- Email: [email protected]
Functional impairment in circulating and intrahepatic NK cells and relative mechanism in hepatocellular carcinoma patients
Clinical Immunology (2008) 129, 428–437
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y c l i m
Functional impairment in circulating and intrahepatic NK cells and relative mechanism in hepatocellular carcinoma patients Lun Cai a,b,1 , Zheng Zhang b,1 , Lin Zhou, Haiyan Wang d , Junliang Fu b , Shuye Zhang b , Min Shi b , Hui Zhang b , Yongping Yang c , Hao Wu d , Po Tien a,⁎, Fu-Sheng Wang b,⁎ a
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100012, China Research Center for Biological Therapy, Beijing 302 Hospital, Beijing 100039, China c The Ninth Department of Clinic, Beijing 302 Hospital, Beijing 100039, China d Department of Infectious Diseases, Beijing You-An Hospital Affiliated to Capital Medical University, 100069, China b
Received 11 July 2008; accepted with revision 13 August 2008 Available online 27 September 2008 KEYWORDS NK cells; Regulatory T cells; Liver; Hepatocellular carcinoma
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. Crown Copyright © 2008 Published by Elsevier Inc. All rights reserved.
Abbreviations: APC, allophycocyanin; PE, phycoerythrin; FITC, fluorescein isothiocyanate; PerCP, peridin chlorophyll protein; PBMCs, peripheral blood mononuclear cells; NK, natural killer; Treg, regulatory T cell; LIL, liver-infiltrating lymphocyte; TIL, tumor-infiltrating lymphocyte; NIL, non-tumor-infiltrating lymphocyte; GrA, granzyme A; GrB, granzyme B; HCC, hepatocellular carcinoma; HC, healthy control. ⁎ Corresponding authors. F.-S. Wang is to be contacted at the Research Center for Biological Therapy, Beijing 302 Hospital, 100039, China. Fax: +86 010 63879735. P. Tien, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. E-mail addresses: [email protected] (P. Tien), [email protected] (F.-S. Wang). 1 L. Cai and Z. Zhang contributed equally to this study. 1521-6616/$ – see front matter. Crown Copyright © 2008 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2008.08.012
Functional impairment in NK cells and relative mechanism in HCC patients
Introduction
Materials and methods
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 [4–6]. Several studies have shown that liver NK cells mediate higher cytotoxic activity against tumor cells than spleen or peripheral blood NK cells in rodents [4–6]. In patients with a variety of tumors, peripheral NK cell cytotoxicity was found to be significantly decreased compared with non-cancer-bearing controls [7–9]. This decrease in NK cell cytotoxicity was more marked in patients with advanced disease stages [10– 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 [16–18]. 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.
Study subjects
429
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 tissues were obtained from 10 HCC patients who underwent surgical resection or liver transplantation according to our previously-described protocols [15]. This study was approved by the Ethics Committee of our unit, and written informed consent was obtained from each subject before sampling. Concurrence of HCV and HIV infections and autoimmune or alcoholic liver disease was excluded for all enrolled individuals.
Cell isolation Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll–Hypaque density gradient centrifugation, either from heparinized blood or a leukapheresis-derived PBMCenriched sample. Tumor-infiltrating lymphocytes (TILs) and non-tumor-infiltrating lymphocytes (NILs) were isolated according to our previously-described protocols [20]. NK cells were isolated by positive selection using an NK Positive Isolation Kit (Miltenyi Biotech, Bergisch Gladbach, Germany). CD4+CD25+ Tregs were isolated by CD4-negative selection followed by CD25-positive selection, using a CD4+CD25+ T cell isolation kit according to the manufacturer's instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). The purity of NK cells and CD4+CD25+ Tregs was more than 92%. Unless otherwise stated, freshly isolated cells were cultured in complete RPMI media containing 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 μg/ml streptomycin.
Table 1 Clinical characteristics of HCC patients enrolled in this study Variable
Results
Cases Age (mean ± SD, year) Sex, male/female Child–Pugh classification, A/B/C α-fetoprotein level, N 400/400–200/b 200 Stage, I/II/III a Therapy, ETCT/TACE/resection/LT
110 52.4 ± 10.6 98/12 72/36/2 30/10/70 20/35/55 2/8/3/3
ETCT, endocare targeted cryoablation therapy; TACE, transcatheter hepatic arterial chemoembolization; LT, liver transplantation. a HCC patient disease stage was evaluated according to the Chinese criterion for diagnosis and staging primary liver cancer constituted by the Chinese Anti-cancer Association in 2001.
430
FACS analysis For NK cell receptor staining, PBMCs, NILs or TILs were incubated with peridin chlorophyll protein (PerCP)conjugated anti-CD3 mAb, allophycocyanin (APC)conjugated anti-CD56 mAb, fluorescein isothiocyanate (FITC)-conjugated anti-CD16 mAb (all from BD Biosciences, San Jose, CA), and phycoerythrin (PE)-conjugated antiCD158a, anti-CD158b, anti-NKG2A, anti-NKG2D, anti-NKp30, or anti-NKp46 antibodies (R and D Systems, Minneapolis, MN). For intracellular staining, the cells were permeabilized and further intracellularly stained with FITC-conjugated anti-granzyme A (GrA), anti-granzyme B (GrB), or antiperforin. Cells were then analyzed using FACSCalibur and Flowjo software (TreeStar, Ashland, OR, USA).
Degranulation of NK cells and IFN-γ detection PBMCs were stimulated with PMA (25 ng/ml) and ionomycin (1 μg/ml) or K562 cells (at the ratio of 10:1) for 1 h. Then, FITC-conjugated anti-CD107a and GolgiStop (BD, Pharmingen, San Jose, CA) were directly added into the medium.
L. Cai et al. After 5 h, cells were collected and stained with surface antibodies and intracellularly with anti-IFN-γ.
Cytolytic assay Cytolytic activity of NK cells to lyse K562 was detected using the carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes, Eugene) staining method [21]. In brief, PBMC effectors were activated with IL-2 (200 U/ml) overnight. K562 targets were labeled with CFSE according to our previously-described protocols [22]. IL-2-activated PBMCs were subsequently incubated with CFSE-labeled K562 targets for 4 h at the ratios of 20:1, 10:1, and 5:1. K562 cells alone were used as controls to measure basic spontaneous cell death. The cells were then collected and further stained with 7-aminoactinomycin D (7-AAD, 1 μg/ml). 7AAD-positive K562 cells were identified as dead cells.
Inhibitory assay of Tregs on NK cells To investigate whether Tregs inhibit NK cell function in vitro, the isolated NK cells (1 × 105 cells/well) were first incubated
Figure 1 Circulating NK cells decreased significantly in HCC patients. (A) Representative dot plots of NK cell subsets from HC subjects and HCC patients are shown. CD56bright and CD56dim NK cell subsets are CD56+CD16− and CD56+CD16+ NK cells, respectively. Values in the quadrant indicate the percentages of CD56bright and CD56dim NK cell subsets among lymphocytes. (B–D) Pool data show the frequency of total NK cells (B), CD56bright (C), and CD56dim NK cells (D) in HCC patients and HC subjects. P values were shown. (E) Pool data show the ratio of CD56bright and CD56dim NK subsets in HCC patients and HC subjects. P values are shown. Each dot in (B–E) represents one subject. Horizontal lines illustrate the median percentiles. HC, healthy controls; Stage I, II, III, different stages of hepatocellular carcinoma (HCC).
Functional impairment in NK cells and relative mechanism in HCC patients with autologous Treg cells in a 96-well U-bottom plate at different ratios (10:0, 10:2, 10:10) for 6 h. The mixed cells were then stimulated with PMA (25 ng/ml) and ionomycin (1 μg/ml) for IFN-γ detection, or acted as effector cells cocultured with K562 targets for detecting cytolytic activity of NK cells, according to the aforementioned protocols.
Statistical analysis All data were analyzed using SPSS version 13.0 for Windows software (SPSS Inc., Chicago, IL). Comparison between various individuals was performed using the Mann–Whitney U test. Comparison between the same individual was performed using the Wilcoxon matched pairs T test. For all tests, two-sided P b 0.05 was considered to be significant.
Results
431
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 NK subsets in these individuals. We found that CD56brightCD16neg NK cells were just expanded in HCC patients on stage III (P b 0.05; Fig. 1C). In contrast, CD56dimCD16pos NK cells were significantly reduced in HCC patients regardless of disease stage, when compared with HCs (P b 0.05; Fig. 1D). Thus, the ratio of CD56bright and CD56dim NK cells was found to be significantly increased in HCC patients (Fig. 1E). However, these subset distributions were similar in HCC patients at various stages (Figs. 1B–E). These data indicate that there may be a redistribution in peripheral NK cell compartments of HCC patients.
Dramatic loss of CD56dimCD16pos NK cells mainly contribute to an overall reduction of circulating NK cell pool in HCC patients
Circulating NK cells displayed normal NK receptor expression and decreased granule production in HCC patients
We first defined the frequency of circulating NK subsets in HCC patients and HCs using flow cytometry (Fig. 1A). It was
We further investigated the concentration of cytoplasmic granules (GrA, GrB, and perforin) that are responsible for
Figure 2 Circulating NK cells exhibited decreased granule production and normal NK receptor expression in HCC patients. (A) Granule production by NK cells was significantly reduced in HCC patients with various disease stages, as compared with HC subjects. GrA, granzyme A; GrB, granzyme B. (B) NK receptor expression (CD158a, CD158b, NKG2A, NKG2D, NKp30, and NKp46) in HCC patients with various disease stages was compatible with those of HC subjects.
432 NK cell cytotoxicity, and the receptor expression levels on NK cell subsets (CD158a, CD158b, NKG2A, NKG2D, NKp46 and NKp30). Our data showed that GrA, GrB and perforin production by total NK cells were substantially decreased in advanced HCC patients (stage II or stage III HCC patients) compared to HCs (Fig. 2A). Significant reductions of GrB and perforin expression were also found in stages II HCC patients compared with stage I HCC patients (Fig. 2A). In addition, we found that no significant differences of CD158a, CD158b, NKG2A, NKG2D, NKp46, and NKp30 expression on total NK cell populations exist between HCC patients HCs (Fig. 2B). There was also no significant difference of these receptors on NK cells in various stages of HCC patients (Fig. 2B). These data indicated that circulating NK cell subsets in HCC patients displayed normal NK receptor expression but were functionally impaired in granule production.
L. Cai et al.
Reduced IFN-γ production and cytotoxicity by circulating NK cells of HCC patients To evaluate the functional properties of NK cells, we identified HCC patient capability of producing IFN-γ and degranulating NK cells. We compared IFN-γ production by NK cells upon stimulation with PMA/ionomycin or MHC-devoid K562 cells between HCC patients and HCs (Fig. 3A). Stimulation with PMA/ionomycin can substantially elicit IFN-γ production by NK cells. We found that almost 50% of NK cells have the ability to produce IFN-γ in HC subjects; whereas only less than 5% of NK cells from HCC patients could produce IFN-γ. K562 cells can represent NK specific responses; however, in HCC patients, K562-induced NK cells produced compatible IFN-γ with HC subjects. Pool data further confirmed this observation (Fig. 3B). Unexpectedly, we also found no significant difference in IFN-γ production by
Figure 3 NK cells displayed an impaired capacity of IFN-γ production and cytotoxicity in HCC patients. (A) Representative dot plots of IFN-γ staining on NK cells from HC subjects and HCC patients upon stimulation with PMA/ionomycin and K562 are shown, respectively. Values in the quadrant indicate the percentages of IFN-γ-producing NK cells. (B) Pool data show that PMA/ionomycinbut not K562-induced IFN-γ production was significantly decreased in HCC patients compared to HC subjects. (C) CD107a expression by NK cells was compatible in HCC patients with HC subjects after PMA and K562 stimulation. (D) Cytotoxic activity of NK cells from HCC patients is lower than that of HC subjects when incubated with K562 targets in different ratios (30:1, 10:1, 3:1). ⁎P values were less than 0.05.
Functional impairment in NK cells and relative mechanism in HCC patients NK cell subsets upon stimulation with PMA/ionomycin and K562 cells in various-staged HCC patients (Fig. 3B). These data indicate that IFN-γ-producing capacity of NK cells was severely impaired in HCC patients. Degranulation of intracellular vesicles by lymphocytes, reflecting their cytotoxic activity, can be measured using the marker CD107a (LAMP-1), as described recently for NK cells [23]. To analyze the CD107a expression on NK cells, NK cells were activated with PMA/ionomycin or K562 cells in HCC patients and HC subjects. We found no significant difference in CD107a expression by NK cells among various-staged HCC patient and healthy subjects (Fig. 3C). These data suggested that circulating NK cells from HCC patients displayed a normal capacity of degranulation, although they contained decreased levels of granule production, as aforementioned. We also defined cytotoxicity of peripheral NK cells against target cells in HCC patients. Upon activation of PBMCs by rIL-2 stimulation in vitro overnight, effectors and target cells were incubated at different E/T ratios. We found that the cytolytic activity of peripheral NK cells against the NK-susceptible K562 targets was markedly reduced in HCC patients as compared with IL-2-activated NK cells from HC individuals (Fig. 3D). We further analyzed the expression of IL-2 receptors on NK cells. It is found that NK cells from HCC patients expressed compatible levels of IL-2 receptors (including IL-2Rα chain, CD25; IL-2Rβ chain, CD122 and
433
IL-2Rγ chain, CD132) with that from HC subjects (data not shown). These data indicated that NK cells from HCC patients processed an intrinsical impairment of cytolytic activity, and excluded the possibility that the low levels of IL-2 receptor expression on NK cells lead to poor responsiveness to IL-2 and subsequent loss of NK cell cytolytic activity in HCC patients.
TILs of advanced-stage HCC patients contained reduced population of NK cells Based on the observation of reduced circulating NK cell subsets in HCC patients, we further investigated liver resident NK cells in HCC patients. Due to lack of healthy livers, we therefore compared the proportion of NK subsets in peripheral blood, TILs, and NILs in HCC patients. It is found that the CD56dimCD16pos NK cell subset accounted for the majority of peripheral NK cells; whereas in both TILs and NILs, the proportion of CD56brightCD16neg NK cell subsets significantly increased (Fig. 4A). Notably, although total peripheral NK cell frequency was found to be decreased in the HCC patient, intrahepatic NK cells were found to be accumulated largely in the site of the same HCC patient. In particular, NILs contained more NK cells than TILs in the HCC patient (Fig. 4A). We further performed a comprehensive analysis in 6 HCC patients on the stage III. It is found that overall NK cell frequency was significantly decreased in TILs
Figure 4 HCC patients show a markedly reduced population of NK cells in TILs. (A) Representative dot plots of NK cell subsets in PBMC, NIL, and TIL from an HCC patient. Values in the quadrant indicate the percentages of total NK cells among lymphocytes. (B) Pool data indicated the frequencies of total, CD56bright, and CD56dim NK subsets in TILs and NILs from HCC patients. TIL, tumor-infiltrating lymphocyte; NIL, non-tumor-infiltrating lymphocyte.
434 compared with NILs (P b 0.05; Fig. 4B). We also found that the reduction of CD56dimCD16pos NK cells was largely responsible for the decrease in total NK cell numbers in TILs (P b 0.05), because the frequency of CD56brightCD16neg NK cells was similar in NILs and TILs in these HCC livers (Fig. 4B). Thus, a more comprehensive analysis of the individual contribution of the 2 NK cell subsets to the total number of liver NK cells was confirmed in the livers of HCC patients. These data demonstrated that intrahepatic NK cell compartment was also dramatically redistributed in HCC patients.
TILs of advanced-stage HCC patients displayed decreased granule production and impaired cytolytic ability compared with NILs We further compared the cytolytic capacity of intrahepatic NK cell subsets between TILs and NILs in these HCC patients. We first detected the content of cytolytic granules (GrA, GrB, and perforin) in liver NK cells of a HCC patient, and found that the proportion of GrA, GrB, and perforin positive NK cells in TILs were significantly lower than those in NILs (Fig. 5A). Pool data further confirmed the observation (Fig. 5B). We also detected CD107a expression by NIL-NK and TIL-NK cells, and failed to find a significant difference in
L. Cai et al. CD107a expression on these two populations of NK cells (data not shown). Moreover, we detected the cytolytic activity of liver-infiltrating NK cells. We found that TIL-NK cells possessed a lower capacity to kill targets than NIL-NK cells at the 30:1 of E/T ratio rather than 10:1 or 3:1 ratios (Fig. 5C). Thus, the decrease of granule proportion in TIL-NK cells in HCC patients might associate with the loss of cytotoxic capacity in the overall NK cells in HCC patients.
NK cell function is suppressed in the presence of CD4+CD25+ Tregs Our previous study indicated that circulating Treg frequency was increased significantly in HCC patients [15]. Whether or not the increased Tregs might impair NK cell anti-tumor immune responses in HCC patients remains unknown. We therefore detected the capacity to produce IFN-γ and kill K562 targets of Treg-treated NK cells in vitro. It is found that Treg-treated NK cells produced less IFN-γ compared with those untreated NK cells. More Tregs displayed stronger suppressive effects on IFN-γ production by NK cells (Fig. 6A). Tanswell assay showed the immunosuppression of Treg on NK cell-derived IFN-γ was mediated by direct cell contacts (data not shown). These data suggest that the increased peripheral
Figure 5 TIL-derived NK cells exhibited a significant decrease in granule production in HCC patients. (A) Representative expression profile of the GrA, GrB, and perforin by TIL- and NIL-derived NK cells in an HCC patient. NK cells are gated from CD3−CD56+ lymphocytes. Values in the quadrant indicate the percentages of GrA-, GrB-, and perforin-expressing NK cells. (B) Pool data indicated that the numbers of GrA-, GrB-, and perforin-expressing NK cells were all reduced in TILs compared with NILs from HCC patients. P values were shown. (C) Cytotoxic activity against K562 targets in TIL-derived NK cells is significantly decreased as compared with NIL-derived NK cells at the indicated ratios (30:1, 10:1, 3:1). ⁎P values were less than 0.05.
Functional impairment in NK cells and relative mechanism in HCC patients
435
Figure 6 NK cells are suppressed by autologous CD4+CD25+ Tregs. (A) Representative dot plots showed that Treg cells from an HCC patient suppressed IFN-γ production by autologous NK cells (n = 5). NK cells and Tregs were mixed at different ratios. Values in the quadrant indicate the percentages of IFN-γ-producing NK cells. (B) Representative dot plots show the inhibitory effects of Treg cells on autologous NK cell cytolytic activity against K562 targets (n = 4). Values in the quadrant indicate the percentages of 7-AAD positive CFSE-labeled K562 cells. (C) Trans-well assay showed that Treg cells suppressed NK cell cytotoxicity against K562 targets in a manner of cell–cell contacts (n = 4). Values out the quadrant indicate the percentages of 7-AAD positive CFSE-labeled K562 cells.
Tregs in HCC patients might contribute to the impaired IFN-γ production by autologous NK cells. We further investigated whether increased Tregs impaired the cytotoxic capacity of NK cells in HCC patients. We found that K562 cells exhibited few spontaneous cell deaths, as indicated by 7-AAD staining. The isolated Tregs also processed a poor capacity to kill K562 targets. In contrast, NK cells could kill more than 40% of K562 targets at the ratio of 10:1. Importantly, we found that Treg (2 × 105) treatment significantly reduced the cytotoxicity of NK cells at the ratio of 10:1 (NK:K562). Addition of more Tregs (10 × 105) largely reduced NK cell cytotoxicity by nearly 50% (Fig. 6B). Notably, separation of Tregs and NK cells in transwell assay significantly led to the loss of immunosuppression of Treg on NK cells killing K562 targets, indicating that Tregs inhibit NK cell anti-tumor activity dependent on direct cell–cell contacts (Fig. 6C). These data indicated that the increased circulating Tregs in HCC patients may diminish the cytotoxicity of NK cells. Thus, our data raised a possibility that the increased Tregs might contribute to the loss of anti-tumor immune responses in HCC patients.
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 functional impairment was found to be associated with the increased Tregs in these HCC patients. These findings revealed that NK cells lose anti-tumor immune responses in HCC patients.
We found a significant reduction of total proportion of peripheral NK cell compartments in HCC patients with different disease stages compared to healthy subjects. This reduction of total NK population mainly results from a substantial decrease in the peripheral CD56dimCD16pos NK cell subset; by contrast, circulating CD56brightCD16neg NK cells increased in HCC patients. Similarly, in the livers of HCC patients we found a significant reduction of CD56dimCD16pos NK cell subsets in tumor regions compared with non-tumor regions in HCC patients, suggesting that local environment is more important for the regulation of NK cell functions in HCC patients. These data indicated that both peripheral and intrahepatic NK cell subsets were largely redistributed in HCC patients. In particular, this decrease of circulating and intra-tumor CD16pos NK cell subset might contribute to reduction of the cytotoxic activity of NK cells since CD16 plays a key role in killing target cells in an ADCC manner [25–27]. More important, we found that both peripheral and tumor-infiltrating NK cells exhibited functional deficiency in producing IFN-γ and killing K562 targets compared with healthy peripheral NK cells and non-tumor-infiltrating NK cells, respectively. These findings suggest that the functional impairment of NK cells might severely hinder the anti-tumor immune responses of HCC patients; however, the mechanisms responsible for impaired NK function remain unknown in HCC patients. We therefore sought to determine the factors leading to the functional impairment of NK cells in HCC patients. One major manner by which NK cells exert their cytolytic function is by releasing cytoplasmic granules containing perforin and granzymes [2,28–31]. Our data supported that the reduced cytoplasmic granules, to some extent, might contribute to impaired cytotoxicity of NK cells in these HCC patients since CD107a expression, as a marker of measuring granule-releasing capacity [23] in HCC patients, was at the compatible levels with healthy subjects. Notably, circulating NK cells in HCC patients displayed
436 normal NK receptor and IL-2 receptor expression, indicating that HCC patients did display an intrinsical impairment of cytolytic activity. Future study should further investigate whether these receptor-mediated signaling was impaired in NK cells of HCC patients. Our previous study showed that peripheral Treg frequency increased significantly in HCC patients. These increased Tregs may impair the effector function of CD8 T cells and promote disease progression of HCC [15]. In this study, we proposed that this increased Treg frequency might impair NK-mediated immune responses. Our data supported the notion that NK cells, being pre-incubated with autologous Tregs, largely lost anti-tumor activity indicated by the decreased capacity of NK cells to produce IFN-γ and kill K562 targets. In addition, this suppression of Tregs on NK cells was dependent on a direct cell-to-cell contact, at least in vitro. Indeed, several studies have indicated that Tregs may execute their suppressive activity through weakening activation signals on NK cells, which is most often described to be independent of immunosuppressive cytokines, as antibodies to TGF-β or IL-10 do not influence Treg-mediated suppression [16,32]. Unexpectedly, we found that this functional impairment of NK cells was independent of disease stage; by contrast, the Treg frequency was found to be associated with disease progression in HCC patients. This differential association with disease progression in HCC patients also suggested that reasons other than suppression from Tregs are most likely responsible for impaired NK cell function in HCC patients. Consequently, this study demonstrates peripheral and tumor-infiltrating NK cell subsets were redistributed, and subsequently result in a severe loss of NK cells in anti-tumor activity of HCC patients. This functional abnormity of NK subsets might associate with the increased Tregs as we previously described in HCC patients. This finding provides an insight into the mechanisms of NK cell function impairment in HCC patients. This study will be of importance in the future development of NK-based therapeutic strategies against tumors.
Acknowledgments
L. Cai et al.
[6]
[7]
[8]
[9]
[10]
[11] [12]
[13]
[14]
[15]
[16]
This study was supported by a grant from the National Key Basic Research Program of China (2006CB504305), the National Youth Foundation of China (30525042).
References [1] J.M. Llovet, A. Burroughs, J. Bruix, Hepatocellular carcinoma, Lancet 362 (2003) 1907–1917. [2] A. Kawasaki, Y. Shinkai, H. Yagita, K. Okumura, Expression of perforin in murine natural killer cells and cytotoxic T lymphocytes in vivo, Eur. J. Immunol. 22 (1992) 1215–1219. [3] M.A. Cooper, M.A. Caligiuri, Isolation and characterization of human natural killer cell subsets, Curr. Protoc. Immunol. Chapter 7 (2004) Unit 7–34. [4] J.J. Subleski, V.L. Hall, T.C. Back, J.R. Ortaldo, R.H. Wiltrout, Enhanced antitumor response by divergent modulation of natural killer and natural killer T cells in the liver, Cancer Res. 66 (2006) 11005–11012. [5] X. Bai, S. Wang, C. Tomiyama-Miyaji, J. Shen, T. Taniguchi, N. Izumi, C. Li, H.Y. Bakir, T. Nagura, S. Takahashi, T. Kawamura, T.
[17]
[18]
[19]
[20]
Iiai, H. Okamoto, K. Hatakeyama, T. Abo, Transient appearance of hepatic natural killer cells with high cytotoxicity and unique phenotype in very young mice, Scan. J. Immunol. 63 (2006) 275–281. D. Vermijlen, C. Seynaeve, D. Luo, M. Kruhoffer, D.L. Eizirik, T.F. Orntoft, E. Wisse, High-density oligonucleotide array analysis reveals extensive differences between freshly isolated blood and hepatic natural killer cells, Eur. J. Immunol. 34 (2004) 2529–2540. M. Jarahian, C. Watzl, Y. Issa, P. Altevogt, F. Momburg, Blockade of natural killer cell-mediated lysis by NCAM140 expressed on tumor cells, Int. J. Cancer 120 (2007) 2625–2634. W. Cao, X. Xi, Z. Hao, W. Li, Y. Kong, L. Cui, C. Ma, D. Ba, W. He, RAET1E2, a soluble isoform of the UL16-binding protein RAET1E produced by tumor cells, inhibits NKG2D-mediated NK cytotoxicity, J. Biol. Chem. 282 (2007) 18922–18928. Y. Kano, K. Soda, T. Nakamura, M. Saitoh, M. Kawakami, F. Konishi, Increased blood spermine levels decrease the cytotoxic activity of lymphokine-activated killer cells: a novel mechanism of cancer evasion, Cancer Immunol. Immunother. 56 (2007) 771–781. V. Jovic, G. Konjevic, S. Radulovic, S. Jelic, I. Spuzic, Impaired perforin-dependent NK cell cytotoxicity and proliferative activity of peripheral blood T cells is associated with metastatic melanoma, Tumori. 87 (2001) 324–329. H.F. Pross, E. Lotzova, Role of natural killer cells in cancer, Nat. Immunol. 12 (1993) 279–292. E.H. Steinhauer, A.T. Doyle, J. Reed, A.S. Kadish, Defective natural cytotoxicity in patients with cancer: normal number of effector cells but decreased recycling capacity in patients with advanced disease, J. Immunol. 129 (1982) 2255–2259. K. Ishiyama, H. Ohdan, M. Ohira, H. Mitsuta, K. Arihiro, T. Asahara, Difference in cytotoxicity against hepatocellular carcinoma between liver periphery natural killer cells in humans, Hepatology 43 (2006) 362–372. M.W. Sung, J.T. Johnson, G. Van Dongen, T.L. Whiteside, Protective effects of interferon-gamma on squamous-cell carcinoma of head and neck targets in antibody-dependent cellular cytotoxicity mediated by human natural killer cells, Int. J. Cancer 66 (1996) 393–399. J. Fu, D. Xu, Z. Liu, M. Shi, P. Zhao, B. Fu, Z. Zhang, H. Yang, H. Zhang, C. Zhou, J. Yao, L. Jin, H. Wang, Y. Yang, Y.X. Fu, F.S. Wang, Increased regulatory T cells correlate with CD8 T-cell impairment and poor survival in hepatocellular carcinoma patients, Gastroenterology 132 (2007) 2328–2339. F. Ghiringhelli, C. Menard, M. Terme, C. Flament, J. Taieb, N. Chaput, P.E. Puig, S. Novault, B. Escudier, E. Vivier, A. Lecesne, C. Robert, J.Y. Blay, J. Bernard, S. Caillat-Zucman, A. Freitas, T. Tursz, O. Wagner-Ballon, C. Capron, W. Vainchencker, F. Martin, L. Zitvogel, CD4+CD25+ regulatory Tcells inhibit natural killer cell functions in a transforming growth factor-betadependent manner, J. Exp. Med. 202 (2005) 1075–1085. M.J. Smyth, M.W. Teng, J. Swann, K. Kyparissoudis, D.I. Godfrey, Y. Hayakawa, CD4+CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of cancer, J. Immunol. 176 (2006) 1582–1587. P. Trzonkowski, E. Szmit, J. Mysliwska, A. Dobyszuk, A. Mysliwski, CD4+CD25+ T regulatory cells inhibit cytotoxic activity of T CD8+ and NK lymphocytes in the direct cellto-cell interaction, Clin. Immunol. 112 (2004) 258–267. Z.W. Sheng, F.S. Wu, Z.M. Ma, Y.Z. Feng, X.R. Zhou, L.S. Teng, The Chinese classification system compared with TNM staging in prognosis of patients with primary hepatic carcinoma after resection, Hepatobiliary Pancreat. Dis. Int. 4 (2001) 561–564. D. Xu, J. Fu, L. Jin, H. Zhang, C. Zhou, Z. Zou, J.M. Zhao, B. Zhang, M. Shi, X. Ding, Z. Tang, Y.X. Fu, F.S. Wang, Circulating and liver resident CD4+CD25+ regulatory T cells actively influence the antiviral immune response and disease
Functional impairment in NK cells and relative mechanism in HCC patients
[21]
[22]
[23]
[24]
[25]
[26]
progression in patients with hepatitis B, J. Immunol. 177 (2006) 739–747. H. Lecoeur, M. Fevrier, S. Garcia, Y. Riviere, M.L. Gougeon, A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity, J. Immunol. Methods 253 (2001) 177–187. Z. Zhang, J.Y. Zhang, E.J. Wherry, B. Jin, B. Xu, Z.S. Zou, S.Y. Zhang, B.S. Li, H.F. Wang, H. Wu, G.K. Lau, Y.X. Fu, F.S. Wang, Dynamic programmed death 1 expression by virus-specific CD8 T cells correlates with the outcome of acute hepatitis B, Gastroenterology 134 (2008) 1938–1949. G. Alter, J.M. Malenfant, M. Altfeld, CD107a as a functional marker for the identification of natural killer cell activity, J. Immunol. Methods 294 (2004) 15–22. C.M. Balch, A.B. Tilden, P.A. Dougherty, G.A. Cloud, T. Abo, Heterogeneity of natural killer lymphocyte abnormalities in colon cancer patients, Surgery 95 (1984) 63–70. R. Gazit, M. Aker, M. Elboim, H. Achdout, G. Katz, D.G. Wolf, S. Katzav, O. Mandelboim, NK cytotoxicity mediated by CD16 but not by NKp30 is functional in Griscelli syndrome, Blood 109 (2007) 4306–4312. O. Mandelboim, P. Malik, D.M. Davis, C.H. Jo, J.E. Boyson, J.L. Strominger, Human CD16 as a lysis receptor mediating direct natural killer cell cytotoxicity, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 5640–5644.
437
[27] J.S. Schleypen, N. Baur, R. Kammerer, P.J. Nelson, K. Rohrmann, E.F. Grone, M. Hohenfellner, A. Haferkamp, H. Pohla, D.J. Schendel, C.S. Falk, E. Noessner, Cytotoxic markers and frequency predict functional capacity of natural killer cells infiltrating renal cell carcinoma, Clin. Cancer Res. 12 (2006) 718–725. [28] T. Koga, H. Harada, T.S. Shi, S. Okada, M.A. Suico, T. Shuto, H. Kai, Hyperthermia suppresses the cytotoxicity of NK cells via down-regulation of perforin/granzyme B expression, Biochem. Biophys. Res. Commun. 337 (2005) 1319–1323. [29] Y. Hayakawa, J.M. Kelly, J.A. Westwood, P.K. Darcy, A. Diefenbach, D. Raulet, M.J. Smyth, Cutting edge: tumor rejection mediated by NKG2D receptor–ligand interaction is dependent upon perforin, J. Immunol. 169 (2002) 5377–5381. [30] V. Screpanti, R.P. Wallin, H.G. Ljunggren, A. Grandien, A central role for death receptor-mediated apoptosis in the rejection of tumors by NK cells, J. Immunol. 167 (2001) 2068–2073. [31] M.J. Smyth, K.Y. Thia, E. Cretney, J.M. Kelly, M.B. Snook, C.A. Forbes, A.A. Scalzo, Perforin is a major contributor to NK cell control of tumor metastasis, J. Immunol. 162 (1999) 6658–6662. [32] M. Terme, N. Chaput, B. Combadiere, A. Ma, T. Ohteki, L. Zitvogel, Regulatory T cells control dendritic cell/NK cell crosstalk in lymph nodes at the steady state by inhibiting CD4+ selfreactive T cells, J. Immunol. 180 (2008) 4679–4686.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y c l i m
Functional impairment in circulating and intrahepatic NK cells and relative mechanism in hepatocellular carcinoma patients Lun Cai a,b,1 , Zheng Zhang b,1 , Lin Zhou, Haiyan Wang d , Junliang Fu b , Shuye Zhang b , Min Shi b , Hui Zhang b , Yongping Yang c , Hao Wu d , Po Tien a,⁎, Fu-Sheng Wang b,⁎ a
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100012, China Research Center for Biological Therapy, Beijing 302 Hospital, Beijing 100039, China c The Ninth Department of Clinic, Beijing 302 Hospital, Beijing 100039, China d Department of Infectious Diseases, Beijing You-An Hospital Affiliated to Capital Medical University, 100069, China b
Received 11 July 2008; accepted with revision 13 August 2008 Available online 27 September 2008 KEYWORDS NK cells; Regulatory T cells; Liver; Hepatocellular carcinoma
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. Crown Copyright © 2008 Published by Elsevier Inc. All rights reserved.
Abbreviations: APC, allophycocyanin; PE, phycoerythrin; FITC, fluorescein isothiocyanate; PerCP, peridin chlorophyll protein; PBMCs, peripheral blood mononuclear cells; NK, natural killer; Treg, regulatory T cell; LIL, liver-infiltrating lymphocyte; TIL, tumor-infiltrating lymphocyte; NIL, non-tumor-infiltrating lymphocyte; GrA, granzyme A; GrB, granzyme B; HCC, hepatocellular carcinoma; HC, healthy control. ⁎ Corresponding authors. F.-S. Wang is to be contacted at the Research Center for Biological Therapy, Beijing 302 Hospital, 100039, China. Fax: +86 010 63879735. P. Tien, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. E-mail addresses: [email protected] (P. Tien), [email protected] (F.-S. Wang). 1 L. Cai and Z. Zhang contributed equally to this study. 1521-6616/$ – see front matter. Crown Copyright © 2008 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2008.08.012
Functional impairment in NK cells and relative mechanism in HCC patients
Introduction
Materials and methods
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 [4–6]. Several studies have shown that liver NK cells mediate higher cytotoxic activity against tumor cells than spleen or peripheral blood NK cells in rodents [4–6]. In patients with a variety of tumors, peripheral NK cell cytotoxicity was found to be significantly decreased compared with non-cancer-bearing controls [7–9]. This decrease in NK cell cytotoxicity was more marked in patients with advanced disease stages [10– 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 [16–18]. 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.
Study subjects
429
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 tissues were obtained from 10 HCC patients who underwent surgical resection or liver transplantation according to our previously-described protocols [15]. This study was approved by the Ethics Committee of our unit, and written informed consent was obtained from each subject before sampling. Concurrence of HCV and HIV infections and autoimmune or alcoholic liver disease was excluded for all enrolled individuals.
Cell isolation Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll–Hypaque density gradient centrifugation, either from heparinized blood or a leukapheresis-derived PBMCenriched sample. Tumor-infiltrating lymphocytes (TILs) and non-tumor-infiltrating lymphocytes (NILs) were isolated according to our previously-described protocols [20]. NK cells were isolated by positive selection using an NK Positive Isolation Kit (Miltenyi Biotech, Bergisch Gladbach, Germany). CD4+CD25+ Tregs were isolated by CD4-negative selection followed by CD25-positive selection, using a CD4+CD25+ T cell isolation kit according to the manufacturer's instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). The purity of NK cells and CD4+CD25+ Tregs was more than 92%. Unless otherwise stated, freshly isolated cells were cultured in complete RPMI media containing 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 μg/ml streptomycin.
Table 1 Clinical characteristics of HCC patients enrolled in this study Variable
Results
Cases Age (mean ± SD, year) Sex, male/female Child–Pugh classification, A/B/C α-fetoprotein level, N 400/400–200/b 200 Stage, I/II/III a Therapy, ETCT/TACE/resection/LT
110 52.4 ± 10.6 98/12 72/36/2 30/10/70 20/35/55 2/8/3/3
ETCT, endocare targeted cryoablation therapy; TACE, transcatheter hepatic arterial chemoembolization; LT, liver transplantation. a HCC patient disease stage was evaluated according to the Chinese criterion for diagnosis and staging primary liver cancer constituted by the Chinese Anti-cancer Association in 2001.
430
FACS analysis For NK cell receptor staining, PBMCs, NILs or TILs were incubated with peridin chlorophyll protein (PerCP)conjugated anti-CD3 mAb, allophycocyanin (APC)conjugated anti-CD56 mAb, fluorescein isothiocyanate (FITC)-conjugated anti-CD16 mAb (all from BD Biosciences, San Jose, CA), and phycoerythrin (PE)-conjugated antiCD158a, anti-CD158b, anti-NKG2A, anti-NKG2D, anti-NKp30, or anti-NKp46 antibodies (R and D Systems, Minneapolis, MN). For intracellular staining, the cells were permeabilized and further intracellularly stained with FITC-conjugated anti-granzyme A (GrA), anti-granzyme B (GrB), or antiperforin. Cells were then analyzed using FACSCalibur and Flowjo software (TreeStar, Ashland, OR, USA).
Degranulation of NK cells and IFN-γ detection PBMCs were stimulated with PMA (25 ng/ml) and ionomycin (1 μg/ml) or K562 cells (at the ratio of 10:1) for 1 h. Then, FITC-conjugated anti-CD107a and GolgiStop (BD, Pharmingen, San Jose, CA) were directly added into the medium.
L. Cai et al. After 5 h, cells were collected and stained with surface antibodies and intracellularly with anti-IFN-γ.
Cytolytic assay Cytolytic activity of NK cells to lyse K562 was detected using the carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes, Eugene) staining method [21]. In brief, PBMC effectors were activated with IL-2 (200 U/ml) overnight. K562 targets were labeled with CFSE according to our previously-described protocols [22]. IL-2-activated PBMCs were subsequently incubated with CFSE-labeled K562 targets for 4 h at the ratios of 20:1, 10:1, and 5:1. K562 cells alone were used as controls to measure basic spontaneous cell death. The cells were then collected and further stained with 7-aminoactinomycin D (7-AAD, 1 μg/ml). 7AAD-positive K562 cells were identified as dead cells.
Inhibitory assay of Tregs on NK cells To investigate whether Tregs inhibit NK cell function in vitro, the isolated NK cells (1 × 105 cells/well) were first incubated
Figure 1 Circulating NK cells decreased significantly in HCC patients. (A) Representative dot plots of NK cell subsets from HC subjects and HCC patients are shown. CD56bright and CD56dim NK cell subsets are CD56+CD16− and CD56+CD16+ NK cells, respectively. Values in the quadrant indicate the percentages of CD56bright and CD56dim NK cell subsets among lymphocytes. (B–D) Pool data show the frequency of total NK cells (B), CD56bright (C), and CD56dim NK cells (D) in HCC patients and HC subjects. P values were shown. (E) Pool data show the ratio of CD56bright and CD56dim NK subsets in HCC patients and HC subjects. P values are shown. Each dot in (B–E) represents one subject. Horizontal lines illustrate the median percentiles. HC, healthy controls; Stage I, II, III, different stages of hepatocellular carcinoma (HCC).
Functional impairment in NK cells and relative mechanism in HCC patients with autologous Treg cells in a 96-well U-bottom plate at different ratios (10:0, 10:2, 10:10) for 6 h. The mixed cells were then stimulated with PMA (25 ng/ml) and ionomycin (1 μg/ml) for IFN-γ detection, or acted as effector cells cocultured with K562 targets for detecting cytolytic activity of NK cells, according to the aforementioned protocols.
Statistical analysis All data were analyzed using SPSS version 13.0 for Windows software (SPSS Inc., Chicago, IL). Comparison between various individuals was performed using the Mann–Whitney U test. Comparison between the same individual was performed using the Wilcoxon matched pairs T test. For all tests, two-sided P b 0.05 was considered to be significant.
Results
431
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 NK subsets in these individuals. We found that CD56brightCD16neg NK cells were just expanded in HCC patients on stage III (P b 0.05; Fig. 1C). In contrast, CD56dimCD16pos NK cells were significantly reduced in HCC patients regardless of disease stage, when compared with HCs (P b 0.05; Fig. 1D). Thus, the ratio of CD56bright and CD56dim NK cells was found to be significantly increased in HCC patients (Fig. 1E). However, these subset distributions were similar in HCC patients at various stages (Figs. 1B–E). These data indicate that there may be a redistribution in peripheral NK cell compartments of HCC patients.
Dramatic loss of CD56dimCD16pos NK cells mainly contribute to an overall reduction of circulating NK cell pool in HCC patients
Circulating NK cells displayed normal NK receptor expression and decreased granule production in HCC patients
We first defined the frequency of circulating NK subsets in HCC patients and HCs using flow cytometry (Fig. 1A). It was
We further investigated the concentration of cytoplasmic granules (GrA, GrB, and perforin) that are responsible for
Figure 2 Circulating NK cells exhibited decreased granule production and normal NK receptor expression in HCC patients. (A) Granule production by NK cells was significantly reduced in HCC patients with various disease stages, as compared with HC subjects. GrA, granzyme A; GrB, granzyme B. (B) NK receptor expression (CD158a, CD158b, NKG2A, NKG2D, NKp30, and NKp46) in HCC patients with various disease stages was compatible with those of HC subjects.
432 NK cell cytotoxicity, and the receptor expression levels on NK cell subsets (CD158a, CD158b, NKG2A, NKG2D, NKp46 and NKp30). Our data showed that GrA, GrB and perforin production by total NK cells were substantially decreased in advanced HCC patients (stage II or stage III HCC patients) compared to HCs (Fig. 2A). Significant reductions of GrB and perforin expression were also found in stages II HCC patients compared with stage I HCC patients (Fig. 2A). In addition, we found that no significant differences of CD158a, CD158b, NKG2A, NKG2D, NKp46, and NKp30 expression on total NK cell populations exist between HCC patients HCs (Fig. 2B). There was also no significant difference of these receptors on NK cells in various stages of HCC patients (Fig. 2B). These data indicated that circulating NK cell subsets in HCC patients displayed normal NK receptor expression but were functionally impaired in granule production.
L. Cai et al.
Reduced IFN-γ production and cytotoxicity by circulating NK cells of HCC patients To evaluate the functional properties of NK cells, we identified HCC patient capability of producing IFN-γ and degranulating NK cells. We compared IFN-γ production by NK cells upon stimulation with PMA/ionomycin or MHC-devoid K562 cells between HCC patients and HCs (Fig. 3A). Stimulation with PMA/ionomycin can substantially elicit IFN-γ production by NK cells. We found that almost 50% of NK cells have the ability to produce IFN-γ in HC subjects; whereas only less than 5% of NK cells from HCC patients could produce IFN-γ. K562 cells can represent NK specific responses; however, in HCC patients, K562-induced NK cells produced compatible IFN-γ with HC subjects. Pool data further confirmed this observation (Fig. 3B). Unexpectedly, we also found no significant difference in IFN-γ production by
Figure 3 NK cells displayed an impaired capacity of IFN-γ production and cytotoxicity in HCC patients. (A) Representative dot plots of IFN-γ staining on NK cells from HC subjects and HCC patients upon stimulation with PMA/ionomycin and K562 are shown, respectively. Values in the quadrant indicate the percentages of IFN-γ-producing NK cells. (B) Pool data show that PMA/ionomycinbut not K562-induced IFN-γ production was significantly decreased in HCC patients compared to HC subjects. (C) CD107a expression by NK cells was compatible in HCC patients with HC subjects after PMA and K562 stimulation. (D) Cytotoxic activity of NK cells from HCC patients is lower than that of HC subjects when incubated with K562 targets in different ratios (30:1, 10:1, 3:1). ⁎P values were less than 0.05.
Functional impairment in NK cells and relative mechanism in HCC patients NK cell subsets upon stimulation with PMA/ionomycin and K562 cells in various-staged HCC patients (Fig. 3B). These data indicate that IFN-γ-producing capacity of NK cells was severely impaired in HCC patients. Degranulation of intracellular vesicles by lymphocytes, reflecting their cytotoxic activity, can be measured using the marker CD107a (LAMP-1), as described recently for NK cells [23]. To analyze the CD107a expression on NK cells, NK cells were activated with PMA/ionomycin or K562 cells in HCC patients and HC subjects. We found no significant difference in CD107a expression by NK cells among various-staged HCC patient and healthy subjects (Fig. 3C). These data suggested that circulating NK cells from HCC patients displayed a normal capacity of degranulation, although they contained decreased levels of granule production, as aforementioned. We also defined cytotoxicity of peripheral NK cells against target cells in HCC patients. Upon activation of PBMCs by rIL-2 stimulation in vitro overnight, effectors and target cells were incubated at different E/T ratios. We found that the cytolytic activity of peripheral NK cells against the NK-susceptible K562 targets was markedly reduced in HCC patients as compared with IL-2-activated NK cells from HC individuals (Fig. 3D). We further analyzed the expression of IL-2 receptors on NK cells. It is found that NK cells from HCC patients expressed compatible levels of IL-2 receptors (including IL-2Rα chain, CD25; IL-2Rβ chain, CD122 and
433
IL-2Rγ chain, CD132) with that from HC subjects (data not shown). These data indicated that NK cells from HCC patients processed an intrinsical impairment of cytolytic activity, and excluded the possibility that the low levels of IL-2 receptor expression on NK cells lead to poor responsiveness to IL-2 and subsequent loss of NK cell cytolytic activity in HCC patients.
TILs of advanced-stage HCC patients contained reduced population of NK cells Based on the observation of reduced circulating NK cell subsets in HCC patients, we further investigated liver resident NK cells in HCC patients. Due to lack of healthy livers, we therefore compared the proportion of NK subsets in peripheral blood, TILs, and NILs in HCC patients. It is found that the CD56dimCD16pos NK cell subset accounted for the majority of peripheral NK cells; whereas in both TILs and NILs, the proportion of CD56brightCD16neg NK cell subsets significantly increased (Fig. 4A). Notably, although total peripheral NK cell frequency was found to be decreased in the HCC patient, intrahepatic NK cells were found to be accumulated largely in the site of the same HCC patient. In particular, NILs contained more NK cells than TILs in the HCC patient (Fig. 4A). We further performed a comprehensive analysis in 6 HCC patients on the stage III. It is found that overall NK cell frequency was significantly decreased in TILs
Figure 4 HCC patients show a markedly reduced population of NK cells in TILs. (A) Representative dot plots of NK cell subsets in PBMC, NIL, and TIL from an HCC patient. Values in the quadrant indicate the percentages of total NK cells among lymphocytes. (B) Pool data indicated the frequencies of total, CD56bright, and CD56dim NK subsets in TILs and NILs from HCC patients. TIL, tumor-infiltrating lymphocyte; NIL, non-tumor-infiltrating lymphocyte.
434 compared with NILs (P b 0.05; Fig. 4B). We also found that the reduction of CD56dimCD16pos NK cells was largely responsible for the decrease in total NK cell numbers in TILs (P b 0.05), because the frequency of CD56brightCD16neg NK cells was similar in NILs and TILs in these HCC livers (Fig. 4B). Thus, a more comprehensive analysis of the individual contribution of the 2 NK cell subsets to the total number of liver NK cells was confirmed in the livers of HCC patients. These data demonstrated that intrahepatic NK cell compartment was also dramatically redistributed in HCC patients.
TILs of advanced-stage HCC patients displayed decreased granule production and impaired cytolytic ability compared with NILs We further compared the cytolytic capacity of intrahepatic NK cell subsets between TILs and NILs in these HCC patients. We first detected the content of cytolytic granules (GrA, GrB, and perforin) in liver NK cells of a HCC patient, and found that the proportion of GrA, GrB, and perforin positive NK cells in TILs were significantly lower than those in NILs (Fig. 5A). Pool data further confirmed the observation (Fig. 5B). We also detected CD107a expression by NIL-NK and TIL-NK cells, and failed to find a significant difference in
L. Cai et al. CD107a expression on these two populations of NK cells (data not shown). Moreover, we detected the cytolytic activity of liver-infiltrating NK cells. We found that TIL-NK cells possessed a lower capacity to kill targets than NIL-NK cells at the 30:1 of E/T ratio rather than 10:1 or 3:1 ratios (Fig. 5C). Thus, the decrease of granule proportion in TIL-NK cells in HCC patients might associate with the loss of cytotoxic capacity in the overall NK cells in HCC patients.
NK cell function is suppressed in the presence of CD4+CD25+ Tregs Our previous study indicated that circulating Treg frequency was increased significantly in HCC patients [15]. Whether or not the increased Tregs might impair NK cell anti-tumor immune responses in HCC patients remains unknown. We therefore detected the capacity to produce IFN-γ and kill K562 targets of Treg-treated NK cells in vitro. It is found that Treg-treated NK cells produced less IFN-γ compared with those untreated NK cells. More Tregs displayed stronger suppressive effects on IFN-γ production by NK cells (Fig. 6A). Tanswell assay showed the immunosuppression of Treg on NK cell-derived IFN-γ was mediated by direct cell contacts (data not shown). These data suggest that the increased peripheral
Figure 5 TIL-derived NK cells exhibited a significant decrease in granule production in HCC patients. (A) Representative expression profile of the GrA, GrB, and perforin by TIL- and NIL-derived NK cells in an HCC patient. NK cells are gated from CD3−CD56+ lymphocytes. Values in the quadrant indicate the percentages of GrA-, GrB-, and perforin-expressing NK cells. (B) Pool data indicated that the numbers of GrA-, GrB-, and perforin-expressing NK cells were all reduced in TILs compared with NILs from HCC patients. P values were shown. (C) Cytotoxic activity against K562 targets in TIL-derived NK cells is significantly decreased as compared with NIL-derived NK cells at the indicated ratios (30:1, 10:1, 3:1). ⁎P values were less than 0.05.
Functional impairment in NK cells and relative mechanism in HCC patients
435
Figure 6 NK cells are suppressed by autologous CD4+CD25+ Tregs. (A) Representative dot plots showed that Treg cells from an HCC patient suppressed IFN-γ production by autologous NK cells (n = 5). NK cells and Tregs were mixed at different ratios. Values in the quadrant indicate the percentages of IFN-γ-producing NK cells. (B) Representative dot plots show the inhibitory effects of Treg cells on autologous NK cell cytolytic activity against K562 targets (n = 4). Values in the quadrant indicate the percentages of 7-AAD positive CFSE-labeled K562 cells. (C) Trans-well assay showed that Treg cells suppressed NK cell cytotoxicity against K562 targets in a manner of cell–cell contacts (n = 4). Values out the quadrant indicate the percentages of 7-AAD positive CFSE-labeled K562 cells.
Tregs in HCC patients might contribute to the impaired IFN-γ production by autologous NK cells. We further investigated whether increased Tregs impaired the cytotoxic capacity of NK cells in HCC patients. We found that K562 cells exhibited few spontaneous cell deaths, as indicated by 7-AAD staining. The isolated Tregs also processed a poor capacity to kill K562 targets. In contrast, NK cells could kill more than 40% of K562 targets at the ratio of 10:1. Importantly, we found that Treg (2 × 105) treatment significantly reduced the cytotoxicity of NK cells at the ratio of 10:1 (NK:K562). Addition of more Tregs (10 × 105) largely reduced NK cell cytotoxicity by nearly 50% (Fig. 6B). Notably, separation of Tregs and NK cells in transwell assay significantly led to the loss of immunosuppression of Treg on NK cells killing K562 targets, indicating that Tregs inhibit NK cell anti-tumor activity dependent on direct cell–cell contacts (Fig. 6C). These data indicated that the increased circulating Tregs in HCC patients may diminish the cytotoxicity of NK cells. Thus, our data raised a possibility that the increased Tregs might contribute to the loss of anti-tumor immune responses in HCC patients.
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 functional impairment was found to be associated with the increased Tregs in these HCC patients. These findings revealed that NK cells lose anti-tumor immune responses in HCC patients.
We found a significant reduction of total proportion of peripheral NK cell compartments in HCC patients with different disease stages compared to healthy subjects. This reduction of total NK population mainly results from a substantial decrease in the peripheral CD56dimCD16pos NK cell subset; by contrast, circulating CD56brightCD16neg NK cells increased in HCC patients. Similarly, in the livers of HCC patients we found a significant reduction of CD56dimCD16pos NK cell subsets in tumor regions compared with non-tumor regions in HCC patients, suggesting that local environment is more important for the regulation of NK cell functions in HCC patients. These data indicated that both peripheral and intrahepatic NK cell subsets were largely redistributed in HCC patients. In particular, this decrease of circulating and intra-tumor CD16pos NK cell subset might contribute to reduction of the cytotoxic activity of NK cells since CD16 plays a key role in killing target cells in an ADCC manner [25–27]. More important, we found that both peripheral and tumor-infiltrating NK cells exhibited functional deficiency in producing IFN-γ and killing K562 targets compared with healthy peripheral NK cells and non-tumor-infiltrating NK cells, respectively. These findings suggest that the functional impairment of NK cells might severely hinder the anti-tumor immune responses of HCC patients; however, the mechanisms responsible for impaired NK function remain unknown in HCC patients. We therefore sought to determine the factors leading to the functional impairment of NK cells in HCC patients. One major manner by which NK cells exert their cytolytic function is by releasing cytoplasmic granules containing perforin and granzymes [2,28–31]. Our data supported that the reduced cytoplasmic granules, to some extent, might contribute to impaired cytotoxicity of NK cells in these HCC patients since CD107a expression, as a marker of measuring granule-releasing capacity [23] in HCC patients, was at the compatible levels with healthy subjects. Notably, circulating NK cells in HCC patients displayed
436 normal NK receptor and IL-2 receptor expression, indicating that HCC patients did display an intrinsical impairment of cytolytic activity. Future study should further investigate whether these receptor-mediated signaling was impaired in NK cells of HCC patients. Our previous study showed that peripheral Treg frequency increased significantly in HCC patients. These increased Tregs may impair the effector function of CD8 T cells and promote disease progression of HCC [15]. In this study, we proposed that this increased Treg frequency might impair NK-mediated immune responses. Our data supported the notion that NK cells, being pre-incubated with autologous Tregs, largely lost anti-tumor activity indicated by the decreased capacity of NK cells to produce IFN-γ and kill K562 targets. In addition, this suppression of Tregs on NK cells was dependent on a direct cell-to-cell contact, at least in vitro. Indeed, several studies have indicated that Tregs may execute their suppressive activity through weakening activation signals on NK cells, which is most often described to be independent of immunosuppressive cytokines, as antibodies to TGF-β or IL-10 do not influence Treg-mediated suppression [16,32]. Unexpectedly, we found that this functional impairment of NK cells was independent of disease stage; by contrast, the Treg frequency was found to be associated with disease progression in HCC patients. This differential association with disease progression in HCC patients also suggested that reasons other than suppression from Tregs are most likely responsible for impaired NK cell function in HCC patients. Consequently, this study demonstrates peripheral and tumor-infiltrating NK cell subsets were redistributed, and subsequently result in a severe loss of NK cells in anti-tumor activity of HCC patients. This functional abnormity of NK subsets might associate with the increased Tregs as we previously described in HCC patients. This finding provides an insight into the mechanisms of NK cell function impairment in HCC patients. This study will be of importance in the future development of NK-based therapeutic strategies against tumors.
Acknowledgments
L. Cai et al.
[6]
[7]
[8]
[9]
[10]
[11] [12]
[13]
[14]
[15]
[16]
This study was supported by a grant from the National Key Basic Research Program of China (2006CB504305), the National Youth Foundation of China (30525042).
References [1] J.M. Llovet, A. Burroughs, J. Bruix, Hepatocellular carcinoma, Lancet 362 (2003) 1907–1917. [2] A. Kawasaki, Y. Shinkai, H. Yagita, K. Okumura, Expression of perforin in murine natural killer cells and cytotoxic T lymphocytes in vivo, Eur. J. Immunol. 22 (1992) 1215–1219. [3] M.A. Cooper, M.A. Caligiuri, Isolation and characterization of human natural killer cell subsets, Curr. Protoc. Immunol. Chapter 7 (2004) Unit 7–34. [4] J.J. Subleski, V.L. Hall, T.C. Back, J.R. Ortaldo, R.H. Wiltrout, Enhanced antitumor response by divergent modulation of natural killer and natural killer T cells in the liver, Cancer Res. 66 (2006) 11005–11012. [5] X. Bai, S. Wang, C. Tomiyama-Miyaji, J. Shen, T. Taniguchi, N. Izumi, C. Li, H.Y. Bakir, T. Nagura, S. Takahashi, T. Kawamura, T.
[17]
[18]
[19]
[20]
Iiai, H. Okamoto, K. Hatakeyama, T. Abo, Transient appearance of hepatic natural killer cells with high cytotoxicity and unique phenotype in very young mice, Scan. J. Immunol. 63 (2006) 275–281. D. Vermijlen, C. Seynaeve, D. Luo, M. Kruhoffer, D.L. Eizirik, T.F. Orntoft, E. Wisse, High-density oligonucleotide array analysis reveals extensive differences between freshly isolated blood and hepatic natural killer cells, Eur. J. Immunol. 34 (2004) 2529–2540. M. Jarahian, C. Watzl, Y. Issa, P. Altevogt, F. Momburg, Blockade of natural killer cell-mediated lysis by NCAM140 expressed on tumor cells, Int. J. Cancer 120 (2007) 2625–2634. W. Cao, X. Xi, Z. Hao, W. Li, Y. Kong, L. Cui, C. Ma, D. Ba, W. He, RAET1E2, a soluble isoform of the UL16-binding protein RAET1E produced by tumor cells, inhibits NKG2D-mediated NK cytotoxicity, J. Biol. Chem. 282 (2007) 18922–18928. Y. Kano, K. Soda, T. Nakamura, M. Saitoh, M. Kawakami, F. Konishi, Increased blood spermine levels decrease the cytotoxic activity of lymphokine-activated killer cells: a novel mechanism of cancer evasion, Cancer Immunol. Immunother. 56 (2007) 771–781. V. Jovic, G. Konjevic, S. Radulovic, S. Jelic, I. Spuzic, Impaired perforin-dependent NK cell cytotoxicity and proliferative activity of peripheral blood T cells is associated with metastatic melanoma, Tumori. 87 (2001) 324–329. H.F. Pross, E. Lotzova, Role of natural killer cells in cancer, Nat. Immunol. 12 (1993) 279–292. E.H. Steinhauer, A.T. Doyle, J. Reed, A.S. Kadish, Defective natural cytotoxicity in patients with cancer: normal number of effector cells but decreased recycling capacity in patients with advanced disease, J. Immunol. 129 (1982) 2255–2259. K. Ishiyama, H. Ohdan, M. Ohira, H. Mitsuta, K. Arihiro, T. Asahara, Difference in cytotoxicity against hepatocellular carcinoma between liver periphery natural killer cells in humans, Hepatology 43 (2006) 362–372. M.W. Sung, J.T. Johnson, G. Van Dongen, T.L. Whiteside, Protective effects of interferon-gamma on squamous-cell carcinoma of head and neck targets in antibody-dependent cellular cytotoxicity mediated by human natural killer cells, Int. J. Cancer 66 (1996) 393–399. J. Fu, D. Xu, Z. Liu, M. Shi, P. Zhao, B. Fu, Z. Zhang, H. Yang, H. Zhang, C. Zhou, J. Yao, L. Jin, H. Wang, Y. Yang, Y.X. Fu, F.S. Wang, Increased regulatory T cells correlate with CD8 T-cell impairment and poor survival in hepatocellular carcinoma patients, Gastroenterology 132 (2007) 2328–2339. F. Ghiringhelli, C. Menard, M. Terme, C. Flament, J. Taieb, N. Chaput, P.E. Puig, S. Novault, B. Escudier, E. Vivier, A. Lecesne, C. Robert, J.Y. Blay, J. Bernard, S. Caillat-Zucman, A. Freitas, T. Tursz, O. Wagner-Ballon, C. Capron, W. Vainchencker, F. Martin, L. Zitvogel, CD4+CD25+ regulatory Tcells inhibit natural killer cell functions in a transforming growth factor-betadependent manner, J. Exp. Med. 202 (2005) 1075–1085. M.J. Smyth, M.W. Teng, J. Swann, K. Kyparissoudis, D.I. Godfrey, Y. Hayakawa, CD4+CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of cancer, J. Immunol. 176 (2006) 1582–1587. P. Trzonkowski, E. Szmit, J. Mysliwska, A. Dobyszuk, A. Mysliwski, CD4+CD25+ T regulatory cells inhibit cytotoxic activity of T CD8+ and NK lymphocytes in the direct cellto-cell interaction, Clin. Immunol. 112 (2004) 258–267. Z.W. Sheng, F.S. Wu, Z.M. Ma, Y.Z. Feng, X.R. Zhou, L.S. Teng, The Chinese classification system compared with TNM staging in prognosis of patients with primary hepatic carcinoma after resection, Hepatobiliary Pancreat. Dis. Int. 4 (2001) 561–564. D. Xu, J. Fu, L. Jin, H. Zhang, C. Zhou, Z. Zou, J.M. Zhao, B. Zhang, M. Shi, X. Ding, Z. Tang, Y.X. Fu, F.S. Wang, Circulating and liver resident CD4+CD25+ regulatory T cells actively influence the antiviral immune response and disease
Functional impairment in NK cells and relative mechanism in HCC patients
[21]
[22]
[23]
[24]
[25]
[26]
progression in patients with hepatitis B, J. Immunol. 177 (2006) 739–747. H. Lecoeur, M. Fevrier, S. Garcia, Y. Riviere, M.L. Gougeon, A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity, J. Immunol. Methods 253 (2001) 177–187. Z. Zhang, J.Y. Zhang, E.J. Wherry, B. Jin, B. Xu, Z.S. Zou, S.Y. Zhang, B.S. Li, H.F. Wang, H. Wu, G.K. Lau, Y.X. Fu, F.S. Wang, Dynamic programmed death 1 expression by virus-specific CD8 T cells correlates with the outcome of acute hepatitis B, Gastroenterology 134 (2008) 1938–1949. G. Alter, J.M. Malenfant, M. Altfeld, CD107a as a functional marker for the identification of natural killer cell activity, J. Immunol. Methods 294 (2004) 15–22. C.M. Balch, A.B. Tilden, P.A. Dougherty, G.A. Cloud, T. Abo, Heterogeneity of natural killer lymphocyte abnormalities in colon cancer patients, Surgery 95 (1984) 63–70. R. Gazit, M. Aker, M. Elboim, H. Achdout, G. Katz, D.G. Wolf, S. Katzav, O. Mandelboim, NK cytotoxicity mediated by CD16 but not by NKp30 is functional in Griscelli syndrome, Blood 109 (2007) 4306–4312. O. Mandelboim, P. Malik, D.M. Davis, C.H. Jo, J.E. Boyson, J.L. Strominger, Human CD16 as a lysis receptor mediating direct natural killer cell cytotoxicity, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 5640–5644.
437
[27] J.S. Schleypen, N. Baur, R. Kammerer, P.J. Nelson, K. Rohrmann, E.F. Grone, M. Hohenfellner, A. Haferkamp, H. Pohla, D.J. Schendel, C.S. Falk, E. Noessner, Cytotoxic markers and frequency predict functional capacity of natural killer cells infiltrating renal cell carcinoma, Clin. Cancer Res. 12 (2006) 718–725. [28] T. Koga, H. Harada, T.S. Shi, S. Okada, M.A. Suico, T. Shuto, H. Kai, Hyperthermia suppresses the cytotoxicity of NK cells via down-regulation of perforin/granzyme B expression, Biochem. Biophys. Res. Commun. 337 (2005) 1319–1323. [29] Y. Hayakawa, J.M. Kelly, J.A. Westwood, P.K. Darcy, A. Diefenbach, D. Raulet, M.J. Smyth, Cutting edge: tumor rejection mediated by NKG2D receptor–ligand interaction is dependent upon perforin, J. Immunol. 169 (2002) 5377–5381. [30] V. Screpanti, R.P. Wallin, H.G. Ljunggren, A. Grandien, A central role for death receptor-mediated apoptosis in the rejection of tumors by NK cells, J. Immunol. 167 (2001) 2068–2073. [31] M.J. Smyth, K.Y. Thia, E. Cretney, J.M. Kelly, M.B. Snook, C.A. Forbes, A.A. Scalzo, Perforin is a major contributor to NK cell control of tumor metastasis, J. Immunol. 162 (1999) 6658–6662. [32] M. Terme, N. Chaput, B. Combadiere, A. Ma, T. Ohteki, L. Zitvogel, Regulatory T cells control dendritic cell/NK cell crosstalk in lymph nodes at the steady state by inhibiting CD4+ selfreactive T cells, J. Immunol. 180 (2008) 4679–4686.