Comparative analyses of regulatory T cell subsets in patients with hepatocellular carcinoma: a crucial role of CD25(-) FOXP3(-) T cells.

Regulatory T cells (Tregs) play pivotal role in cancer-induced immunoediting. Increment of CD25(high+) FOXP3+ natural Tregs has been reported in patients with hepatocellular carcinoma (HCC); however, the involvement of other type of Tregs remain elusive. We aimed to clarify whether FOXP3- Tregs are increased and functionally suppressive or not in patients with HCC. We enrolled 184 hepatitis C-infected patients with chronic liver diseases or HCC, 57 healthy subjects and 27 HCC patients with other etiology. Distinct Treg subsets were phenotypically identified by the expression of CD4, CD25, CD127 and forkhead/winged helix transcription factor (FOXP3). Their gene profiles, frequency and suppressor functions against T cell proliferation were compared among the subjects. To examine the molecules involving in Treg differentiation, we cultured naive CD4+ T cells in the presence of HCC cells and dendritic cells. We determined two types of CD4+ CD127- T cells with comparable regulatory ability; one is CD25(high+) cells expressing FOXP3 (CD25(high+) FOXP3+ Tregs) and the other is CD25- cells without FOXP3- expression (CD25- FOXP3- cells). The peripheral or intrahepatic frequency of CD25- FOXP3- Tregs in HCC patients is higher than those in other groups, of which significance is more than CD25(high+) FOXP3+ cells. Of importance, CD25- FOXP3- Tregs, but not CD25(high+) FOXP3+ cells, dynamically change in patients accompanied by the ablation or the recurrence of HCC. CD25- FOXP3- T cells with CD127- IL-10+ phenoype are inducible in vitro from naive CD4(+) T cells, in which programmed cell death 1 ligand 1, immunoglobulin-like transcript 4 and human leukocyte antigen G are involved.. In conclusion, CD25- FOXP3- Tregs with suppressive capacity are increased in patients with HCC, suggesting their distinct roles from CD25+ FOXP3+ Tregs.

Comprehensive immunological analyses of colorectal cancer patients in the phase I/II study of quickly matured dendritic cell vaccine pulsed with carcinoembryonic antigen peptide.

Dendritic cell (DC) vaccine has been used to treat patients with advanced colorectal cancer (CRC). The results of vaccine-induced clinical responses have not always been satisfactory partially because of DC incompetence. In order to evaluate the feasibility of novel mature DCs for therapeutic adjuvants against CRC, we conducted clinical trials with carcinoembryonic antigen (CEA) peptide-loaded DC quickly generated with a combination of OK432 (Streptococcuspyogenes preparation), prostanoid, and interferon-α (OPA-DC). In the ten patients enrolled in this study, the OPA-DC vaccine was well tolerated and administered four times every 2 weeks except for two patients, who were switched to other treatments due to disease progression. Among the eight evaluable patients, one displayed stable disease (SD), while the remaining seven showed progressive disease (PD). In the SD patient, natural killer (NK) cell frequency and cytolytic activity were increased. In the same patient, the frequency of CEA-specific cytotoxic T cells (CTLs) increased stepwise with repetitive vaccinations; however, most of the CTLs exhibited central memory phenotype. In those with PD, NK cells proliferated well regardless of failure of response, whereas CTLs failed to do so. We concluded that the OPA-DC vaccine is well tolerated and has immune-stimulatory capacity in patients with CRC. Additional modulation is needed to attain significant clinical impact.

[A patient who developed diaphragmatic hernia 20 months after percutaneous radiofrequency ablation for hepatocellular carcinoma].

A 50-year-old man underwent radiofrequency ablation (RFA) for a hepatocellular carcinoma, located in the S8 area adjacent to the diaphragm. 20 months later, he was admitted because of severe right hypochondralgia and dyspnea. Computed tomography revealed a prolapsed large intestine through a defect on the right diaphragm, and emergency surgery was performed. Patients who have undergone RFA for a hepatic tumor adjacent to the diaphragm should be carefully followed up for possible diaphragmatic hernia, even after a long postoperative interval.

Impaired cytokine response in myeloid dendritic cells in chronic hepatitis C virus infection regardless of enhanced expression of Toll-like receptors and retinoic acid inducible gene-I.

Dendritic cells utilize various sets of Toll-like receptors (TLR) or cytosolic sensors to detect pathogens and evoke immune responses. In patients with hepatitis C virus (HCV) infection, a higher prevalence of various infectious diseases is reported; suggesting that innate immunity against pathogens is impaired. The aim of this study was to clarify whether the TLR and retinoic acid inducible gene-I (RIG-I) system in myeloid dendritic cells is preserved or not in chronic HCV infection. The expression of TLRs, RIG-I and its relatives were compared in myeloid dendritic cells between 39 patients and 52 healthy volunteers. The induction of type-I interferon (IFN) and inflammatory cytokines was examined in response to agonists for TLR2 (palmitoyl-3-cysteine-serine-lysine-4), TLR3/RIG-I (polyinosine-polycytidylic acid) or TLR4 (lipopolysaccharide). The relative expressions of TLR2, TLR4, RIG-I, and LGP2 from the patients were significantly higher than those from the volunteers, whereas TLR3 and MDA-5 expressions did not differ. In search for factors regulating TLR/RIG-I expression, it was shown that IFN-alpha, polyinosine-polycytidylic acid and lipopolysaccharide induced TLR3, TLR4 and RIG-I, but TNF-alpha, HCV core or HCV non-structural proteins did not. For the functional analyses, myeloid dendritic cells from the patients induced significantly less amounts of IFN-beta, TNF-alpha and IL-12p70 in response to polyinosine-polycytidylic acid or lipopolysaccharide. It is noteworthy that the expression of TRIF and TRAF6, which are essential adaptor molecules transmitting TLR3 or TLR4-dependent signals, is reduced in the patients. Thus, innate cytokine responses in myeloid dendritic cells are impaired regardless of enhanced expressions of TLR2, TLR4, and RIG-I in HCV infection.

Involvement of dendritic cell frequency and function in virological relapse in pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C patients.

A combination of pegylated interferon alpha (PEG-IFNalpha) and ribavirin has been used widely. Enhancement of immune response against hepatitis C virus (HCV) is known to be involved in the efficacy of the combination therapy. The aim of the study was to elucidate whether the frequency or function of immunocompetent blood cells is related to the outcome of the therapy. Twenty-five chronic hepatitis C patients with high viral load of HCV genotype 1 who underwent 48 weeks of PEG-IFNalpha2b and ribavirin therapy were examined. During the treatment, frequencies of dendritic cell subsets, helper T cell subsets, and NK cells were phenotypically determined. In some patients, the ability of dendritic cells to stimulate allogeneic CD4(+)T cells was examined at the end and after the therapy. Among the 25 patients, 11 showed a sustained virological response, 11 a transient response, and 3 no response. In comparison with sustained virological responders, non-sustained virological responders showed impaired dendritic cell function at the end and after the treatment. The transient responders showed a decline of plasmacytoid dendritic cell frequency from Weeks 1-12 and impaired dendritic cell function as well. Even in patients who attained negative serum HCV RNA at Week 12, the transient responders showed a significant decrease of plasmacytoid dendritic cell frequency and impaired dendritic cell function. In conclusion, in PEG-IFNalpha and ribavirin combination therapy for chronic hepatitis C patients, the early-phase plasmacytoid dendritic cell frequency and/or end-of-treatment dendritic cell function are related to the virological outcome of the therapy.

Enhanced ability of peripheral invariant natural killer T cells to produce IL-13 in chronic hepatitis C virus infection.

BACKGROUND/AIMS: Human invariant natural killer T (iNKT) cells express a TCR Valpha24-JalphaQ paired with Vbeta11 and are activated by a surrogate ligand, alpha-galactosylceramide (alphaGalCer). The iNKT cells are involved in the regulation of anti-viral immune responses; however, little is known about their roles in hepatitis C virus (HCV) infection. METHODS: We compared the frequency of peripheral iNKT cells and their cytokine producing capacity reactive to alphaGalCer between chronically HCV-infected patients and healthy subjects. Cytokine production of freshly isolated iNKT cells were analyzed by ELISPOT. Activated iNKT cells were obtained by culture with alphaGalCer-loaded dendritic cells (DCs) and re-stimulated with them for the measurement of cytokine production. RESULTS: The frequencies of iNKT cells were not different between HCV-infected patients and healthy subjects. The number of fresh IFN-gamma-producing iNKT cells reactive to alphaGalCer was not different between the patients and controls, whereas fresh iNKT cells produced negligible amounts of Th2 cytokines regardless of HCV infection. In response to alphaGalCer, expanded iNKT cells from the patients secreted IFN-gamma comparable in amount to controls, whereas they released significantly more IL-13 than cells from controls. CONCLUSIONS: Activated iNKT cells from HCV-infected patients gain more ability to secrete IL-13 than those from healthy subjects.

Reduced expression and functional impairment of Toll-like receptor 2 on dendritic cells in chronic hepatitis C virus infection.

BACKGROUND: Dendritic cells (DCs) utilize Toll-like receptors (TLRs) to sense virus and initiate immune responses. We aimed at elucidating the roles of TLRs on DCs in hepatitis C virus (HCV) infection. METHODS: Monocyte-derived DCs were obtained from 32 healthy volunteers (HV) and 30 chronically HCV-infected patients (CH). TLR2, TLR3 and TLR4 expressions on immature DCs were quantified by real-time quantitative RT-PCR. We stimulated DCs with specific TLR ligands and examined DC maturation, cytokine production and ability to stimulate allogeneic CD4(+) T cells. RESULTS: TLR2 expression on immature DCs was lower in the CH group, whereas those of TLR3 or TLR4 were not different between the groups. Each TLR ligand induced DC maturation and stimulated them to release comparable levels of IL-12p70, IL-6, IL-10, TNF-alpha and IFN-beta between the groups. TLR2 and TLR4 ligands enhanced DC ability to stimulate T cell proliferation, with the degree due to the TLR2 ligand being lower in the CH group. CONCLUSIONS: In HCV infection, the TLR2 expression on DCs is reduced and TLR2-stimulated DCs show lesser ability to proliferate T cells than healthy counterparts, suggesting that the TLR2 system is involved in HCV-induced immunopathogenesis.

Impaired function of dendritic cells circulating in patients infected with hepatitis C virus who have persistently normal alanine aminotransferase levels.

Hepatitis C virus (HCV) induces chronic liver disease in hosts which can eventually progresses to liver cirrhosis and hepatocellular carcinoma. However, progression of liver disease is slower in patients with persistently normal levels of alanine aminotransferase (ALT) than in those with active hepatitis. Although distinct immune responses against HCV have been proposed in asymptomatic infection, the role of circulating dendritic cells (DC) in the pathogenesis of these patients remains obscure. To address this issue, we compared the number and function of myeloid DC (MDC) and plasmacytoid DC (PDC) between uninfected individuals and HCV-infected patients with or without elevated ALT levels. Numbers of DC and DC progenitors were significantly lower in patients with chronic active hepatitis than in control subjects. However, no differences were found in the number of DC between normal controls and HCV-infected patients with persistently normal ALT levels. MDC from patients with active hepatitis were less able to polarize naive CD4 T cells into the Th1 phenotype, while their MDC and PDC primed more CD4 T cells producing IL-10 than those from normal controls. Such dysfunction of DC was also observed in patients with persistently normal ALT levels. In conclusion, circulating DC decrease in number predominantly in HCV-infected patients with active hepatitis, and the function of DC is impaired even in those with normal ALT levels.

Quick generation of fully mature dendritic cells from monocytes with OK432, low-dose prostanoid, and interferon-alpha as potent immune enhancers.

Dendritic cells (DCs) are one of the promising tools for enhancing antigen-specific immune responses in clinical settings. Many studies have been performed thus far to verify the efficacy of the DC vaccine in cancer patients; however, the responses have not always been satisfactory, partly because of DC incompetence. To obtain DCs potentially applicable for vaccination of cancer patients, our group sought to establish the strategy of DC generation mainly by modulating culture periods and maturation stimuli. Novel mature DCs that can be generated from monocytes within 3 days by using a combination of OK432 (Streptococcus pyogenes preparation), low-dose prostaglandin E2 (PGE2), and interferon-alpha (OPA-DCs) were developed. They strongly express CD83, CD86, and CCR7 and have potent ability to migrate to CCL21. In addition, they were able to activate natural killer and T helper 1 (TH1) cells and to induce peptide-antigen-specific cytotoxic T lymphocytes more significantly than monocyte-derived DCs stimulated with a conventional cytokine cocktail of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and PGE2 (monocyte-conditioned medium [MCM]-mimic DCs). The profound ability of OPA-DCs to stimulate these effectors is attributable to their higher expression of IL-12p70, IL-23, and IL-27 than MCM-mimic DCs, which was supported by the findings that the neutralization of IL-12p70 and IL-23 reduced the TH1 priming ability of OPA-DCs. Even when from advanced gastric or colonic cancer patients, OPA-DCs displayed abilities of migration and TH1 induction comparable to those from healthy subjects. Therefore, OPA-DCs may serve as a feasible vaccine with the potential to enhance TH1-dominant and cytolytic immune responses against cancers.

Juvenile hepatocellular carcinoma with congestive liver cirrhosis.

A case of juvenile hepatocellular carcinoma (HCC) with congestive liver cirrhosis is reported. The patient was a 21-year-old woman. She had been diagnosed as having transposition of the great arteries, type 2, in 1978. She underwent the Mustard operation, but suffered from chronic heart failure. In 1995, she experienced abdominal pain and underwent examination. The laboratory data were normal, except for elevated total bilirubin (5.2 mg/dl). Blood examinations were performed at frequent intervals, and the total bilirubin level fluctuated between 0.9 and 8.1 mg/dl over the next 4 years, but the transaminase level remained normal. In 1999, she experienced abdominal pain again and was admitted to our hospital. Computed tomography showed four space-occupying lesions in the liver; 45 mm, 20 mm, 12 mm, and 10 mm in size. She was diagnosed as having HCC, and transcatheter arterial chemoembolization and percutaneous ethanol injection therapy were performed. Histology of the cancerous and the noncancerous liver tissue revealed HCC, moderately differentiated type, in cirrhotic liver with congestion. This patient had no background factors of liver disease, except for liver congestion, associated with the chronic heart failure. Because most patients with cardiac cirrhosis die of cardiac disease, only a small number of these patients develop liver failure. However, the incidence of HCC in patients with congestive liver disease is likely to increase in the future, as survival time is prolonged with the advances in treatment for chronic heart failure. Therefore, patients with congestive liver disease should be followed, taking into account the possibility of HCC.

Reduced numbers and impaired ability of myeloid and plasmacytoid dendritic cells to polarize T helper cells in chronic hepatitis C virus infection.

Hepatitis C virus (HCV) infection induces a wide range of chronic liver injuries. The mechanism by which HCV evades the immune surveillance system remains obscure. Blood dendritic cells (DCs) consist of myeloid and plasmacytoid subsets that play distinct roles in the regulation of antivirus immune responses; however, their roles in the pathogenesis of HCV infection are yet to be determined. We compared the numbers and functions of myeloid and plasmacytoid DCs between 43 patients with chronic hepatitis and 26 age-matched healthy volunteers. Absolute numbers of myeloid DCs, plasmacytoid DCs, and DC progenitors in the periphery were significantly lower in patients with chronic hepatitis than in healthy volunteers. Myeloid and plasmacytoid DCs from the patients had impaired abilities to stimulate allogeneic CD4 T cells and to produce interleukin (IL)-12 p70 and interferon- alpha , compared with those from healthy volunteers. After exposure to naive CD4 T cells, myeloid DCs from the patients were less able to drive the T helper type 1 response, whereas myeloid and plasmacytoid DCs from the patients primed more IL-10-producing cells than did those from healthy volunteers. In conclusion, in chronic HCV infection, both types of blood DCs are reduced and have an impaired ability to polarize T helper cells.

Pseudotype hepatitis C virus enters immature myeloid dendritic cells through the interaction with lectin.

Dendritic cells (DC) are the most potent antigen-presenting cells that regulate immune responses. One of the mechanisms for hepatitis C virus (HCV) persistence is the ability of HCV to suppress DC function. Direct HCV infection to blood DC has been implicated for DC dysfunction. To clarify the susceptibility of each DC subset to HCV, we used pseudotype vesicular stomatitis virus (VSV) coated with chimeric HCV envelope glycoproteins (E1 and E2). We demonstrate that pseudotype VSV enters myeloid DC (MDC) but not plasmacytoid DC (PDC). The highest efficiency of pseudotype VSV entry to MDC was observed when MDC were cultured with GM-CSF. Such efficiency decreased when MDC are matured with the treatment of IL-4, CpG oligodeoxynucleotide, or CD40 ligand. Mannan inhibited pseudotype VSV entry to MDC, but Ca(2+) chelators failed to do so. These results show that pseudotype VSV possessing HCV-E1 and E2 enters immature MDC through the interaction with lectins in a Ca(2+)-independent manner.