Lysosomal processing of sulfatide analogs alters target NKT cell specificity and immune responses in cancer.

In a structure-function study of sulfatides that typically stimulate type II NKT cells, we made an unexpected discovery. We compared analogs with sphingosine or phytosphingosine chains and 24-carbon acyl chains with 0-1-2 double bonds (C or pC24:0, 24:1, or 24:2). C24:1 and C24:2 sulfatide presented by the CD1d monomer on plastic stimulated type II, not type I, NKT cell hybridomas, as expected. Unexpectedly, when presented by bone marrow-derived DCs (BMDCs), C24:2 reversed specificity to stimulate type I, not type II, NKT cell hybridomas, mimicking the corresponding ß-galactosylceramide (ßGalCer) without sulfate. C24:2 induced IFN-γ-dependent immunoprotection against CT26 colon cancer lung metastases, skewed the cytokine profile, and activated conventional DC subset 1 cells (cDC1s). This was abrogated by blocking lysosomal processing with bafilomycin A1, or by sulfite blocking of arylsulfatase or deletion of this enyzme that cleaves off sulfate. Thus, C24:2 was unexpectedly processed in BMDCs from a type II to a type I NKT cell-stimulating ligand, promoting tumor immunity. We believe this is the first discovery showing that antigen processing of glycosylceramides alters the specificity for the target cell, reversing the glycolipid's function from stimulating type II NKT cells to stimulating type I NKT cells, thereby introducing protective functional activity in cancer. We also believe our study uncovers a new role for antigen processing that does not involve MHC loading but rather alteration of which type of cell is responding.

Frequency and role of NKp46 and NKG2A in hepatitis B virus infection.

BACKGROUND AND AIM: Natural Killer (NK) cells are involved in the control of viral infection. However, the role of NK cells in chronic hepatitis B (CHB) remains unclear. This study investigated the frequencies and roles of NK cells in CHB, with a focus on activating receptor NKp46 and inhibitory receptor NKG2A. PATIENTS/METHOD: Peripheral blood lymphocytes were obtained from 71 CHB patients and 37 healthy subjects (HS). The expressions of NKp46 and NKG2A were analyzed using flow cytometry. The role of NKp46-ligand was assessed using an in vitro co-culture system. Cytotoxicity and IFN-γ production in NK cells were evaluated using RT-PCR and flow cytometry. RESULTS: CHB patients were classified into treatment-naïve patients with low HBV DNA titer (CHB-L; n = 28), high HBV DNA titer (CHB-H; n = 24) by the cut-off level of serum HBV DNA 4 log copies/ml, and patients receiving nucleos(t)ide analogue (CHB-NA; n = 19). The expressions of NKp46 and NKG2A were higher in CHB-H than in HS/CHB-L/CHB-NA. HepG2.2.15 had higher NKp46-ligand expression than HepG2. When NK cells from HS were co-cultured with HepG2.2.15, inhibition of the NKp46 and NKp46-ligand interaction by anti-NKp46 antibody significantly reduced cytolysis of HepG2.2.15 and IFN-γ production. However, those reductions were not observed in co-culture with HepG2. Additionally, NK cells that highly expressed NKp46 also highly expressed NKG2A (NKp46highNKG2Ahigh subset). The frequencies of NKp46highNKG2Ahigh subset in CHB-H were higher than those in HS/CHB-L/CHB-NA. Among treatment-naïve CHB patients, the frequencies of NKp46highNKG2Ahigh subset were positively correlated with serum ALT (P<0.01, r = 0.45) and HBV DNA (P<0.01, r = 0.59) levels. The expressions of Fas-L, STAT1, TRAIL and CD107a were higher and IFN-γ expression was lower in the NKp46highNKG2Ahigh subset than in the other subsets. CONCLUSION: The NKp46 and NKp46-ligand interaction contributes to NK cell activation. A novel NK cell subset, the NKp46highNKG2Ahigh subset, may be associated with liver injury and HBV replication.

[Squamous cell carcinoma of the anal canal treated with chemoradiotherapy in a patient with HIV].

Since the introduction of combination antiretroviral therapy (ART), the life expectancy has increased for patients infected with human immunodeficiency virus (HIV). This has been associated with reductions in the incidences of some AIDS-defining malignancies, such as Kaposi sarcoma and non-Hodgkin lymphoma, but has coincided with an increased incidence of non-AIDS-defining malignancies, such as anal cancer. However, anal cancers are rare in patients with HIV in Japan. We report the case of an HIV-infected patient with anal cancer treated with chemoradiotherapy. A 37-year-old man receiving ART for HIV infection presented with a 1-month history of left inguinal lymphadenopathy and anal pain. Magnetic resonance imaging and computed tomography revealed a 56-mm mass, left inguinal lymphadenopathy, and left external iliac lymphadenopathy. The mass had infiltrated from the anal canal to the right levator ani and corpus spongiosum. Colonoscopy revealed a tumor with an ulcer in the anal canal. Histological examination of the tumor biopsy specimens confirmed the diagnosis of squamous cell carcinoma. The patient was diagnosed with anal cancer (T4N2M1 stage IV), and he received 5-fluorouracil (1000mg/m(2) on days 1-4 and 29-32) plus mitomycin C (10mg/m(2) on days 1 and 29) and concurrent radiotherapy (total dose, 59.4Gy in 33 fractions) along with ART. The treatment-related adverse events were grade 4 leukopenia and neutropenia, grade 3 thrombocytopenia, and grade 2 radiation dermatitis. Moreover, CD4 suppression was observed:the CD4 count decreased from 190 cells/µl before chemoradiotherapy to 138 cells/µl after 3 months, but increased to 210 cells/µl after 1 year. Because of the grade 4 leukopenia and neutropenia, the dose of 5-fluorouracil was reduced to 800mg/m(2) on days 29-32. A complete response was confirmed on magnetic resonance imaging, and colonoscopy confirmed the disappearance of the anal cancer. The patient is living with no signs of recurrence at 2 years after chemoradiotherapy. When treating HIV-infected patients with anal cancer by chemoradiotherapy and ART, clinicians should be aware of the possibility of CD4 suppression.

S100A8 Production in CXCR2-Expressing CD11b+Gr-1high Cells Aggravates Hepatitis in Mice Fed a High-Fat and High-Cholesterol Diet.

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease with a spectrum of presentations. S100A8 has been suggested to play a pivotal role as an endogenous immune-activator in inflammatory diseases. In this study, we investigated the involvement of S100A8 in the development of NAFLD. We used a diet model of NAFLD, in which mice were fed either a high-fat and high-cholesterol diet (HFHCD) or a normal diet (ND) as a control. We also assessed liver tissues from patients with NAFLD, including patients with nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). HFHCD-fed mice, but not ND-fed mice, developed steatohepatitis. S100A8 expression was significantly elevated in the livers of HFHCD-fed mice compared with the controls. S100A8 was exclusively expressed in CXCR2-expressing CD11b(+)Gr-1(high) cells, which significantly increased in the livers of HFHCD-fed mice. These cells were F4/80 negative and did not possess a suppressor function. TNF-α expression was enhanced by S100A8 in primary liver leukocytes or a hepatocyte cell line and significantly elevated in the livers of HFHCD-fed mice. TNF-α was primarily produced from CD11b(+)F4/80(+) cells in liver leukocytes in response to S100A8. TNF-α deficiency attenuated hepatitis in HFHCD-fed mice. S100A8 was significantly more expressed in the liver tissues of patients with NASH than in those of patients with NAFL. In conclusion, these results suggest that S100A8 is primarily produced from CXCR2-expressing CD11b(+)Gr-1(high) cells, and it upregulates TNF-α production in CD11b(+)F4/80(+) cells through cellular cross-talk, which is an important mechanism in the development of NAFLD.

Invariant natural killer T cell deficiency leads to the development of spontaneous liver inflammation dependent on γδT cells in mice.

BACKGROUND: Immune tolerance is maintained in the liver, and perturbation of tolerance can lead to immune-mediated liver diseases such as autoimmune hepatitis (AIH). Invariant natural killer T (iNKT) cells and γδT cells have been shown to maintain immune homeostasis as regulatory cells and to play pathogenic roles in immune-mediated diseases as effector cells. We hypothesized that iNKT cells and γδT cells are involved in the maintenance of hepatic immune tolerance and immune-mediated liver disease. METHODS: We measured liver inflammation and the cytokine profiles of liver mononuclear cells in BALB/c wild-type (WT) mice and BALB/c Jα18-deficient (KO) mice lacking iNKT cells. We also examined the role of γδT cells in AIH using liver tissue from AIH patients and control subjects. RESULTS: Spontaneous liver inflammation, hepatocyte damage, and anti-nuclear-antibody production occurred in Jα18 KO mice but not in WT mice. Furthermore, liver mononuclear cells from Jα18 KO mice, but not those from WT mice, produced interleukin-17 (IL-17). γδT cells were the primary producers of the cytokine, and they were more abundant in the livers of Jα18 KO mice than in those of WT mice. In Jα18 KO mice, the administration of anti-γδT-cell-receptor antibody abolished liver inflammation, hepatocyte damage, and IL-17 production. γδT cells accumulated in the livers of AIH patients but not in those of the control subjects. CONCLUSIONS: Our results suggest a protective role for iNKT cells, a pathologic role for γδT cells, and an association between these cells in the pathogenesis of AIH.

Differential alteration of CD56(bright) and CD56 (dim) natural killer cells in frequency, phenotype, and cytokine response in chronic hepatitis C virus infection.

BACKGROUND: Natural killer (NK) cells play an important role in immune responses to virus infection. The cell population consists of CD56(bright) (bright-subset) and CD56(dim) (dim-subset) subsets that possess armed functions of cytokine production and cytolysis, respectively. How these subsets are involved in chronic hepatitis C virus infection (CHC) remains obscure. METHODS: We investigated the frequency, phenotype, and cytokine response of these subsets in blood from CHC patients and healthy subjects (HS). RESULTS: Dim-subset, but not bright-subset, showed lower frequency in the patients than in HS. Bright-subset from the patients more frequently expressed the NKG2A/CD94 inhibitory receptor than that from HS, while both subsets from the patients expressed lower levels of the NKG2D activating receptor. Both subsets from the patients displayed a significantly higher level of the signal transducer and activator of transcription (STAT) 1, compared with the HS. Upon stimulation with interferon-α, bright-subset activated less STAT4, required for interferon-γ production, and dim-subset activated more STAT1, required for cytolysis, in the patients than in HS. CONCLUSIONS: These results indicate alterations of NK cell subsets in frequency, phenotype, and cytokine response in CHC, which might be associated with the immune pathogenesis of CHC.

Absence of invariant natural killer T cells deteriorates liver inflammation and fibrosis in mice fed high-fat diet.

BACKGROUND: Invariant natural killer T (iNKT) cells have been suggested to play critical roles in a wide range of immune responses by acting in a proinflammatory or anti-inflammatory manner. Nonalcoholic steatohepatitis (NASH) is a chronic liver disease progressing to advanced cirrhosis and hepatocellular carcinoma. Despite the abundance of iNKT cells in the liver, their role in the pathogenesis of NASH remains obscure. Here, we investigated their role in the development of diet-induced steatosis/steatohepatitis. METHODS: We used BALB/c wild-type mice and Jα18-deficient (KO) mice lacking iNKT cells fed either a normal diet or a high-fat diet (HFD). The liver and blood were collected from these mice to examine liver inflammation, steatosis, and fibrosis at the indicated time points. RESULTS: KO mice fed the HFD, compared with control mice fed the HFD, exhibited a clearly higher serum alanine aminotransferase level and a greater number of hepatic inflammatory foci, although there was no significant difference in hepatic lipid retention between these groups of mice. The HFD enhanced hepatic messenger RNA expression of inflammatory cytokines and chemokines in KO but not in control mice. The HFD also increased the proportion of hepatic CD4 T cells and CD8 T cells that composed hepatic inflammatory foci in KO mice, but not in the controls. Prolonged feeding with the HFD augmented liver fibrosis in KO but not in control mice. CONCLUSIONS: These findings indicate that iNKT cells play a protective role against liver inflammation progressing to fibrosis, but not against steatosis, enhanced by dietary excess fat, suggesting a key role of these cells in NASH pathogenesis.

Altered interferon-alpha-signaling in natural killer cells from patients with chronic hepatitis C virus infection.

BACKGROUND & AIMS: Natural killer (NK) cells play an important role in the immune response against virus infection. Interferon (IFN)-alpha, an essential component in therapy against hepatitis C virus (HCV) infection, regulates NK cell function. However, it remains obscure how chronic HCV infection (CHC) modifies intracellular IFN-alpha signaling in NK cells. We investigated IFN-alpha signaling in NK cells in patients with CHC. METHODS: Peripheral blood mononuclear cells were obtained from patients with CHC and healthy subjects (HS) as controls. RESULTS: The expression level of signal transducer and activator of transcription (STAT) 1, a key molecule of IFN-alpha signaling, was clearly higher in NK cells from the CHC patients than in those from HS. The phosphorylation level of STAT1 with IFN-alpha stimulation was significantly greater in NK cells from the CHC patients than in those from the HS, while that of STAT4 was significantly less. These phosphorylation levels of STAT1 and STAT4 positively and negatively correlated with the STAT1 level in NK cells, respectively. The IFN-alpha induced messenger RNA level of the suppressor of cytokine signaling 1, which is a downstream gene of phosphorylated-STAT1, was clearly greater in NK cells from the CHC patients than in those from the HS, while that of IFN-gamma, which is a downstream gene of phosphorylated-STAT4, was clearly lower. CONCLUSIONS: These results indicate altered IFN-alpha signaling in NK cells in CHC patients, suggesting that this alteration is associated with the persistence of HCV infection and resistance to IFN-alpha therapy.