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)
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+CD25−Foxp3− Tregs in liver-cancer-bearing mice, 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. 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). 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. 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. 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. 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. 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.
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. 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. 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.
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, 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. 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. 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. 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. Moreover, IDO has been implicated as an immunosuppressive effector mechanism of Tregs. Tregs could condition DCs to express IDO, which then indirectly suppress T-cell responses. 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. 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, and in patients with previously untreated metastatic melanoma as well. 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.