Elsevier

Clinical Immunology

Volume 129, Issue 3, December 2008, Pages 428-437
Clinical Immunology

Functional impairment in circulating and intrahepatic NK cells and relative mechanism in hepatocellular carcinoma patients

https://doi.org/10.1016/j.clim.2008.08.012Get rights and content

Abstract

Functional defects in natural killer (NK) cells have been proposed to be responsible for the failure of anti-tumor immune responses. Whether and how NK cells are impaired in hepatocellular carcinoma (HCC) patients remain unknown. In this study, we found that HCC patients displayed a dramatic reduction in peripheral CD56dimCD16pos NK subsets compared with healthy subjects. A significant reduction of CD56dimCD16pos NK subsets was also found in tumor regions compared with non-tumor regions in the livers of these HCC patients. Both these peripheral and tumor-infiltrating NK cells exhibited poorer capacity to produce IFN-γ and kill K562 targets, which was further found to be associated with increased CD4+CD25+ T regulatory cells as we previously-described in HCC patients. Addition of Tregs from HCC patients efficiently inhibited the anti-tumor ability of autologous NK cells in vitro. These findings are helpful for understanding the mechanism of NK cell-mediated anti-tumor immune responses in HCC patients.

Introduction

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, especially among Asian populations [1]. Despite recent advances in new therapeutic modalities, a significant number of HCC patients show frequent recurrence and progress to an advanced stage with few curative options [1]. In this regard, due to the particular resistance of these tumors to cytostatic agents leading to disappointing results using conventional chemotherapy, alternative hepatocellular carcinoma treatment strategies focus on the development of immunomodulatory approaches. Therefore, the identification and manipulation of immune cells or immune molecules may offer new strategies for improving and broadening therapeutic options specifically for advanced HCC.

Natural killer (NK) cells are the major component of innate immunity [2]. According to the intensity of CD56 expression (dim or bright) and the presence or absence of CD16, NK cells were divided into two subsets: CD56brightCD16neg and CD56dimCD16pos. The former provisionally produces cytokines and the latter primarily functions in terms of cytotoxicity levels [3]. NK cells predominantly reside in the liver, in contrast to a relatively small percentage in the peripheral blood [[4], [5], [6]]. Several studies have shown that liver NK cells mediate higher cytotoxic activity against tumor cells than spleen or peripheral blood NK cells in rodents [4], [5], [6]. In patients with a variety of tumors, peripheral NK cell cytotoxicity was found to be significantly decreased compared with non-cancer-bearing controls [7], [8], [9]. This decrease in NK cell cytotoxicity was more marked in patients with advanced disease stages [10], [11], [12]. In particular, NK cells from the liver perfusates of HCC patients displayed a reduced cytotoxicity against the HCC cell line after in vitro IL-2 stimulation, compared with peripheral NK cells and healthy NK cells from donor liver perfusates [13]. This evidence suggests that functional defects in NK cells might be responsible for the failure of anti-tumor immune responses [14]. However, NK cell characteristics in human livers have not been extensively investigated because of the limited availability of appropriate human samples. It is unclear what mechanisms are responsible for impaired NK cytotoxicity, particularly in HCC patients. Our previous study found that circulating regulatory T cell (Treg) frequency increased significantly in HCC patients. Increased Tregs may impair the effector function of CD8 T cells, and thereby promote HCC disease progression [15]. Whether and how these increased Tregs might impair NK cell anti-tumor immune responses in HCC patients remain unknown. Indeed, several studies have indicated that Tregs may execute their suppressive activity through weakening activation signals of NK cells [16], [17], [18].

In this study, we analyzed the numeric, phenotypic, and functional characteristics of peripheral and liver resident NK cell subsets in HCC patients. Our data indicated that NK subsets from HCC patients displayed a dramatic redistribution, and severe impairments in cytotoxic activity and IFN-γ production. This loss of anti-tumor immune responses in NK cells was found to be correlated with the high amount of suppressive Treg cells as we previously-described in HCC patients. These findings are helpful for understanding the mechanism for the failure of NK cell-mediated anti-tumor immune responses.

Section snippets

Study subjects

A total of 110 HCC patients and 69 healthy controls (HCs) were enrolled in this study. HCC diagnosis and stage was determined according to standard imaging or biopsy examination as described previously by our team [15], [19]. These patients did not receive any anti-tumor therapy before sampling. The controls were age- and sex-matched healthy individuals. The clinical background of the HCC patients is shown in Table 1. Peripheral blood samples were obtained from these study subjects. Liver

Dramatic loss of CD56dimCD16pos NK cells mainly contribute to an overall reduction of circulating NK cell pool in HCC patients

We first defined the frequency of circulating NK subsets in HCC patients and HCs using flow cytometry (Fig. 1A). It was found that peripheral proportions of total NK cells were significantly decreased in HCC patients with various stages (mean, 14.29% ± 0.96%) compared to those in HCs (mean, 23.41% ± 1.59%) (Fig. 1B). To gain a more comprehensive understanding of the individual contribution of the two NK cell subsets to the total number of NK cells, we studied the proportions of these two different

Discussion

Functional impairment of NK cells has been identified in cancer patients [24]. However, little information is currently available regarding the mechanisms responsible for functional deficiency of NK cells and their association with disease progression, in particular in HCC patients. This study indicates that the frequency of both peripheral blood and liver CD56dim NK cells decreased, and their function with regard to IFN-γ production and cytotoxicity was impaired in HCC patients. This

Acknowledgments

This study was supported by a grant from the National Key Basic Research Program of China (2006CB504305), the National Youth Foundation of China (30525042).

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    L. Cai and Z. Zhang contributed equally to this study.

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