key: cord-0988313-ufkojrk8
authors: Kawarabayashi, Nobuaki; Seki, Shuhji; Hatsuse, Kazuo; Ohkawa, Takashi; Koike, Yuji; Aihara, Tsukasa; Habu, Yoshiko; Nakagawa, Ryusuke; Ami, Katsunori; Hiraide, Hoshio; Mochizuki, Hidetaka
title: Decrease of CD56(+)T cells and natural killer cells in cirrhotic livers with hepatitis C may be involved in their susceptibility to hepatocellular carcinoma
date: 2003-12-30
journal: Hepatology
DOI: 10.1053/jhep.2000.19362
sha: 526b64360333235e7fe21066940e039be2cd302e
doc_id: 988313
cord_uid: ufkojrk8

CD56(+)T cells and CD56(+)natural killer (NK) cells are abundant in the human liver. The aim of this study was the further characterization of these cells in the liver with or without hepatitis C virus (HCV) infection. Liver mononuclear cells (MNC) were isolated from liver specimens obtained from the patients during abdominal surgery. In addition to a flow cytometric analysis, liver MNC and PBMC were cultured with the immobilized anti‐CD3 Ab, IL‐2, or a combination of IL‐2 and IL‐12 and their IFN‐γ production and the antitumor cytotoxicity were assessed. The liver MNC of HCV (−) patients contained 20% CD56(+)T cells whereas the same proportions decreased to 11% in chronic hepatitis livers and to 5% in cirrhotic livers. The proportion of NK cells also decreased in the cirrhotic livers. On the other hand, the populations of these cells in PBMC did not significantly differ among patient groups. The IFN‐γ production and the cytotoxicity against K562 cells, Raji cells, and a hepatocellular carcinoma, HuH‐7 cells, greatly decreased in the cirrhotic liver MNC. In contrast, the cytotoxicity in PBMC did not significantly differ among the patient groups and was lower than that in the liver MNC of HCV (−) patients. CD56(+)T cells and NK cells but not regular T cells purified from liver MNC cultured with cytokines showed potent cytotoxicities against HuH‐7 cells. These results suggest that a decreased number of CD56(+)T cells and NK cells in cirrhotic livers may be related to their susceptibility to hepatocellular carcinoma.

Chronic viral hepatitis patients, especially hepatitis C patients, often fall victim to liver cirrhosis and subsequent hepatocellular carcinoma (HCC). 1 It is now believed that the HCV infection in hepatocytes itself is not cytopathic whereas the cellular immune response to infected hepatocytes may indeed cause hepatocyte injury. 1 It has been suggested that nonspecific NK cell activation 1,2 and viral antigen-specific ac-tivation of either CD4 ϩ T cells or cytotoxic CD8 ϩ T cells may be responsible for hepatocyte injury. 1, [3] [4] [5] [6] Consistent with this hypothesis, it has also been reported that liver lymphocytes expressed T helper 1 cytokine, IFN-␥, and IL-2 messenger RNA, and the serum levels of these cytokines were elevated in the patients with HCV. 7, 8 On the other hand, livers from mice and humans have recently been reported to contain not only a large population of NK cells but also of T cells with NK cell markers. [9] [10] [11] [12] [13] Namely, mouse NK1.1 antigen ϩ T (NKT) cells and human CD56 ϩ T cells are abundant in the livers. NKT cells in mice were activated by IL-12 and inhibited tumor metastases in the liver of mice 10, 11, [14] [15] [16] (for a review, see Seki et al., 16 ). Human peripheral blood CD56 ϩ T cells were also activated in vitro by IL-12 and thus acquired an antitumor cytotoxicity against NK-resistant tumors. 13 Furthermore, strongly activated mouse liver NKT cells destroyed the syngeneic hepatocytes. 17 Therefore, the possibility has been raised that human CD56 ϩ T cells may also play an important role in both hepatocyte injury in chronic viral hepatitis and antitumor immunity in the liver. In the present study, we show that human liver MNC activated by anti-CD3 Ab or a combination of IL-2 and IL-12 (or IL-2 alone) both produced IFN-␥ and killed tumors more effectively than did PBMC. Furthermore, human liver CD56 ϩ T cells and CD56 ϩ NK cells gradually decreased in parallel with the progress of the hepatitis C and diminished in livers with cirrhosis. These liver MNC from cirrhotic livers could not effectively produce IFN-␥ and could not effectively kill not only K562 cells and Raji cells but also a human HCC cell line, HuH-7 cells, thus suggesting that the decrease in CD56 ϩ T cells and NK cells may be one of the mechanisms explaining why HCC frequently originates from cirrhotic livers.

Patients and Liver Specimens. Liver specimens were obtained during surgery from the patients listed in Table 1 after obtaining their informed consent. Liver specimens obtained from the anti-HCV Aband HBs antigen (Ag)-negative patients with cancers other than HCC and were regarded as the HCV (Ϫ) liver specimens. Other liver specimens were from anti-HCV Ab-positive patients with chronic hepatitis C or with liver cirrhosis. Peripheral blood samples were also obtained during surgery. All liver specimens were obtained from areas other than tumor nodules.

Reagents. Anti-CD3 Ab (UCHT1, mouse IgG1) were purchased from PharMingen (San Diego, CA). Recombinant human IL-2 and IL-12 were purchased from PEPRO TECH EC (London, UK). Recombinant human IL-15 was purchased from Genzyme (Cambridge, MA).

The liver specimens were cut into small pieces with scissors and then treated with collagenase (0.5 mg/mL) and DNAse (100 g/mL) (Sigma Chemical Co., St Louis, MO) at 37°C for 20 minutes. Treated samples of the liver were pressed through a 200-gauge stainless mesh and then were suspended in RPMI 1640 medium. After washing 3 times with medium, the cells were resuspended in osmolarity-and pH-adjusted 33% Percoll solution containing 100 U/mL heparin and then were centrifuged at 2,000 rpm for 15 minute at room temperature. 14, 16 The pellet was resuspended in a red blood cell lysis solution, then washed twice in 5% FBS RPMI. PBMC were obtained from blood samples using a Lymphocyte Separation Medium (ICN Biomedicals Inc., Aurora, OH).

Flow Cytometric Analysis and Cell Culture. Hepatic MNC or PBMC were stained with FITC-conjugated anti-NKR-P1 (CD161, DX12, PharMingen) mAb, phycoerythrin (PE)-conjugated anti-CD56 mAb (NKH-1, Beckman Coulter), phycoerythrin-cyanin 5.1 (PE-Cy5)conjugated anti-␣␤TCR mAb (Beckman Coulter), and gated lymphocytes were analyzed by 3-color flow cytometric analysis using EPICS XL (Beckman Coulter). One hundred microliters (10 g/mL) of anti-CD3 mAb (UCHT1, mouse IgG1) was incubated overnight at 4°C in flat-bottomed 96-well plates to immobilize Ab, and then the plates were washed 3 times before starting the culture. Liver MNC or PBMC (2 ϫ 10 5 ) in 200 L of RPMI 1640 containing 10% human serum were cultured with immobilized anti-CD3 Ab in 5% CO 2 at 37°C in flat-bottomed 96-well plates. MNC were also incubated with human IL-12 (20 ng/mL) and IL-2 (100 ng/mL) or IL-2 alone in flat-bottomed 96-well plates. After the 48-hour culture, the supernatants were harvested and stocked in -80°C for enzyme-linked immunosorbent assay (ELISA). After the 5 days of culture, the cells were harvested and then subjected to cytotoxic assays.

Assays for IFN-␥ Levels. IFN-␥ in MNC culture supernatants were evaluated using the cytokine-specific ELISA kit (Endogen, Inc.).

Cytotoxic Assay. NK-sensitive K562 cells, NK-resistant Raji cells and a human HCC cell line, HuH-7 cells were used as targets. HuH-7 cells established from HCC tissue from HBsAg-negative HCC patients 18 were provided by Cancer Cell Repository, Institute of Development, Aging and Cancer, Tohoku University, Japan and were maintained in 10% FBS RPMI 1640. K562 cells or Raji cells were labeled with 100 Ci Na 2 ( 51 Cr)O 4 for 60 minutes at 37°C in RPMI 1640 medium containing 10% FBS, washed 3 times with medium, and then were subjected to cytotoxicity assays. The labeled K562 cells or Raji cells (2 ϫ 10 3 to 1 ϫ 10 4 /well) were incubated in a total volume of 200 L with effector cells in RPMI 1640 in round-bottomed 96-well microtiter plates (Effector/Target [E/T]) ratio, 10:1). The plates were centrifuged after incubation for 4 hours, after which the supernatants were harvested and counted with a gamma counter. In the case of HuH-7 cells, 10 3 cells were incubated in 10% FBS RPMI 1640 medium in a flat-bottomed 96-well microtiter plate for 4 days before the cytotoxic assay and then were labeled with Na 2 ( 51 Cr)O 4 (1 Ci/well) overnight in 5% CO 2 at 37°C in RPMI 1640 medium before undergoing the cytotoxic assays. The plates were washed 3 times with medium, and adherent HuH-7 cells were incubated with effector cells for 4 hours, and thereafter the supernatants were harvested and counted with a gamma counter. The cytotoxicity was calculated as a percentage of releasable counts after the subtraction of spontaneous release. The spontaneous release was less than 15% of the maximum release.

Cell Sorting and Culture. Liver MNC from patients without HCC were cultured with IL-12 (20 ng/mL), IL-2 (100 ng/mL), and IL-15 (5 ng/mL) in a flat-bottomed 96-well plate thereafter in a flat-bottomed 24-well plate for 3 weeks. Cytokines and 10% FBS containing complete medium were changed twice a week. After staining cultured liver MNC with anti-CD56 Ab and anti-␣␤TCR Ab, CD56 ϩ NK cells, CD56 ϩ T cells, and regular CD56 Ϫ T cells were purified by a cell sorter (EPICS ELITE, Beckman Coulter) and subjected to cytotoxic assays.

Analysis of Class I Expression of Tumor Cells. K562 cells (0.5 ϫ 10 6 ), Raji cells, and HuH-7 cells were stained with FITC-anti-human HLA-A, B, C Ab (mouse IgG2a, Beckman Coulter) at 4°C for 20 minutes and analyzed by EPICS XL. FITC-conjugated isotype control Ab (mouse IgG2a, Beckman Coulter) was also used.

Statistical Analysis. The differences between 2 groups were analyzed by the Mann-Whitney U test, and the differences among the 3 groups were analyzed by an ANOVA analysis with the Scheffe' F using the Stat View program on an Apple computer (Cupertino, CA). Differences were considered to be significant when P was Ͻ.05.

patients contained not only regular CD56 Ϫ T cells (41%) but also CD56 ϩ T cells (20.4%), and approximately 80% of CD56 ϩ T cells were CD161 ϩ (Fig. 1 ). PBMC contained 3.5% CD56 ϩ T cells, and approximately half of them expressed CD161 (Fig. 1 ). CD161 is also referred to as NKR-P1A 19 and is expressed in most of NK cells but not in a majority of regular CD56 Ϫ T cells. Most of CD56 ϩ T cells in the liver and PBMC were CD8 ϩ V␣24 Ϫ (data not shown). The hepatic MNC also contained a large population of CD3 Ϫ CD56 ϩ NK cells (31.0%) (Fig. 1) . Although we showed that approximately 14% were NK cells in PBMC in our recent report, 13 (Fig. 1 ). This discrepancy was the result of the fact that all patients in this study were cancer patients, and some of them had substantially higher populations of NK cells in PBMC. The numbers of liver MNC obtained were approximately 3.0 ϫ 10 6 /g from HCV (Ϫ) livers, 3.5 ϫ 10 6 /g from HCV (ϩ) hepatitis livers, and 2.0 ϫ 10 6 /g from cirrhotic livers.

With Cirrhosis. The proportion of CD56 ϩ T cells in the HCV (Ϫ) livers were 20.4% although it decreased to 11% (P Ͻ .01) and 5% (P Ͻ .01) in the livers with chronic hepatitis and with cirrhosis, respectively (Fig. 2) . Although the proportional decrease of NK cells in the livers with chronic hepatitis was not statistically significant, the proportion of NK cells also significantly decreased in the cirrhotic livers (31% vs. 18%, P Ͻ .05) (Fig. 2) . In contrast, the number of regular CD56 Ϫ T cells did not decrease (Fig. 2) . However, proportions of these cells in PBMC did not significantly differ among PBMC from HCV (Ϫ) patients ( Fig. 1) , hepatitis patients, and cirrhosis patients (data not shown).

ther Immobilized Anti-CD3 Ab or IL-2. HCV (Ϫ) liver MNC produced approximately 12 ng/mL of IFN-␥ by anti-CD3 Ab stimulation for 48 hours whereas PBMC produced 5 ng/mL of IFN-␥ (Fig. 3, left column) . The liver MNC also produced greater amounts of IFN-␥ in response to IL-2 than did PBMC (Fig. 3, left) . However, the IFN-␥ amounts produced by the stimulation of IL-2 and IL-12 did not significantly differ between the liver MNC and PBMC (Fig. 3, left) . In addition, HCV (Ϫ) liver MNC stimulated with either anti-CD3 Ab, IL-2, or a combination of IL-2 and IL-12 acquired a more potent cytotoxicity against K562 tumor cells than did PBMC (Fig. 3, right) .

Patients in Response to Anti-CD3 Ab or IL-2. The liver MNC from cirrhotic livers produced significantly lower amounts of IFN-␥ than did normal liver MNC when they were stimulated with either anti-CD3 Ab or IL-2 whereas cirrhotic liver MNC stimulated with IL-2 and IL-12 produced amounts of IFN-␥ comparable with those from HCV (Ϫ) liver MNC (Fig. 4) . Although anti-CD3-stimulated IFN-␥ production from PBMC of cirrhosis patients tended to decrease as compared with that from the PBMC of HCV (Ϫ) patients, the differences were not statistically significant, and cytokine-stimulated IFN-␥ production did not significantly differ among PBMC from HCV (Ϫ) patients, hepatitis patients, and cirrhosis patients (data not shown). 

ulated With Anti-CD3 Ab or Cytokines. Liver MNC from cirrhotic livers acquired a lower cytotoxicity against NK-sensitive K562 cells after either by CD3 or by IL-2 stimulation than MNC from HCV (Ϫ) livers or chronic hepatitis livers (Fig. 5,  left) . IL-2-and IL-12-stimulated cirrhotic liver MNC also showed lower cytotoxicities against K562 cells than those from the MNC of chronic hepatitis livers (Fig. 5, left) . Either CD3 or cytokine-stimulated liver MNC from cirrhotic livers also showed a much lower cytotoxicity against NK-resistant Raji cells (Fig. 5, right) . On the other hand, anti-CD3 or cytokine-stimulated antitumor cytotoxicities did not significantly differ among PBMC from HCV (Ϫ) patients, hepatitis patients, and cirrhosis patients (data not shown).

Cells. Furthermore, cultured MNC from cirrhotic livers showed a lower cytotoxicity against a human HCC cell line, HuH-7 cells than did those from HCV (Ϫ) livers (Fig. 6) .

Cytotoxic Against HuH-7 Cells. Because IL-15 reportedly activate NK cells and sustain their survival, 20 liver MNC from livers without HCC were cultured with a combination of IL-2 (100 ng/mL), IL-12 (20 ng/mL), and IL-15 (5 ng/mL) for 3 weeks to obtain more numbers of liver MNC. After the culture, the proportion of CD56 ϩ T cells increased to 50% to 60% in liver MNC whereas the proportion of NK cells decreased to approximately 10% and approximately 30% were regular CD56 Ϫ T cells. CD56 ϩ T cells, CD56 Ϫ T cells, and CD56 ϩ NK cells were purified by a cell sorter from cultured liver MNC and cytotoxicities against K562 cells, Raji cells, and HuH-7 cells of these populations were examined. The result showed that CD56 ϩ T cells and NK cells but not regular CD56 Ϫ T cells exerted potent cytotoxicities against tumors (Fig. 7) .

These results suggest that NK cells as well as CD56 ϩ T cells in the liver MNC were thus the main cytotoxic effectors against HuH-7 cells.

The K562 cells lacked any MHC class I (HLA-A, B, C) expression. However, the Raji cells showed a strongly positive MHC class I expression whereas HuH-7 cells showed only a weakly positive MHC class I expression (Fig. 8) .

The presence of HCV had been expected in the patients with non-A, non-B hepatitis since the 1970s, [21] [22] [23] and the cDNA of this RNA virus was identified in 1989. 24, 25 Chronic hepatitis C, which is a parenterally transmitted liver disease, is now one of the major causes of liver cirrhosis and HCC. 1 Initially, hepatitis C patients are usually asymptomatic for FIG. 4 . Comparison of IFN-␥ production among liver MNC from nonhepatitis livers, chronic hepatitis livers, and cirrhotic livers. The liver MNC of each group were stimulated for 48 hours with anti-CD3 Ab, IL-2, or IL-2 and IL-12 in a flat-bottomed 96-well plate with 10% human pooled sera containing RPMI 1640, and supernatants were subjected to ELISA. The data represent the means Ϯ SE from 12 nonhepatitis livers, 12 chronic hepatitis livers, and 5 cirrhotic livers.

The data represent the means Ϯ SE from 12 nonhepatitis livers, 12 chronic hepatitis livers, and 5 cirrhotic livers. The cytotoxicities against NK-resistant Raji cells (right panels). The data represent the means Ϯ SE from 4 nonhepatitis livers, 4 chronic hepatitis livers, and 3 cirrhotic livers. The liver MNC of each group were cultured for 5 days with anti-CD3 Ab, IL-2, or IL-2 and IL-12 in a flat-bottomed 96-well plate with 10% human pooled sera containing RPMI 1640 and subjected to cytotoxic assays against K562 cells or Raji cells (E/T ratio, 10:1). HEPATOLOGY Vol. 32, No. 5, 2000 relatively long periods (1 or 2 decades) while thereafter some patients progress into liver cirrhosis and HCC. 1 Cellular immunity by NK cells and T cells has been suggested to play an important role in the hepatocyte injury of hepatitis C. [1] [2] [3] [4] [5] [6] However, the conditions and functions of NK cells and T cells with the NK cell marker, CD56, in the liver with or without hepatitis C have not been well defined.

In the present study, we showed that liver MNC displayed the potent capacity to produce IFN-␥ and also exerted antitumor cytotoxicity by CD3 stimulation or Th1 cytokine stimulations. The capacity of liver MNC was much larger than PBMC. It is also important to note that CD56 ϩ T cells in the liver (most of which were CD161 ϩ ) 19,26 progressively decreased in parallel with the progress of the hepatitis. Normal liver specimens contained 20.4% CD56 ϩ T cells in total liver MNC whereas cirrhotic liver MNC had only 5% of CD56 ϩ T cells. In addition, although less dramatically than CD56 ϩ T cells, the proportion of CD56 ϩ NK cells also significantly decreased in cirrhotic liver MNC. Consistent with these findings, the IFN-␥ production and antitumor cytotoxicity of liver MNC, in general, were observed to steadily decrease as the disease progressed. Furthermore, MNC of cirrhotic livers were poorly cytotoxic against not only K562 cells and Raji cells but also against HuH-7 cells, and this was the result of the decrease of CD56 ϩ T cells and NK cells in the liver as evidenced by the cytotoxic assays of purified each population from liver MNC cultured with cytokines.

Mouse liver NKT cells have been reported to decrease in number in CCl 4 -induced experimental liver cirrhosis in mice, and the cytotoxicity of liver MNC against a mouse HCC cell line, MH134, was also found to be markedly disturbed. 27 However, the depletion of Kupffer cells did not affect the number of NKT cells. 27 Because some liver MNC firmly adhered to parenchymal hepatocytes, it was suggested that hepatocytes play a role as stromal-like cells for NKT cells. 27 In fact, hepatocytes were found to express IL-7 messenger RNA, which was reported to be an important cytokine for NKT cell development. 28 The present results in human liver MNC were consistent with these findings in mice. In addition, we and others reported that the adult mouse liver contains pluripotent hematopoietic stem cells that can produce all lineage leukocytes. 29, 30 O'Farrelly et al., also reported that human livers contain pluripotent stem cells and RAG 1-positive T cell precursors. 31, 32 These findings suggest that liver T cells with NK cell markers in the livers of mice and humans may FIG. 7 . NK cells and CD56 ϩ T cells but not CD56 Ϫ T cells cultured with cytokines were cytotoxic against tumors. Liver MNC from patients without HCC were cultured with IL-2, IL-12, and IL-15 for 3 weeks and each cell population was purified by a cell sorter and was subjected to a cytotoxic assay. The data represent the means Ϯ SE from 3 independent experiments. thus develop in situ from their liver or bone marrow precursors whereas liver cirrhosis may inhibit the development or proliferation of these cells in the liver.

We and others recently reported that mouse NKT cells in the liver were specifically activated in vivo by IL-12 to produce IFN-␥ and acquired a potent antitumor cytotoxicity and inhibited tumor growth and metastases in the livers. 10, 11, [14] [15] [16] [17] CD56 ϩ T cells in the PBMC of humans were also strongly activated by IL-2 and IL-12 in vitro and acquired a potent antitumor cytotoxicity against NK-resistant tumors. 13 In the present study, we extended our study into these cells in human livers, especially in HCV-infected livers. Normal liver MNC activated by various stimuli produced a large amount of IFN-␥ and acquired a potent cytotoxicity against tumor cells including HuH-7 cells. Because the present study showed that the liver NK cells as well as CD56 ϩ T cells were mainly responsible for the antitumor cytotoxicity in vitro, these cells in the livers are therefore considered to play a crucial role in the antitumor immunity of the liver.

CD4 ϩ T cells and CD8 ϩ T cells, either from the liver or peripheral blood of HCV-infected patients have been reported to recognize HCV Ags 1-6 and were also activated to produce Th1 cytokines, such as IFN-␥ and IL-2. 7,8 NK cells in the livers of chronic viral hepatitis patients were also suggested to be activated. 1, 2 In addition, mouse NKT cells were reported to be activated in livers with viral hepatitis. 33 We also showed that human liver NK cells and CD56 ϩ T cells exerted potent cytotoxicities against tumors including HuH-7 cells in an MHCunrestricted manner. However, it should be noted that liver MNC from cirrhotic livers or PBMC of nonhepatitis patients, when stimulated with IL-2 and IL-12, produced IFN-␥ comparable with those from HCV (Ϫ) liver MNC. Therefore, regular T cells also have the capacity to produce IFN-␥ under certain conditions. Taken together, it is suggested that Ag specific regular T cells, NK cells, and CD56 ϩ T cells are all activated to produce Th1 cytokines and eradicate virusinfected hepatocytes so as to inhibit the replication of hepatitis virus. In fact, IFN-␥ receptor mutant mice have been reported to be susceptible to coronavirus-induced hepatitis. 34 However, it should be noted that because HCV replicates at a rapid rate but lacks proofreading ability, it thus has a large genetic diversity. 1 It may therefore avoid effective surveillance of the host cellular immunity unless it is eradicated in the acute phase of hepatitis C.

On the other hand, 80% of CD56 ϩ T cells in the human liver were NKRP-1 (CD161) ϩ whereas most of them were V␣24 Ϫ . NKT cells in mice mainly use V␣14 gene products for their T cell receptors 35, 36 and V␣24 ϩ T cells in humans have been suggested to be the counterparts of mouse NKT cells because the V␣14 gene of mice and the V␣24 gene of humans have a sequence homology, 36 and both responded CD1 dependently to ␣-galactosylceramide to produce IFN-␥ and acquire an antitumor cytotoxicity. [37] [38] [39] However, V␣24 ϩ T cells were rarely found in both human PBMC and the liver MNC in the present study, and liver MNC and PBMC stimulated with ␣-galactosylceramide did not produce IFN-␥ and did not acquire antitumor cytotoxicity (our unpublished observation, March, 2000) . It is therefore suggested that although mouse V␣14 ϩ T cells and human V␣24 ϩ T cells are indeed a counterpart of each other, the role in host immune responses of human V␣24 ϩ T cells might be more limited than that of mouse V␣14 ϩ T cells. Because NK1.1 Ag of mice and CD161 in humans are both belong to the NKR-P1 family, 26 we proposed that CD56 ϩ T cells (more precisely, CD161 ϩ CD56 ϩ T cells) are a functional counterpart of NKT cells in mice as evidenced by their common properties of the tissue localization, IFN-␥ production, and also their antitumor function. 16 In other words, human CD56 ϩ T cells constitute more heterogeneous populations than mouse NKT cells as was also pointed out by Doherty et al. 40 Because Kupffer cells, which were activated with various bacterial stimuli produced IL-12 and activated NKT cells as well as NK cells 41, 42 and the in vivo depletion of Kupffer cells in mice suppressed the antitumor activity of liver MNC, 42 Kupffer cells may also play an important role in the antitumor immunity of the liver.

Liver MNC of chronic hepatitis patients activated with IL-2 and IL-12 exerted an even stronger cytotoxicity than did HCV (Ϫ) liver MNC, thus suggesting that they are activated by an HCV infection even though NK cells and CD56 ϩ T cells slightly decreased in comparison with those of HCV (Ϫ) livers. It is also noteworthy that IL-2-activated liver MNC exerted even a stronger cytotoxicity against K562 cells than did IL-2 and IL-12-activated liver MNC. This is probably a result of the fact that IL-12 inhibits the moderate or high dose of IL-2-induced proliferation of NK cells. 43 In fact, the proportion of NK cells decreased when PBMC were cultured with IL-2 and IL-12 13 and also when liver MNC were cultured with a combination of IL-2, IL-12, and IL-15 as shown in the present study.

MHC class I expression of tumors has been reported to be inversely correlated with susceptibility to NK cell-mediated lysis. [44] [45] [46] The enhancement of MHC class I expression of tumor cells by IFN-␥ induced the resistance of tumors to NK cell-mediated cytolysis 44 while a decrease of surface MHC class I expression of NK-resistant tumors (including Raji cells) by citric acid treatment makes them susceptible to NK cell-mediated lysis. 46 Because K562 cells lacked an MHC class I expression 46 while HuH-7 cells were MHC class I weakly positive and Raji cells were MHC class I strongly positive, it is reasonable to assume that cytokine-activated NK cells effectively killed K562 cells and HuH-7 cells whereas CD56 ϩ T cells effectively killed Raji cells. It is therefore suggested that the degree of MHC class I expression of HCC may determine its susceptibility to either NK cells or CD56 ϩ T cells.

We recently found by sorting and culture experiments of PBMC that the IFN-␥ production from CD3-stimulated CD56 ϩ T cells was much greater than that from regular T cells and IFN-␥ production from either NK cells or CD56 ϩ T cells stimulated with IL-2, IL-12, and IL-15 was much greater than that from regular T cells (T. Ohkawa et al., submitted), thus indicating that the abundance of CD56 ϩ T cells and NK cells and the decrease of these cells in the liver are responsible for the potent IFN-␥ production in the MNC from livers without cirrhosis and the decreased IFN-␥ production in the MNC from cirrhotic livers, respectively.

In conclusion, NK cells and CD56 ϩ T cells in humans are therefore considered to play an important role not only in the innate antitumor immunity of the liver but possibly also in the hepatocyte injury in hepatitis C patients, and the decrease of CD56 ϩ T cells and NK cells in cirrhotic livers may therefore be one of the important mechanisms explaining why HCC frequently originates in cirrhotic livers.

Schiff's Diseases of the Liver

Isolation, phenotyping, and functional analysis of lymphocytes from human liver

T-lymphocyte response to hepatitis C virus in different clinical courses of infection

Compartmentalization of T lymphocytes to the site of disease: intrahepatic CD4 ϩ T cells specific for the protein NS4 of hepatitis C virus in patients with chronic hepatitis C

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes recognize epitopes in the core and envelope proteins of HCV

HLA B44-restricted cytotoxic T lymphocytes recognizing an epitope on hepatitis C virus nucleocapsid protein

Immunoregulatory cytokines in chronic hepatitis C virus infection: pre-and posttreatment with interferon alfa

Progressive liver injury in chronic hepatitis C infection correlates with increased intrahepatic expression of Th1-associated cytokines

Major histocompatibility complex class I related molecules control the development of CD4 ϩ and CD4 -8 -subsets of natural killer1.1 ϩ T cell receptor-␣␤ ϩ cells in the liver of mice

Cytotoxic NK1.1 Ag ϩ ␣␤ T cells with intermediate TCR induced in the liver of mice by IL-12

Liver NK1.1 ϩ CD4 ϩ ␣␤ T cells as a major effector in inhibition of tumor metastasis

Mouse liver T cells: their change with aging and in comparison with peripheral T cells

Cytotoxic ␥␦ or ␣␤ T cells with an NK cell marker, CD56, induced from human peripheral blood lymphocytes by the combination of IL-12 and IL-2

Antimetastatic effect of NK1 ϩ T cells on experimental hematogenous tumor metastasis in the liver and lung

Requirement for V␣14 NK1 ϩ T cells in IL-12-mediated rejection of tumors

The liver as a crucial organ in the first line of host defense: the roles of Kupffer cells, NK cells and NK1.1 Ag ϩ T cells in T helper 1 immune responses

Involvement of the NK1.1 ϩ T cells and their IFN-␥ production in the generalized Shwartzman reaction

Hormonal control of ␣-fetoprotein secretion in human hepatoma cell lines proliferating in chemically defined medium

Human NKR-P1A. A disulfide-linked homodimer of the C-type lectin superfamily expressed by a subset of NK and T lymphocytes

A potential role for interleukin-15 in the regulation of human natural killer cell survival

Long-incubation post-transfusion hepatitis without serological evidence of exposure to hepatitis-B virus

Transfusion-associated hepatitis not due to viral hepatitis type A or B

Posttransfusion non-A, non-B hepatitis in chimpanzees. Physicochemical evidence that the tubule-forming agent is a small, enveloped virus

Cloning of a cDNA associated with acute and chronic hepatitis C infection generated from patients serum RNA

Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome

Association with FcR gamma is essential for activation signal through NKR-P1 (CD161) in natural killer (NK) cells and NK1.1 ϩ T cells

Supportive cellular elements for hepatic T cell differentiation: T cells expressing intermediate levels of the T cell receptor are cytotoxic against syngeneic hepatoma, and are lost after hepatocyte damage

A comparison of proliferative response to IL-7 and expression of IL-7 receptors in intermediate TCR cells of the liver, spleen, and thymus

c-kit ϩ stem cells and thymocyte precursors in the livers of adult mice

Presence of hematopoietic stem cells in the adult liver

RAG2 and pre-T cell receptor alpha chain expression by adult human hepatic T cells: evidence for extrathymic T cell maturation

In vitro evidence for the presence of hematopoietic stem cells in the adult human liver

Clearance of adenovirus-infected hepatocytes by MHC Class I-restricted CD4 ϩ CTLs in vivo

Exacerbated viral hepatitis in IFN-␥ receptor-deficient mice is not suppressed by IL-12

Extrathymic differentiation of a T cell bearing invariant V alpha 14J alpha 281 TCR

An invariant T cell receptor alpha chain is used by a unique subset of major histocompatibility complex class I-specific CD4 ϩ and CD4 Ϫ 8 Ϫ T cells in mice and humans

CD1d-restricted and TCR-mediated activation of v-alpha14 NKT cells by glycosylceramides

CD1d-mediated recognition of an alpha-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution

CD1d-restricted recognition of synthetic glycolipid antigens by human natural killer T cells

Innate and adaptive lymphoid cells in the human liver

LPS induces NK1.1 ϩ ␣␤ T cells with potent cytotoxicity in the liver of mice via production of IL-12 from Kupffer cells

Activation of mouse liver NK cells and NK1.1 ϩ T cells by bacterial superantigen primed Kupffer cells

Interleukin 12: a proinflammatory cytokine with immunomodulatory functions that bridge innate resistance and antigen-specific adaptive immunity

YAC-1 MHC class I variants reveal an association between decreased NK sensitivity and increased H-2 expression after interferon treatment or in vivo passage

NK susceptibility varies inversely with target cell class I HLA antigen expression

Analysis of mechanisms by which NK cells acquire increased cytotoxicity against class I MHC-eliminated targets