Human CD14+CTLA-4+ regulatory dendritic cells suppress T-cell response by cytotoxic T-lymphocyte antigen-4-dependent IL-10 and indoleamine-2,3-dioxygenase production in hepatocellular carcinoma

Authors

  • Yanmei Han,

    1. National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai, China
    Search for more papers by this author
    • These authors contributed equally to this work.

  • Zhubo Chen,

    1. National Key Laboratory of Medical Molecular Biology and Department of Immunology, Chinese Academy of Medical Sciences, Beijing, China
    Search for more papers by this author
    • These authors contributed equally to this work.

  • Yuan Yang,

    1. Third Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital, Shanghai, China
    Search for more papers by this author
    • These authors contributed equally to this work.

  • Zhengping Jiang,

    1. National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai, China
    Search for more papers by this author
  • Yan Gu,

    1. National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai, China
    Search for more papers by this author
  • Yangfang Liu,

    1. National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai, China
    Search for more papers by this author
  • Chuan Lin,

    1. Third Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital, Shanghai, China
    Search for more papers by this author
  • Zeya Pan,

    1. Third Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital, Shanghai, China
    Search for more papers by this author
  • Yizhi Yu,

    1. National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai, China
    Search for more papers by this author
  • Minghong Jiang,

    1. National Key Laboratory of Medical Molecular Biology and Department of Immunology, Chinese Academy of Medical Sciences, Beijing, China
    Search for more papers by this author
  • Weiping Zhou,

    1. Third Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital, Shanghai, China
    Search for more papers by this author
    • These two authors share senior authorship.

  • Xuetao Cao

    Corresponding author
    1. National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai, China
    2. National Key Laboratory of Medical Molecular Biology and Department of Immunology, Chinese Academy of Medical Sciences, Beijing, China
    • Address reprint requests to: Xuetao Cao, M.D., Ph.D., National Key Laboratory of Medical Immunology & Institute of Immunology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China. E-mail: caoxt@immunol.org; fax: +86 21 6538 2502.

    Search for more papers by this author
    • These two authors share senior authorship.


  • This work was supported by grants from the MOST 125 Major Project of China (2012ZX10002014-001 and 2012ZX10002010), the National Key Basic Research Program of China (2011CB965202, 2013CB530503, and 2013CB944903), the National Natural Science Foundation of China (31170843), and the Foundation for the Author of National Excellent Doctoral Dissertation of China (201071).

  • Potential conflict of interest : Nothing to report.

Abstract

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with limited therapeutic options. HCC-induced immunosuppression often leads to ineffectiveness of immuno-promoting therapies. Currently, suppressing the suppressors has become the potential strategy for cancer immunotherapy. So, figuring out the immunosuppressive mechanisms induced and employed by HCC will be helpful to the design and application of HCC immunotherapy. Here, we identified one new subset of human CD14+CTLA-4+ regulatory dendritic cells (CD14+DCs) in HCC patients, representing ∼13% of peripheral blood mononuclear cells. CD14+DCs significantly suppress T-cell response in vitro through interleukin (IL)-10 and indoleamine-2,3-dioxygenase (IDO). Unexpectedly, CD14+DCs expressed high levels of cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death-1, and CTLA-4 was found to be essential to IL-10 and IDO production. So, we identified a novel human tumor-induced regulatory DC subset, which suppresses antitumor immune response through CTLA-4-dependent IL-10 and IDO production, thus indicating the important role of nonregulatory T-cell-derived CTLA-4 in tumor-immune escape or immunosuppression. Conclusions: These data outline one mechanism for HCC to induce systemic immunosuppression by expanding CD14+DCs, which may contribute to HCC progression. This adds new insight to the mechanism for HCC-induced immunosuppression and may also provide a previously unrecognized target of immunotherapy for HCC. (Hepatology 2014;59:567–579)

Abbreviations
1-MT

1-methytryptophan

7-AAD

7-amino-actinomycin D

Abs

antibodies

Ags

antigens

APCs

antigen-presenting cells

BDCA-3

blood dendritic cell antigen 3

CCA

cholangiocarcinoma

CD14+DC

CD14+CTLA-4+ regulatory dendritic cell

CFSE

carboxyfluorescein succinimidyl ester

CTLA-4

cytotoxic T-lymphocyte antigen-4

DAPI

4′,6-diamidino-2-phenylindole

DC

dendritic cell

ELISA

enzyme-linked immunosorbent assay

ERK

extracellular signal-regulated kinase

FasL

Fas ligand

FCM

flow cytometry

FITC

fluorescein isothiocyanate

GITRL

glucocorticoid-induced tumor necrosis factor-related ligand

HCC

hepatocellular carcinoma

HLA

human leukocyte antigen

IDO

indoleamine-2,3-dioxygenase

IL

interleukin

LPS

lipopolysaccharide

M2

type 2 macrophages

mAb

monoclonal Ab

mDC

mature dendritic cell

MDSCs

myeloid-derived suppressor cells

MNCs

mononuclear cells

MoDC

monocyte-derived dendritic cell

PBMCs

peripheral blood mononuclear cells

PD-1

programmed death-1

PE

phycoerythrin

pERK1/2

phosphorylated ERK1/2

TGF-β

transforming growth factor beta

Th

T helper

Treg

regulatory T cell

Tumor, especially at the advanced stage, can develop variable strategies to suppress antitumor immune responses, for example, production of immunosuppressive factors, activation of negative costimulatory signals, and inducing, recruiting, or expanding various immune regulatory cells.[1, 2] Among the tumor-related regulatory cells, regulatory T cells (Tregs) are the most extensively studied, which is believed to play essential roles in tumor-induced immune suppression.[3, 4] Tregs use various potential suppressive mechanisms, which can be grouped into four basic modes of action: suppression by inhibitory cytokines, such as interleukin (IL)-10 and transforming growth factor beta (TGF-β); suppression by perforin and granzymes-induced cytolysis; suppression by metabolic disruption; and suppression by targeting dendritic cells (DCs) through cytotoxic T-lymphocyte antigen-4 (CTLA-4) and indoleamine-2,3-dioxygenase (IDO).[5, 6] Our group previously identified one novel population of CD4+CD69+CD25Foxp3 Tregs in liver-cancer-bearing mice[7], which suppress CD4 T-cell response via membrane-bound TGF-β. In addition, type 2 macrophages (M2) have been shown to be important in the immunosuppressive tumor microenvironment.[8] However, whether there exist new populations of immunosuppressive immune cells in the tumor-bearing host, what the relation is of the presence of the new subsets with the progression of the host, and what the mechanism is of the new subsets for tumor-induced immunosuppression need to be further investigated.

Healthy human blood contains a few DCs, representing ∼1% of peripheral blood mononuclear cells (PBMCs).[9] Human blood DCs can be divided into CD1c(BDCA-1)+ and CD141(BDCA-3)+ myeloid DCs as well as CD123highCD303(BDCA-2)+CD304(BDCA-4)+ plasmacytoid DCs.[10] Gene expression profiles and emerging functional data have determined that these CD1c+ and CD141+ myeloid DCs appeared as human equivalents of mouse lymphoid resident CD8+ and CD8 DC subsets, respectively.[11] Recent studies show that human blood CD1c+ myeloid DCs are naturally regulatory DCs, which secrete IL-10 and display an immunoregulatory phenotype and function in response to Escherichia coli.[12, 13] CD1c+ DCs represent the most prevalent human liver DC subset, which might contribute to hepatic tolerance by promoting T-cell hyporesponsiveness through IL-10.[14] Recently, resident CD141+CD14+ regulatory DCs have been identified in human skin, which produce IL-10 and induce Tregs to maintain skin homostasis, and, interestingly, these cells could be induced in vitro by vitamin D3 from CD1c+CD14 human healthy blood DCs.[15] However, little is known about the phenotype and function of human DCs in pathological states, especially in cancer patients. When compared to human regulatory DCs, mouse regulatory DCs have been investigated more extensively.[16, 17] For example, we found that tumor could educate DCs to differentiate into a regulatory CD11bhighIalow DC subset, which suppresses T-cell response by arginase I, suggesting that regulatory DCs also contribute to tumor-induced immunosuppression.[18]

Hepatocellular carcinoma (HCC) is one of the most common malignancies with limited therapeutic options, which causes the third-leading cancer deaths worldwide. Because HCC has been shown to be immunogenic, T-cell-based immunotherapy is considered a promising treatment. However, HCC-induced immunosuppressive environments, such as Tregs, myeloid-derived suppressor cells (MDSCs), and inhibitor receptors, often lead to poor effects of immuno-promoting therapy.[19] For example, increased intratumoral Tregs have been proven to be related to poor prognosis of HCC patients, and intratumoral macrophages are involved in their induction.[20] Instead, suppressing the suppressors of antitumor immunity has become a potential strategy of cancer immunotherapy. Therefore, figuring out the complicated immunosuppressive mechanisms induced and employed by HCC becomes the first and essential step to design effective immunotherapy for HCC. In this study, we identified one new population of regulatory DCs in HCC patients, with a unique phenotype of CD14+CD11bhighCTLA-4+, representing ∼13% of PBMCs. These CD14+DCs suppress CD4 T-cell response by CTLA-4-dependent IL-10 and IDO production. So, our discovery of the tumor-induced new subset of human regulatory DCs may provide new mechanistic explanation for immunosuppression in HCC and also outline a previously unidentified target potentially for treatment of HCC.

Materials and Methods

Samples

Blood sample, HCC tissue, peritumor liver tissue, and distal liver tissue (distal end) were from HCC patients (n = 65) from Eastern Hepatobiliary Surgery Hospital (Shanghai, China). All samples were freshly collected, delivered on ice, and processed within 1 hour. Details of these HCC patients are shown in Table 1. One hundred twenty-seven blood samples of HCC patients, including 65 samples described above, five blood samples of intrahepatic cholangiocarcinoma (CCA) patients, and nine of cavernous hemangioma of liver patients were also from Eastern Hepatobiliary Surgery Hospital. Eight blood samples of gastric carcinoma patients and six of colorectal cancer patients were from Changhai Hospital (Shanghai, China). All samples were collected with the informed consent of the patients, and the experiments were approved by the ethics committee of Second Military Medical University (Shanghai, China). Whole-blood staining with bead quantitation was performed. PBMCs of healthy human donors (n = 28) or various patients were prepared by density-gradient isolation using Ficoll-Paque (Sigma-Aldrich, St. Louis, MO). Viability of the isolated PBMCs was >99% by 7-amino-actinomycin D (7-AAD) labeling.

Table 1. General Characteristics of the 65 Patients With HCC
VariableResults
  1. Data are expressed as the number of patients except age, and the percentage is also shown for the last three items.

  2. Abbreviations: HBV, hepatitis B virus; HCV, hepatitis V virus; AFP, a-fetoprotein.

Age, years (range/mean)17-79/51
Gender (male/female)59/6
Virus: HBV/HCV56/9
AFP (>100/<100 ng/mL)38/27
Child-Pugh classification (A/B/C)51/12/2
Tumor factors 
Tumor size (<3.0/>3.0 cm)7/58
Number of tumors (single/double/multiple)56/7/2
Cases with chronic hepatitis, %32 (21/65)
Cases with liver cirrhosis, %66 (43/65)
Cases with metastasis, %3 (2/65)

Reagents

The antibodies (Abs) for flow cytometry (FCM), including Abs against human CD11b (ICRF44), CD11c (3.9), CD1c (L161), human leukocyte antigen (HLA)-DR (L243), CD68 (Y1/82A), CD80 (2D10), CD86 (IT2.2), CD83 (HB15e), CD14 (HCD14), blood dendritic cell antigen 3 (BDCA-3; M80), Fas ligand (FasL; NOK-1), CTLA-4 (L3D10), CD25 (BC96), programmed death-1 (PD-1; MIH4), and IL-10 (JES3-9D7), were from BioLegend (San Diego, CA). Ab against human glucocorticoid-induced tumor necrosis factor-related ligand (GITRL; MM0312-4Q25) was from Abcam (Cambridge, MA). Phorbol myristate acetate, ionomycin, carboxyfluorescein succinimidyl ester (CFSE), and U0126 were from Sigma-Aldrich. Intracellular staining kit of IL-10 and enzyme-linked immunosorbent assay (ELISA) kits of IL-10, TGF-β, and IL-12p70 were from R&D Systems (Minneapolis, MN). The ELISA kit of IDO was from Uscn Life Science Inc. (Wuhan, China). Neutralizing anti-IL-10 monoclonal Ab (mAb) and anti-CTLA-4 mAb were from LifeSpan BioSciences, Inc. (Seattle, WA).

Isolation of Mononuclear Cells From Liver and Tumor Tissue

Tumor and liver tissues from HCC patients were digested with collagenase IV, DNase, and hyaluronidase (Sigma-Aldrich) for 3 hours, and then the single-cell suspension was passed through a 40-μm cell strainer (BD Falcon; BD Biosciences, San Jose, CA). Mononuclear cells (MNCs) were purified by centrifugation through a Percoll gradient. Briefly, cells were collected, washed with phosphate-buffered saline, and resuspended in 40% Percoll (Sigma-Aldrich). Then, the cell suspension was gently overlaid onto 70% Percoll and centrifuged for 20 minutes at 750×g. MNCs were collected from the interface.[7, 21]

FCM

Cells were analyzed by FCM with a FACS LSR II (BD Biosciences), and data were analyzed with FACSDiva software, as described previously.[7]

For intracellular staining of IL-10, cells were stained with Abs against cell-surface antigen (Ag), and then fixed and permeabilized for 20 minutes. Cells were washed with permeabilization buffer and incubated with anti-IL-10 phycoerythrin (PE) for 70 minutes at 4°C. Then, cells were washed and analyzed by FCM.

For intracellular staining of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2; pT202/pY204), cells were fixed with prewarmed BD Cytofix buffer, permeabilized with BD Phosflow Perm buffer (BD Biosciences), and then labeled with Alexa Fluor 647-conjugated pERK1/2 (pT202/pY204) mAb. Then, cells were washed and analyzed by FCM.[7]

To purify CD14+DCs (CD11bhighCD11chigh cells) from PBMCs of HCC patients, cells were stained with anti-CD11b fluorescein isothiocyanate (FITC) and anti-CD11c PE, and CD11bhighCD11chigh cells were sorted by a MoFlo high-speed cell sorter (Dako; Carpinteria, CA). Purity was confirmed by FCM to be >97%.

Preparation of Monocyte-Derived DCs and Matured DCs In Vitro

DCs were prepared as previously described.[22] Briefly, CD14+ cells were sorted from human healthy donor PBMCs by a MoFlo high-speed cell sorter and were then cultured for 4-5 days with 50 ng/mL of granulocyte macrophage colony-stimulating factor and 1,000 U/mL IL-4 (R&D Systems). Cells were collected as monocyte-derived DCs (MoDCs). For preparation of mature DCs (mDCs), cells were further stimulated with 300 ng/mL of lipopolysaccharide (LPS; Sigma-Aldrich) for 24 hours.

Fluorescence Confocal Microscopy

Purified CD14+DCs and MoDCs after stimulation with LPS for 24 hours were stained with 4′,6-diamidino-2-phenylindole (DAPI), anti-CD11c PE, and then cells were visualized with a Leica TCS SP2 confocal laser microscope (Leica Microsystems GmbH, Wetzlar, Germany). All cell images were obtained using a 40× dry objective lens on the confocal microscope with Leica Confocal Software.

Detection of Cytokines

Cytokines in supernatants were determined by ELISA kits according to the manufacturer's instructions (R&D or Uscn Life Science Inc.).

Assay for Phagocytic Ability

Phagocytosis of FITC-conjugated dextran (FITC-dextran) was analyzed by FCM as described previously.[23] Briefly, purified CD14+DCs, MoDCs, and monocytes were incubated at 37°C for 4 hours with FITC-dextran (Invitrogen, Carlsbad, CA). After being washed twice, cells were resuspended for detection by FCM. Cells incubated with FITC-dextran at 4°C were used as negative control.

Mixed Lymphocyte Reactions

For analyzing the ability of CD14+DCs to promote T-cell proliferation, CD4 T cells were sorted from healthy human donor PBMCs, labeled with CFSE (Sigma-Aldrich), and then cocultured with purified allogeneic CD14+DCs, MoDCs, or mDCs for 5 days at the ratio of 10:1 (T cells/DCs). Degree of T-cell proliferation was gauged from the serial diminution of the CSFE label, as determined by FCM.[7]

For analyzing the immune-suppressive ability of CD14+DCs, CD4 T cells were sorted from healthy human donor PBMCs, labeled with CFSE, and cocultured with allogeneic mDCs for 5 days at the ratio of 10:1. Purified CD14+DCs or MoDCs were added into the coculture system. The ratio of mDCs and CD14+DCs or MoDCs was 1:1. Degree of CD4 T-cell proliferation was determined by FCM, as described above.[7]

Statistical Analysis

Data were analyzed for statistical significance using the Student t test. Statistical significance was determined as P values <0.05.

Results

Identification of CD11bhighCD11chigh Cells in PBMCs of HCC Patients

For identifying a new population of regulatory cells induced by tumor, we screened the expression of different markers on PBMCs from HCC patients by FCM. Interestingly, we found a population of CD11bhighCD11chigh cells appearing in PBMCs from HCC patients, but not from healthy donors (Fig. 1A). The population of CD11bhighCD11chigh cells also expressed HLA-DR, CD1c, CD14, CD86 (Fig. 1B), and BDCA-3 (Supporting Fig. 1), but not CD80 (data not show). Compared to CD11blowCD11chigh cells, CD11bhighCD11chigh cells expressed a lower level of CD83, but a higher level of CD68 (Fig. 1B). Most CD11b+CD11c+ cells from healthy PMBCs were CD14+CD68+ monocytes, which barely expressed CD1c and CD83 and expressed relatively less HLA-DR and CD86 (Fig. 1C). Also, circulating CD1c+ DCs from healthy blood, identified by CD1c+CD11c+CD20, highly expressed HLA-DR, CD86, and CD83, but expressed a low level of CD68 and CD14 (Supporting Fig. 2A). Therefore, we identified a new population of CD11bhighCD11chighCD14+CD1c+BDCA-3+HLA-DR+CD80CD86+CD83low cells in HCC PBMCs. Considering that these peripheral cells express high levels of DC markers CD11c, HLA-DR, and CD1c, and also the monocyte marker, CD14, we designated these cells as CD14+DCs.

Figure 1.

Phenotype of CD11bhighCD11chigh cells from PBMCs of HCC patients. (A) PBMCs of HCC patients or healthy donors were analyzed for CD11b and CD11c expression by FCM. (B) The phenotypes of CD11bhighCD11chigh cells and CD11blowCD11chigh cells from HCC PBMCs were analyzed by FCM. (C) The phenotype of CD14+CD68+ monocytes in CD11b+CD11c+ cells from healthy PBMCs was analyzed by FCM. Dot plots show one of the representative results (n = 10).

Phenotype and Morphology of CD14+DCs

To determine the characteristics and functions of CD14+DCs identified from HCC patients, we chose healthy human MoDCs as main control cells. Unexpectedly, we found that CD14+DCs expressed CTLA-4, PD-1, and GITRL, as well as high levels of FasL and CD25 (Fig. 2A,B), whereas MoDCs did not express these molecules, except for FasL, and monocytes as well as circulating CD1c+ DCs from healthy PBMCs did not express these molecules either (Fig. 2A and Supporting Fig. 2B).

Figure 2.

Functional molecule expression and morphological features of CD14+DCs. (A) FasL, CTLA-4, CD25, PD-1, and GITRL expression on CD14+DCs, MoDCs, and normal monocytes were analyzed by FCM. Dot plots show one of the representative results (n = 10). (B) Mean fluorescence intensity of FasL, CTLA-4, CD25, PD-1, and GITRL expressed by CD14+DCs in PBMCs from various tumor patients are shown (n = 100). (C) Optical microscopy of CD14+DCs and MoDCs with or without LPS stimulation and confocal microscopy of them stained with DAPI and anti-CD11c PE after stimulation are shown (n = 5). Image shows one of the representative results.

Next, morphological features of CD14+DCs were analyzed. Behavior and morphology of CD14+DCs were similar with DCs, to a certain degree. Just as MoDCs, purified CD14+DCs got together when cultured in vitro with stimulation by LPS or without (Fig. 2C). Fluorescence confocal microscopy assay showed that CD14+DCs and MoDCs had similar cell size, and, more interesting, CD14+DCs displayed a few dendritic protrusions after stimulation with LPS, although much fewer than DCs (Fig. 2C). These data support that these cells are just like DCs.

Distribution of CD14+DCs

Then, we detected the distribution of CD14+DCs, determined by FCM as CD11bhighCD11chighHLA-DR+CD14+ cells. Frequencies of CD14+DCs in PBMCs under various settings are shown in Table 2. Only PBMCs of liver cancers, including HCC and intrahepatic CCA, contained high frequencies of CD14+DCs (∼13%), whereas PBMCs of gastric carcinoma, colorectal carcinoma, and cavernous hemangioma of liver contained very few CD14+DCs as that from healthy humans (∼3%).

Table 2. CD14+DC Proportions in PBMCs From Variable Disorders
DiseaseNumberAge, Years (Range/Mean)Gender (Male/Female)Proportions of CD14+DCs (Range/Mean)
HCC12717-70/49117/109.3-15.7/13.2%
Intrahepatic CCA542-61/535/08.4-14.3/12.4%
Gastric carcinoma845-68/546/20.9-6.7/2.8%
Colorectal cancer643-64/616/01.2-5.6/3.5%
Cavernous hemangioma of liver937-53/471/80.3-3.9/1.9%
Healthy2821-35/2724/40.3-4.1/2.1%

We also detected whether CD14+DCs existed in tumor tissues. We collected tumor tissue, peritumor liver tissue, and liver tissue far from tumor (distal end) from 65 HCC patients. The general characteristics of these 65 HCC patients are shown in Table 1. According to the HCC staging criteria made by the HCC Professional Committee of the Chinese Anti-Cancer Association in 2001, these 65 HCC patients were separated into four tumor stages: Ia; Ib; II; and III. The number of positive samples and frequency ranges of CD14+DCs in PBMCs or in MNCs of tumor and liver tissue were sorted out by tumor stages (shown in Table 3). We found that all HCC PBMCs contained high levels of CD14+DCs, and there was no obvious difference of their frequencies among different tumor stages. CD14+DCs also infiltrated in some, but not all, tumor tissues, peritumor liver tissues, and distal liver tissues. Therefore, CD14+DCs not only exist in PBMCs, but also in liver cancer tissues and liver tissue of HCC patients.

Table 3. CD14+DC Proportions in MNCs From the 65 HCC Patients
Tumor StageaNAge (Range/Mean)Gender (Male/Female)PBMCs (Positiveb/Rangec)Tumor (Positive/Range)Peritumor (Positive/Range)Distal End (Positive/Range)
  1. a

    According to the Chinese general rules for primary liver cancer.

  2. b

    The number of samples where we could detect CD14+DCs.

  3. c

    The range of the percentage of CD14+DCs from the samples where we could detect CD14+DCs (positive samples).

Ia (size, <3.0 cm)737-57/465/27/8.5%-14.7%4/5.7%-14.8%5/4.3%-17.8%5/3.9%-15.2%
Ib (size, >3.0 cm)4617-65/5342/446/7.3%-15.1%10/1.3%-12.6%14/3.7%-12.3%21/3.2-14.2%
II845-79/518/08/9.6%-14.9%-1/2.6%1/4.6%
III442-67/564/04/10.3%-16.2%---

Cytokine Profile of CD14+DCs

Then, we screened the cytokine expression of CD14+DCs, comparing this to MoDCs. Interestingly, intracellular staining showed that ∼11.5% of CD14+DCs spontaneously expressed IL-10, and ∼87.4% expressed IL-10 after LPS stimulation, whereas only very few MoDCs had this ability (Fig. 3A). ELISA assay of IL-10 confirmed the results of IL-10 intracellular staining (Fig. 3B). IDO has been found to be related to the regulatory functions of DCs,[24] so we detected IDO production of these cells by ELISA. MoDCs scarcely produced IDO with or without LPS stimulation, but CD14+DCs produced plenty of IDO spontaneously, independent of LPS stimulation (Fig. 3B). In addition, we also found that CD14+DCs could produce few TGF-β, and there was no difference in IL-12p70 production between CD14+DCs and MoDCs (Fig. 3B). Therefore, the data show that CD14+DCs could produce high levels of IL-10 and IDO, as well as IL-12p70 and few TGF-β.

Figure 3.

Cytokine expression as well as phagocytic and T-cell-promoting ability of CD14+DCs. (A) Intracellular IL-10 expression of CD14+DCs and MoDCs after stimulation with or without LPS for 24 hours were analyzed by FCM. Dot plots represent one of the independent experiments with similar results (n = 5). (B) CD14+DCs and MoDCs were purified and stimulated with or without 300 ng/mL of LPS for 24 hours. Supernatants were collected for ELISA assay of IL-10, IDO, TGF-β, and IL-12p70. Means ± standard error of the mean (SEM; n = 5; 3 patients per group). ***P < 0.01. (C) Poor phagocytic ability of CD14+DCs. Purified CD14+DCs, MoDCs, and monocytes (Mo) were incubated at 37°C for 4 hours with FITC-dextran, then cells were collected for FCM analysis. Cells incubated with FITC-dextran at 4°C were used as control. Histograms represent one of the independent experiments with similar results (n = 3). (D) Poor ability of CD14+DCs to promote T-cell proliferation. CD4 T cells were sorted from healthy human donor PBMCs, labeled with CFSE, and cocultured with purified allogeneic CD14+DCs, MoDCs, or mDCs for 5 days. Degree of T-cell proliferation was gauged from the serial diminution of the CSFE label, as determined by FCM; one representative histogram result is also shown. Means ± SEM (n = 4; 3 patients per group). ***P < 0.01.

CD14+DCs Suppress T-Cell Responses by IL-10 and IDO

Phagocytic ability assay showed that CD14+DCs possessed similar poor phagocytic ability with MoDCs, much weaker than monocytes (Fig. 3C). We also detected the antigen-presenting ability of CD14+DCs, and found that CD14+DCs had much less ability to promote CD4 T-cell proliferation than mDCs and MoDCs (Fig. 3D).

Considering that these tumor-induced cells express inhibitor molecules, such as CTLA-4, PD-1, IL-10, and IDO, we predict that CD14+DCs probably possess immune regulatory functions, thus helping tumor escape of immune attack and promoting tumor progression. Next, we detected whether CD14+DCs could inhibit CD4 T-cell response. CFSE-labeled healthy human PBMC-derived CD4 T cells cocultured with allogeneic mDCs for 5 days displayed potent proliferative response (∼47.9%; Fig. 4A). If CD14+DCs were added into the coculture system, the degree of CD4 T-cell proliferation obviously dropped to ∼14.5%, whereas if MoDCs were included, CD4 T-cell proliferation was not obviously affected (Fig. 4A,B). We also detected whether CD14+DCs could induce T-cell apoptosis. CD4 T cells after coculture were further labeled with 7-AAD. We found that ∼38.4% CD4 T cells after coculture with CD14+DCs went into apoptosis, wherease only ∼9.3% of CD4 T cells after coculture with MoDCs did so (Fig. 4A). Thus, we proved that CD14+DCs suppressed CD4 T-cell response, at least partly, by inducing CD4 T-cell apoptosis.

Figure 4.

CD14+DCs suppress T-cell proliferation by IL-10 and IDO. (A) CD14+DCs suppress T-cell proliferation by inducing T-cell apoptosis. Purified healthy CD4 T cells were labeled with CFSE, then were cocultured with allogeneic mDCs for 5 days. Purified CD14+DCs or MoDCs were added into the coculture system. Degrees of T-cell proliferation gauging from the serial diminution of the CSFE label were analyzed by FCM. CFSE+ CD4 T cells after proliferation were further labeled with 7-AAD, the degree of apoptosis was determined by FCM. Histograms represent one of five independent experiments. (B) CD14+DCs suppress T-cell proliferation by IL-10 and IDO. Purified healthy CD4 T cells were labeled with CFSE, then cocultured with allogeneic mDCs for 5 days. Purified CD14+DCs or MoDCs were added into the coculture system. Degree of CD4 T-cell proliferation was determined by FCM. In some experiments, 0.4-μm transwells were used to separate CD14+DCs from CD4 T cells/mDCs (transwell), or CD14+DCs were fixed with 1% glutaraldehyde before adding to the coculture system (fixation), or neutralizing anti-IL-10 mAb or 1-MT was included into the coculture system. Isotype indicates isotype control for anti-IL-10 mAb. Means ± standard error of the mean (n = 5; 3 patients per group). ***P < 0.01.

Next, we detected the immune-suppressive mechanism of CD14+DCs. When 0.4-μm transwells were used to separate CD14+DCs from CD4 T cells/mDCs, the immune-suppressive function was not affected, but when CD14+DCs were fixed by 1% glutaraldehyde to exclude the functions of soluble factors from CD14+DCs, their suppressive effect was almost totally inhibited (Fig. 4B), suggesting that soluble factors, but not cell-surface Ags were responsible for their suppressive effect. Considering that CD14+DCs could produce high levels of IL-10 and IDO, we predict that their suppressive effect might depend on IL-10 and/or IDO. When neutralizing anti-IL-10 mAb was added into the coculture system, the immune-suppressive effect was partly inhibited (Fig. 4B). Also, when IDO-specific inhibitor 1-methytryptophan (1-MT) was added, the immune-suppressive effect was almost totally inhibited (Fig. 4B). As expected, when neutralizing anti-IL-10 mAb and 1-MT were added together, the suppressive effect was also totally inhibited (Fig. 4B). Therefore, we proved that CD14+DCs suppressed CD4 T-cell response by IL-10 and IDO.

CTLA-4 Is Essential for IL-10 and IDO Production in CD14+DCs Cells

We have found that CD14+DCs expressed CTLA-4, and CTLA-4 plays important roles in the functions of Tregs, so we detected whether CTLA-4 displayed essential roles in the functions of CD14+DCs. We found that neutralizing anti-CTLA-4 mAb not only inhibited IL-10 production of CD14+DCs induced by LPS (Fig. 5A), but also inhibited IDO production (Fig. 5B). Therefore, CTLA-4 is essential to the functions of CD14+DCs through its interfering with the production of IL-10 and IDO. Considering that CD14+DCs also express CD86, one of the major ligands of CTLA-4, we propose here that CD14+DCs might induce themselves reciprocally to produce IL-10 and IDO through the CD86/CTLA-4 pathway (Fig. 5D).

Figure 5.

CTLA-4 is essential for IL-10 and IDO production of CD14+DCs. (A and B) CD14+DCs were stimulated with LPS for 24 hours. Supernatants were collected for ELISA assay of IL-10 (A) and IDO (B). In some experiments, neutralizing anti-CTLA-4 mAb (1 μg/mL) or its isotype control, ERK inhibitor U0126 (10 μM), or its vehicle control (dimethyl sulfoxide) were added into the culture. Means ± standard deviation (n = 8). *P > 0.05; ***P < 0.01. (C) PBMCs from HCC patients were stimulated with or without LPS for 24 hours. CD14+DCs were gated, and intracellular expression of pERK1/2 (pT202/pY204) was determined by FCM. Dot plots represent one of the independent experiments (n = 4). (D) One mechanism of Treg function: Binding of CTLA-4 on Tregs to CD80 and CD86 on DCs induce DCs to up-regulate IDO expression, which then suppresses T-cell responses (left). Model for the role of CTLA-4 in CD14+DCs: Binding of CTLA-4 on one CD14+DC to CD86 on other CD14+DC might induce themselves reciprocally to express IDO and IL-10, which then suppress T-cell responses (right).

Previous studies have shown that activation of extracellular signal-regulated kinase (ERK) modulates IL-10 production of DCs and macrophages subsequent to TLR stimulation.[25, 26] So, we investigated whether activation of ERK was involved in the production of IL-10 and IDO by CD14+DCs. We found that CD14+DCs constitutively expressed pERK, and LPS stimulation only slightly promoted ERK activation (Fig. 5C). Using U0126, a chemical inhibitor of ERK, we found that ERK activation was not involved in IL-10 production, but was involved in IDO production of CD14+DCs (Fig. 5A,B).

Discussion

Here, we identified one new population of CD11bhighCD11chighHLA-DR+CD14+CD1c+BDCA3+ regulatory DCs in the PBMCs of HCC patients. Although they also express monocyte marker CD14, and macrophage marker CD68,[27] we classified these cells to DCs, because these cells express human DC markers CD1c, BDCA-3, CD11c, and HLA-DR, their morphology are like DCs, and they showed some ability to promote CD4 T-cell proliferation. Healthy human blood DCs do not express CD14,[9, 15] so we named these HCC-induced cells as CD14+DCs.

We found that healthy human blood scarcely contained CD14+DCs, suggesting that they are not naturally occurring cells. CD14+DCs only existed in great quantity in the PBMCs of patients bearing liver cancers, including HCC and intrahepatic CCA, but not in the PBMCs of patients with other kinds of cancers, such as gastric carcinoma and colorectal cancer, or nontumor liver disease, such as cavernous hemangioma. However, at present, we cannot conclude that only liver cancer could induce the generation of CD14+DCs, because we only detected a few kinds of human cancers, and cannot exclude the possibility that other nonliver cancers could also induce the generation of CD14+DCs. Furthermore, the liver has been generally regarded as a classical immunosuppressive organ, which can induce the generation of immunosuppressive cells distributed systemically, so the microenvironment of liver cancers probably could amplify liver-based immunoregulation or -suppression strategies, resulting in much more enhanced global immunosuppressive environments than other nonliver cancers, thus contributing to the induction of circulating monocytes to regulatory CD14+DCs. In addition, in cancer patients, including liver cancer patients, many kinds of immunosuppressive cells, including Tregs and MDSCs, could be induced in peripheral blood and organs, such as spleen, and then these immunosuppressive cells could be recruited to the tumor tissue, contributing to the immunosuppressive status of cancer patients in this way utilized by cancer.

Moreover, we found that CD14+DCs could infiltrate in some tumor mass or liver tissue of HCC patients. It suggests that CD14+DCs are probably induced in peripheral blood, not in tumor tissue, and they could be recruited to tumor tissue to suppress local antitumor immune responses. It has been recognized long ago that tumor is a kind of systemic disease that not only affects tumor-bearing tissue or organs, but also the entire system of the body, especially the immune system, resulting in the systemic immunosuppressive status of the tumor-bearing host. Here, we propose that CD14+DCs might represent one of the important systemic changes induced by HCC, which contributes to, at least partly, the tumor-induced systemic immunosuppressive status, helping tumor escape of immune attack and promoting tumor progression. In addition, how CD14+DCs are induced is an important question in need of an answer. It has been proven that the immunosuppressive molecule, vitamin D3, could induce CD14 human blood DCs to CD14+ regulatory DCs in vitro.[15] Perhaps, some soluble factors from liver cancer tissues are responsible for this induction, which needs to be determined in the future.

As one of the important immune-suppressive cytokines, IL-10 plays central roles in various physio- and pathological immune responses and inflammatory processes.[28] IL-10 is expressed not only by cells of the adaptive immune system, including T helper (Th)1, Th2, Th17, and Tregs, as well as CD8 T and B cells, but also by cells of the innate immune system, including DCs, monocytes, macrophages, mast cells, natural killer cells, eosinophils, and neutrophils.[28] For example, recently identified regulatory B cells mainly depend on IL-10 to restrain excessive inflammatory responses.[29, 30] Here, we found that CD14+DCs could produce plenty of IL-10 in response to LPS, and neutralizing anti-IL-10 mAb could attenuate the immune-suppressive effect of CD14+DCs, although not absolutely, suggesting that they suppress T-cell responses partly by IL-10 production. Our data further prove the important roles of IL-10 in tumor immunosuppression.

IDO is one well-known molecule that contributes to tumor-induced immunosuppression. Recent studies have determined that IDO is overexpressed in both tumor cells and antigen-presenting cells (APCs) in tumor-draining lymph nodes.[31, 32] IDO catalyzes the rate-limiting step of tryptophan degradation along the kynurenine pathway, and both the reduction of local tryptophan concentration and the production of immunomodulatory tryptophan metabolites contribute to the immunosuppressive functions of IDO.[33] Moreover, IDO has been implicated as an immunosuppressive effector mechanism of Tregs.[34] Tregs could condition DCs to express IDO, which then indirectly suppress T-cell responses.[35] In this study, we found that CD14+DCs expressed high levels of IDO independently on LPS stimulation, and IDO inhibitor 1-MT almost suppressed the immunoregulatory function totally, showing that IDO might be more important than IL-10 in the mediation of suppressive effect of CD14+DCs.

Unexpectedly, CD14+DCs expressed high levels of CTLA-4 and PD-1, two key inhibitory receptors, mainly expressed on Tregs and activated T cells, that critically affect peripheral T-cell function.[36] Many studies have demonstrated that CTLA-4 is required of Tregs to suppress immune responses by affecting APCs.[37, 38] Ipilimumab, a fully human mAb against CTLA-4, improved overall survival in patients with previously treated metastatic melanoma,[39] and in patients with previously untreated metastatic melanoma as well.[40] Ipilimumab was approved by the U.S. Food and Drug Administration for treatment of metastatic melanoma in 2011. Here, we also found that CTLA-4 is essential to the function of CD14+DCs, because blockage of CTLA-4 inhibited them to produce IL-10 and IDO, suggesting the important role of non-Treg-derived CTLA-4. Therefore, ipilimumab might not only interfere with the function of Tregs, but also impair the immunosuppressive function of CD14+DCs. Previous studies have shown that binding of CTLA-4 on Tregs to CD80 and CD86 on DCs can induce DCs to up-regulate IDO protein expression and functional enzymatic activity,[35, 41] representing one mechanism of Treg function (Fig. 5D, left). CD14+DCs express CTLA-4 and CD86 together, so we propose that the binding of CTLA-4 on one CD14+DC to CD86 on other CD14+DCs might induce themselves reciprocally to express IDO and IL-10 (Fig. 5D, right). Therefore, it seems that CD14+DCs do the jobs of two kinds of cells: Tregs and DCs, displaying a more economical, effective way used by tumor to suppress antitumor immune responses.

In summary, we identified new regulatory CD14+CTLA-4+ DCs in PBMCs and tumor mass of HCC patients. They expressed two important immune inhibitory molecules (CTLA-4 and PD-1). After LPS stimulation, these cells produced plenty of IL-10 and IDO. Moreover, CD14+DCs suppress CD4 T-cell response by IL-10 and IDO, which depends on CTLA-4, suggesting the important role of non-Treg-derived CTLA-4. CD14+DCs might represent one of the important systemic changes induced by HCC, which partly contribute to the tumor-induced systemic immunosuppressive status helping tumor immune escape and progression; thus, our results provide a previously unrecognized target of immunotherapy for HCC.

Acknowledgment

The authors sincerely thank Ms. Jianqiu Long and Ms. Xiaoting Zuo for their technical assistance.

Ancillary