Increase of CD4 + CD25 + regulatory T-cells in the liver of patients with hepatocellular carcinoma Xiu Hua Yang 1,  , Satoshi Yamagiwa 1 , Takafumi Ichida 2, * , Yasunobu Matsuda 1 , Satoshi Sugahara 1 , Hisami Watanabe 3 , Yoshinobu Sato 4 , Toru Abo 5 , David A. Horwitz 6 , Yutaka Aoyagi 1 1 Division of Gastroenterology and Hepatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan 2 Division of Gastroenterology and Hepatology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan 3 Division of Cellular and Molecular Immunology, Center of Molecular Bioscience, University of the Ryukyu, Okinawa, Japan 4 Division of Surgery, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan 5 Division of Immunology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan 6 Division of Rheumatology and Immunology, Department of Internal Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA See Editorial, pages 178–181 Background/ Aims: The immune response to tumor-specific antigens is typically unable to control the growth and spread of malignant cells. Accumulating evidence indicates that the suppressive effects of CD4 + CD25 + regulatory T-cells are at least partially responsible for the failure of immune-mediated elimination of tumor cells. Methods: We have studied 25 patients with hepatocellular carcinoma (HCC). The liver tissues with HCC were separat- ed into the marginal region of tumor (peri-tumor region) and the non-tumor region distant from the tumor. CD4 + CD25 + T-cells were quantified in the blood and the liver by flow cytometry and immunohistochemistry, and their effect on T-cell proliferation and activation was determined. Results: We found a significant increase in both the proportion and absolute numbers of CD4 + CD25 + T-cells in the peri-tumor regions, but not in unaffected areas (9.5 ± 4.5 vs. 4.6 ± 2.8%, P = 0.011). CD4 + CD25 + T-cells isolated from peri-tumor regions displayed phenotype markers characteristic of regulatory T-cells, and expressed Foxp3 mRNA. CD8 + T-cells in peri-tumor regions were inversely proportional to CD4 + CD25 + T-cells in the same region (P < 0.001). Moreover, isolated CD4 + CD25 + T-cells inhibited autologous CD8 + T-cell proliferation. Conclusions: Our results suggest that CD4 + CD25 + T-cells in the marginal region of HCC may play a critical role in controlling CD8 + cytotoxic T-cell activity and, thereby, contribute to the progression of HCC. Ó 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Regulatory T-cells; Tumor immunity; Hepatocellular carcinoma; Intrahepatic lymphocytes; Cytotoxic T lymphocytes 1. Introduction The response of T-cells to self and non-self antigens is controlled by a network of regulatory T (Treg) cells. Many subsets of Treg cells have been described and recently much progress has been made in understand- ing their ontogeny, function, and mechanism of action [1–3]. CD4 + cells that constitutively express CD25, the interleukin-2-receptor a-chain, are generally 0168-8278/$32.00 Ó 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2006.01.036 Received 30 January 2005; received in revised form 14 January 2006; accepted 25 January 2006; available online 9 March 2006 * Corresponding author. Tel.: + 81 55 948 3111; fax: + 81 55 948 5088. E-mail address: takafumi@med.juntendo.ac.jp (T. Ichida). Abbreviations: Ab, antibody; CH, chronic hepatitis; CTL, cytotoxic T lymphocytes; FITC, fluorescein isothiocyanate; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; mAb, mono- clonal antibody; MNCs, mononuclear cells; PBL, peripheral blood lymphocytes;LC, livercirrhosis; LDLT, livingdonor liver transplantation.   Present address: General Surgery Department, The First Hospital Affiliated Harbin medical University, Harbin, China. www.elsevier.com/locate/jhep Journal of Hepatology 45 (2006) 254–262