the lung are considered to confer protection from respiratory viruses. Several groups demonstrated that the route of priming was likely to have an implication for the trafficking of antigen-specific CTLs. Therefore, we investigated whether the route of immunization with adenoviral vaccine influenced the recruitment of virus-specific CTLs in the lung that should provide potent protection from influenza A virus. Mice were immunized with recombinant adenovirus expressing the matrix (M1) protein of influenza A virus via various immunization routes involving intraperitoneal, intranasal, intramuscular, or intravenous administration as well as subcutaneous administration in the hind hock. We found that the immunization route dramatically impacted the recruitment of M1-specific IFN-γ(+) CD8(+) T cells both in the lung and the spleen. Surprisingly, hock immunization was most effective for the accumulation in the lung of IFN-γ-producing CD8(+) T cells that possessed M1-specific cytolytic activity. Further, antigen-driven IFN-γ(+) CD8(+) T cells in the lung, but not in the spleen, were likely to be correlated with the resistance to challenge with influenza A virus. These results may improve our ability to design vaccines that target virus-specific CTL responses to respiratory viruses such as influenza A virus.

Highly efficient antiviral CD8+ T-cell induction by peptides coupled to the surfaces of liposomes.

In previous studies, we have demonstrated that liposomes with differential lipid components display differential adjuvant effects when antigens (Ags) are chemically coupled to their surfaces. When ovalbumin was coupled to liposomes made by using unsaturated fatty acids, it was found to be presented not only to CD4(+) T cells but also to CD8(+) T cells and induced cytotoxic T lymphocytes (CTLs) which effectively eradicated the tumor from mice. In this study, we coupled liposomes to immunodominant CTL epitope peptides derived from lymphocytic choriomeningitis virus (LCMV) and evaluated its potency as an antiviral vaccine. The intramuscular immunization of mice with the peptide-liposome conjugates along with CpG resulted in the efficient induction of antiviral CD8(+) T-cell responses which conferred complete protection against not only LCMV Armstrong but also a highly virulent mutant strain, clone 13, that establishes persistent infections in immunocompetent mice. The intranasal vaccination induced mucosal immunity effective enough to protect mice from the virus challenge via the same route. Complete protection was achieved in mice even when the Ag dose was reduced to as low as 280 ng of liposomal peptide. This form of vaccination with a single CTL epitope induced Ag-specific memory CD8(+) T cells in the absence of CD4(+) T-cell help, which could be shown by the complete protection of CD4-knockout mice in 10 weeks as well as by the analysis of recall responses. Thus, surface-linked liposomal peptide might have a potential advantage for the induction of antiviral immunity.

Synthetic peptides coupled to the surface of liposomes effectively induce SARS coronavirus-specific cytotoxic T lymphocytes and viral clearance in HLA-A*0201 transgenic mice.

We investigated whether the surface-linked liposomal peptide was applicable to a vaccine based on cytotoxic T lymphocytes (CTLs) against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV). We first identified four HLA-A*0201-restricted CTL epitopes derived from SARS-CoV using HLA-A*0201 transgenic mice and recombinant adenovirus expressing predicted epitopes. These peptides were coupled to the surface of liposomes, and inoculated into mice. Two of the liposomal peptides were effective for peptide-specific CTL induction, and one of them was efficient for the clearance of vaccinia virus expressing epitopes of SARS-CoV, suggesting that the surface-linked liposomal peptide might offer an effective CTL-based vaccine against SARS.

IL-23 enhances host defense against vaccinia virus infection via a mechanism partly involving IL-17.

To investigate roles of IL-23 in viral infection, we have engineered recombinant vaccinia virus (VV) expressing IL-12 (VV-IL-12) and expressing IL-23 (VV-IL-23). We found VV-IL-23 was less virulent in BALB/c mice than wild-type VV (VV-WT), indicating that IL-23 enhances resistance to VV. VV-specific CTL activity in VV-IL-23-infected mice was slightly higher than activity in VV-WT-inoculated mice, although antiviral Ab production and NK activity were not increased. IL-12/23p40-deficient mice survived the infection with VV-IL-23, indicating that IL-23 promotes VV resistance independently of IL-12. The mechanism of the IL-23-mediated resistance was distinct from that of the IL-12-regulated resistance because IFN-gamma-deficient mice did not eliminate VV-IL-12, but did eradicate VV-IL-23. These data indicate that IFN-gamma is essential for the IL-12-mediated resistance, but dispensable for the IL-23-regulated resistance. Because IL-17 is a key in the IL-23-regulated resistance to bacteria, we hypothesized an involvement of IL-17 in the resistance to VV. Treatment with an anti-IL-17 mAb resulted in a significant increase of viral titers in VV-IL-23-infected IFN-gamma-deficient mice. In addition, VV-IL-17 was less virulent than VV-WT in BALB/c mice, and IL-17-deficient mice were more sensitive to VV-WT than control mice. However, the effect of neutralization with an anti-IL-17 mAb was limited, and IL-17-deficient mice survived the infection with VV-IL-23. Taken together, these data suggest that the IL-23/IL-17 axis plays a certain but subdominant role in the IL-23-mediated resistance to VV. Unveiling of an alternative pathway in the IL-23-regulated resistance might provide a novel strategy against infectious pathogens without side effects of autoimmunity.

Autoantibody response to microsomal epoxide hydrolase in hepatitis C and A.

Autoimmune responses were observed in a large proportion of hepatitis C cases and are suspected to be part of viral pathogenesis. The AN6520 antigen (AN-Ag) is a normal cellular protein mainly expressed in liver that was found associated with non-A, non-B hepatitis. To elucidate its pathogenic role in hepatitis C, we developed an IgM capture assay using purified AN-Ag and confirmed that the antibody response to AN-Ag is associated almost exclusively with hepatitis C cases (29%). Screening of a human liver expression library revealed that AN-Ag is mainly the microsomal epoxide hydrolase (mEH), a drug-metabolizing enzyme that plays an important role in the metabolism of some mutagenic and carcinogenic epoxides. Using the purified recombinant human mEH as an antigen, we now found that antibodies against this protein are associated with nearly 82% of hepatitis C virus infections and surprisingly with 46% of patients with hepatitis A. The appearance of AN-Ag/mEH in the incubation period of hepatitis C as previously reported and the antibody responses shown here indicate that this enzyme may be a marker for or even a cause of some of the pathology associated with hepatitis C and A.

Immunogenic variation between multiple HLA-A*0201-restricted, Hepatitis C Virus-derived epitopes for cytotoxic T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) play a critical role in the immune control of Hepatitis C Virus (HCV) infection. In the current study, a number of HLA-A*0201-restricted CTL epitopes derived from HCV were evaluated by examining the peptide-binding affinity for major histocompatibility complex (MHC) class I molecules, the stability of peptide-MHC complexes, killing activities of peptide-induced CTLs, and frequencies of intracellular interferon (IFN)-gamma-positive CD8+ T cells. Among 24 peptides tested, 15 peptides induced high or medium killing activities of peptide-specific CTLs. Thirteen of the 15 peptides exhibited high or medium binding affinities for HLA-A*0201 molecules, indicating that the high binding affinity for MHC class I molecules is an important factor for immunogenicity. In contrast, the stability of peptide-MHC class I complexes was not correlated with killing activities of peptide-induced CTLs. Furthermore, only a limited number of peptides could induce high or medium frequencies of IFN-gamma-producing CD8+ T cells, which were generally considered to play a crucial role for the clearance of HCV. Analyses of the immunogenicity of CTL epitopes such as in the current study should provide important information about the design of an efficient HCV vaccine that induces vigorous, sustained, and broad HCV-specific CTL responses.

Adjuvant activities of novel cytokines, interleukin-23 (IL-23) and IL-27, for induction of hepatitis C virus-specific cytotoxic T lymphocytes in HLA-A*0201 transgenic mice.

Searching the sequence databases has revealed two novel cytokines: interleukin-23 (IL-23) and IL-27. These cytokines are quite similar to, but clearly distinct from IL-12 in their structures and T-cell stimulatory fashions. In contrast to IL-12, however, little is known about the roles of IL-23 and IL-27 in the immune regulation. Previously, we evaluated the prime-boost immunization consisting of priming and the first boosting with the hepatitis C virus (HCV)-core expression plasmid, followed by a second boosting with recombinant adenovirus expressing HCV core for induction of HCV core-specific cytotoxic T lymphocytes (CTLs) in BALB/c mice. The present study demonstrates that HCV-specific CTL induction was greatly enhanced by coinoculation of an IL-12 expression plasmid in the prime-boost immunization, indicating the potent adjuvant activity of IL-12. We investigated whether similar adjuvant effects could be exerted by either IL-23 or IL-27 in a prime-boost immunization with HLA-A*0201 transgenic mice. Coadministration of either an IL-23 or an IL-27 expression plasmid, as well as an IL-12 expression plasmid, in a prime-boost immunization enhanced induction of HCV-specific CTLs and led to dramatic increases in the numbers of gamma interferon (IFN-gamma)-producing, HCV-specific CD8+ cells. Further, preinjections of IL-12, IL-23, or IL-27 expression plasmids before immunization resulted in great increases in the number of IFN-gamma-producing, HCV-specific CD8+ cells in response to immunization with recombinant adenovirus. These data revealed that both IL-23 and IL-27, as well as IL-12, are potent adjuvants for epitope-specific CTL induction. The two novel cytokines might offer new prophylactic and therapeutic strategies against infectious pathogens such as HCV.

Enhanced induction of hepatitis C virus-specific cytotoxic T lymphocytes and protective efficacy in mice by DNA vaccination followed by adenovirus boosting in combination with the interleukin-12 expression plasmid.

We evaluated the prime-boost immunization consisting of hepatitis C virus (HCV)-core expression plasmid (pCEP4-core) and replication-defective adenovirus expressing HCV-core (Adex1SR3ST) for core-specific CTL induction in mice. Compared to a single booster, double boosters after priming enhance CTL induction. The prime-double boosts immunization involving pCEP4-core priming followed by pCEP4-core and Adex1SR3ST boostings (pC/pC/aC) can induce core-specific CTLs as well as other combinations: pC/aC/aC; aC/pC/pC; aC/aC/aC, whereas pC/pC/pC does not induce CTLs. Furthermore, co-administration of interleukin-12 (IL-12) expression plasmid leads to the highly efficient CTL induction and clearance of HCV-core expressing vaccinia virus challenged. Thus, the prime-double boosts immunization together with IL-12 may be promising for HCV vaccine.

Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by immunization with dendritic cells transduced with replication-defective recombinant adenovirus.

We studied the potential of dendritic cells (DCs) in priming hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTLs) in mice. Recombinant adenovirus expressing HCV core (Adex1SR3ST) was employed to express core in DCs. Core-specific CTLs are effectively elicited by injecting Adex1SR3ST-transduced DCs, whereas injection of Adex1SR3ST does not result in effective priming. Further, Adex1SR3ST-transduced DCs more efficiently prime core-specific CTLs than Adex1SR3ST-transduced macrophages, or DCs treated with an anthrax toxin fusion protein reported previously. Upon challenge with recombinant HCV-core-expressing vaccinia virus, vaccinia titers are significantly reduced in mice immunized with Adex1SR3ST-transduced DCs. Thus, adenovirus-transduced DCs may be a promising candidate for a CTL-based vaccine against HCV.

Downregulation of the proteasome subunits, transporter, and antigen presentation in hepatocellular carcinoma, and their restoration by interferon-gamma.

BACKGROUND: In our previous study, expressions of human histocompatibility leukocyte antigens class I molecules (HLA-I) and the transporter associated with antigen processing (TAP) 1/2 genes were investigated in seven hepatocellular carcinoma (HCC) cell lines. Two cell lines, Hep-3B and HuH-7, showed a reduced level of TAP, which might cause the low surface expression of HLA-I. In order to understand the downregulation mechanism of antigen presentation in tumors, the two cell lines were further investigated. METHODS: Expressions of HLA-I and antigen presentation-related genes were analyzed by flow cytometry and polymerase chain reaction, respectively. Antigen presentation was tested in 51Cr-release assays. RESULTS: Flow cytometric analyses revealed low surface expression of HLA-I on Hep-3B and HuH-7 cells. Introduction of HLA-A2 gene did not result in a high surface expression of HLA-A2. This suggested the downregulation of HLA-I expression might be related to defects in the antigen presentation machinery. We then examined expression levels of various antigen presentation-related genes. Hep-3B and HuH-7 demonstrated low expression of the low-molecular-weight protein (LMP) 2, LMP7, TAP1, and HLA-I heavy-chain transcripts. The downregulation of these genes was dissolved by treatment with gamma-interferon. Furthermore, allo-specific cytotoxic T lymphocyte (CTL) lines failed to recognize Hep-3B and HuH-7 cells, while they killed IFN-gamma-treated Hep-3B and HuH-7 cells. CONCLUSIONS: Our results suggest that defects in the antigen presentation-related molecules might cause downregulation of HLA-I expression, antigen presentation, and subsequently, escape from specific CTL killing. The downregulation could be restored by IFN-gamma treatment, suggesting the potential use of IFN-gamma for therapeutic purposes.