studies

1240A AASLD ABSTRACTS HEPATOLOGY, October, 2015
way member expression profiles with activation of β-catenin
in tissues from HCV cirrhotic patients not on VD supplementation,
while those supplemented showed restoration of TGF-β
pathway member expression profiles with lower expression
of β-catenin. (3) Strikingly, we observed a threefold higher
incidence of HCC in Smad3 mice deprived of VD compared
to mutant and WT mice fed a high-VD diet. (4) RPPA analyses
revealed an activation of β-catenin, Stat5A, and Bcl2-XL, with
reduced levels of the tumor suppressor PDCD4 in Smad3 +/-
mice deprived of VD diet. (5) VD treatment suppressed HCC
cell proliferation and significantly reduced expression levels
of TLR7 and TLR-mediated inflammation associated genes in
Smad3 -/- MEFs. (6) Functional analyses revealed that loss of
Smad3 and shRNA for VD receptor, induced nuclear localization
and activation of β-catenin. (5) Luciferase and ChIP assays
showed that TGF-β negatively regulates TLR7 transcriptional
activity through Smad3 binding at Smad Binding Elements at
the TLR7 promoter. Conclusions: We conclude that VD suppresses
proliferation of hepatocellular cancer cells by restoring
TGF-β signaling. VD deficiency promotes tumor growth in the
context of Smad3 disruption potentially through regulation of
TLR7 expression and β-catenin nuclear localization. VD could
therefore be a strong candidate for hepatocellular cancer prevention
in the context of aberrant Smad3 signaling.
Disclosures:
Hidekazu Tsukamoto - Consulting: Suntory Ltd.; Grant/Research Support: The
Toray Co.
The following authors have nothing to disclose: Ji-Hyun Shin, Jian Chen, Nina M.
Muñoz, Lior Katz, Andrea C. Cortes, Vivek Shukla, Sangbae Kim, H. Franklin
Herlong, Keigo Machida, Kirti Shetty, Asif Rashid, Wilma S Jogunoori, Aiwu R.
He, Lynt B. Johnson, Ju-Seog Lee, Jon White, Lopa Mishra
2119
Chemokine CCL3/CCR5-recruited, Cytotoxic CD4 + T
Cells Eradicate Hepatoma Cells in a Model of Anticancer
Chemotherapy with High-Dose Cyclophosphamide
Tatsushi Naito 1,2 , Tomohisa Baba 2 , Kazuyoshi Takeda 3 , Soichiro
Sasaki 2 , Naofumi Mukaida 2 , Yasunari Nakamoto 1 ; 1 Second
Department of Internal Medicine, University of Fukui, Fukui, Japan;
2 Division of Molecular Bioregulation, Cancer Research Institute,
Kanazawa University, Kanazawa, Japan; 3 Department of immunology,
Juntendo University, School of Medicine, Tokyo, Japan
BACKGROUND: The mortality rate of hepatocellular carcinoma
(HCC) is high because of frequent recurrence due to
the high carcinogenic potentials of background chronic liver
inflammation and cirrhosis. Thus, novel therapeutic strategy
is required including immune therapy. Today, accumulating
evidences indicate the potential role of CD4 + T cells with direct
cyototoxicity (CD4 + CTLs) in antitumor immunity. Correlation
between decrease of CD4 + CTLs and high mortality is also
reported in patients with HCC. However, the importance of
CD4 + CTLs in patients treated with anticancer chemotherapy
remains to be elucidated. Hence, we examined the role of
CD4 + CTLs, in chemotherapy-induced eradication of hepatoma
by means of an animal HCC model. METHODS and RESULTS:
We inoculated a murine hepatoma cell line, BNL 1ME A.7R.1
(BNL), subcutaneously into Balb/c mice and gave single intraperitoneal
injection of 150 mg/kg cyclophosphamide (CTX)
after tumor formation. CTX treatment eradicated tumors in wildtype
(WT) mice, whereas none of the tumors disappeared in
T cell lacking nude mice. Next, we depleted CD4 + or CD8 +
cells by antibody to clarify which subset was involved in the
efficacy of CTX. We found CD4 + but not CD8 + cell depletion
abrogated CTX-induced tumor eradiation. Consistently, CTX
treatment increased intratumoral CD4 + CTLs, which expressed
cytolytic granule molecule, CD107a and granzyme B observed
by flowcytometry and immunofluorescence analysis. To delineate
the underlying mechanism of increase in CD4 + CTLs, congenic
CD45.1 splenocytes were transferred to tumor-bearing
CD45.2 mice one day after CTX treatment and tumors were
analyzed with time by flowcytometry. Transferred CD4 + cells
were recruited into tumors on day 3, and the recruited cells
expressed CD107a without antigen presentation at draining
lymph nodes and proliferation in tumor tissues. Moreover, CTX
administration enhanced mRNA expression of CC chemokine
CCL3 in tumor tissues, and CTX-mediated tumor regression
was attenuated in mice deficient in CCR5 gene, the receptor
for this chemokine. Consistently, CTX-induced accumulation of
intratumoral CD107a-expressing CD4 + T cells was less in mice
receiving CCR5-deficient mouse-derived splenocytes than those
receiving WT mouse-derived splenocytes. CONCLUSIONS: CC
chemokine CCL3/CCR5 plays an important role in intratumoral
CD4 + CTL migration in the process of CTX-induced tumor eradication.
The results suggest that the CC chemokine-dependent
mechanism may be a plausible strategy to enhance the antitumor
cytotoxicity in the treatment of HCC with chemotherapy.
Disclosures:
The following authors have nothing to disclose: Tatsushi Naito, Tomohisa Baba,
Kazuyoshi Takeda, Soichiro Sasaki, Naofumi Mukaida, Yasunari Nakamoto
2120
IFN-α stimulates IFN-γ expression in type I NKT cells
and enhances the inhibition of HCV replication in
human hepatocyte chimeric mice
Eisuke Miyaki 1 , Michio Imamura 1 , Nobuhiko Hiraga 1 , Takuro
Uchida 1 , Hiromi Kan 1 , Masataka Tsuge 1 , Hiromi Abe 1 , C. Nelson
Hayes 1 , Hiroshi Aikata 1 , Chise Tateno 2 , Kazuaki Chayama 1 ;
1 Department of Gastroenterology and Metabolism, Applied Life
Sciences, Institute of Biomedical & Health Sciences, Hiroshima
University, Hiroshima, Japan; 2 PhoenixBio Co., Ltd., Higashihiroshima,
Japan
Background & Aims: Interferon (IFN) inhibits hepatitis C virus
(HCV) replication through up-regulation of IFN-stimulated gene
(ISG) expression. However, the effect of IFN on immune cells is
not well known. In this study, we analyzed the immune cell-mediated
antiviral effects of IFN-α using HCV-infected mice. Methods:
Genotype 1b HCV-infected urokinase-type plasminogen
activator (uPA)-severe combined immunodeficiency (SCID) mice
with transplanted human hepatocytes were injected intra-peritoneally
with 2 x 10 7 human peripheral blood mononuclear
cells (PBMCs) obtained from a healthy volunteer. The mice then
received daily intra-peritoneal injections with 1000 IU/g of
IFN-α for seven days, during which mouse serum HCV RNA,
human albumin and cytokine levels were analyzed. Flow cytometric
analysis of liver infiltrating human immune cells and
histopathological examination were performed. Results: Seven
days of IFN-α treatment without human PBMC injection in mice
reduced serum HCV RNA levels by 1.23 ± 0.15 log whereas
in combination with PBMCs, HCV RNA levels were reduced
by 2.5 ± 0.45 log (p