Jintang Sun

HIF-dependent induction of adenosine receptor A2b skews human dendritic cells to a Th2-stimulating phenotype under hypoxia

Hypoxia is a common characteristic of many pathological and physiological conditions that can markedly change cellular metabolism and cause the accumulation of extracellular adenosine. Recent studies have shown that adenosine can modulate the function of certain immune cell types through binding with different adenosine receptors. Our previous studies have shown that hypoxia has an effect on the biological activity of dendritic cells (DCs) by inducing their differentiation towards a Th2 polarising phenotype. However, the mechanisms underlying this suppression remain unclear. In this study, we have demonstrated that hypoxic mDCs predominantly express adenosine receptor A2b. The A2b receptor antagonist MRS1754 was able to increase the production of IL‐12p70 and TNF‐α by hypoxic mDCs and elevate the amount of Th1 cytokine IFN‐γ production in a mDCs‐T‐cell co‐culture system. We also found that the effect of hypoxia on IL‐12p70 production was mediated via increased intracellular cAMP levels through the up‐regulation of A2b adenosine receptor and the preferential expression of adenosine A2b receptors in hypoxic mDCs was HIF‐1α dependent. Therefore, the hypoxic mDCs could provide a useful tool for researching the function of A2bR in human DCs. Our results offer new insights into understanding the molecular mechanisms underlying the biological activities of DCs in local‐tissue hypoxic microenvironments.

Fucoidan stimulation induces a functional maturation of human monocyte-derived dendritic cells.

Fucoidan is a complex sulfated polysaccharide with a wide variety of biological activities for modulating immune cell functions. However, the effects of fucoidan on maturation process and activation of human monocyte-derived dendritic cells (DCs) remain to be elucidated. The present study demonstrated that the level of special marks and polarization phenotype of DCs was altered by fucoidan. Human monocytes were cultured with GM-CSF and IL-4 for 5 days followed by another 2 days in the presence of fucoidan or LPS. Then DCs were harvested on day 7 and were examined using functional assays. We demonstrated that fucoidan up-regulated the expression of HLA-DR and co-stimulatory molecules of DCs. However the endocytic activity was impaired markedly. Fucoidan induces their Th1-promoting tumor necrosis factor alpha (TNF-alpha) and interleukin-12 (IL-12) secretion, and enhances their allostimulatory capacity. In an allogeneic MLR assay, DCs treated with fucoidan were potent in the secretion of IL-12p70, TNF-alpha and IFN-gamma. Naive T cells stimulated by fucoidan-treated DCs differentiated towards a helper T cell type 1 (Th1) response depending on IL-12 secretion. These results suggest that fucoidan may induce immature DCs maturation and drive their differentiation towards a Th1-polarizing phenotype. Moreover, our data suggest that DCs appear to be a potential target for the immunomodulatory capacity of fucoidan and fucoidan may be used on DC-based vaccines for cancer immunotherapy.

Hypoxia skews dendritic cells to a T helper type 2-stimulating phenotype and promotes tumour cell migration by dendritic cell-derived osteopontin

It is well recognized that tissue microenvironments are involved in regulating the development and function of dendritic cells (DC). Oxygen supply, which varies in different tissues, has been accepted as an important microenvironmental factor in regulating the biological functions of several immune cells and as being involved in tumour progression and metastasis. However, little is known about the effect of hypoxia on the biological functions of DC and the effect of these hypoxia‐conditioned DC on tumour metastasis. In this study, we analysed the transcriptional profiles of human monocyte‐derived immature DC (imDC) and mature DC (mDC) cultured under normoxia and hypoxia by microarray, and found a body of potential targets regulating the functions of DC during hypoxia. In addition, the phagocytic ability of hypoxic imDC markedly decreased compared with that of normoxic imDC. Importantly, hypoxic DC poorly induced the proliferation of allogeneic T cells, but polarized allogeneic CD4+ naive T cells into a T helper type 2 (Th2) response. Moreover, hypoxic DC secreted large amounts of osteopontin, which were responsible for the enhanced migration of tumour cells. Therefore, our study provides new insights into the biological functions of DC under hypoxic conditions and one of mechanisms underlying tumour immune escape during hypoxia.

Synergy between the ectoenzymes CD39 and CD73 contributes to adenosinergic immunosuppression in human malignant gliomas

BACKGROUND The importance of ectoenzymes CD39 and CD73 in mediating adenosinergic immunosuppression has been recognized, but their roles in human malignant glioma-associated immunosuppression remain largely unknown. METHODS In this study, the ectoenzyme characteristics of malignant glioma cells and infiltrating CD4(+) T lymphocytes isolated from newly diagnosed malignant glioma patients were investigated. The ectoenzyme activities of both cell populations were determined by nucleotide hydrolysis assay. The immunosuppressive property of the CD39-CD73 synergic effect was evaluated via responder T-cell proliferation assay. RESULTS We observed that CD39(-)CD73(+) glioma cells and infiltrating CD4(+)CD39(high)CD73(low) T lymphocytes exhibited 2 distinct but complementary ectoenzyme phenotypes, which were further verified by enzyme activity assay. The nucleotide hydrolysis cascade was incomplete unless CD39 derived from T lymphocytes and CD73 collaborated synergistically. We demonstrated that increased suppression of responder CD4(+) T-cell proliferation suppression was induced by CD4(+)CD39(+) T cells in the presence of CD73(+) glioma cells, which could be alleviated by the CD39 inhibitor ARL67156, the CD73 inhibitor APCP, or the adenosine receptor A2aR antagonist SCH58261. In addition, survival analysis suggested that CD73 downregulation was a positive prognostic factor related to the extended disease-free survival of glioblastoma patients. CONCLUSIONS Our data indicate that glioma-derived CD73 contributes to local adenosine-mediated immunosuppression in synergy with CD39 from infiltrating CD4(+)CD39(+) T lymphocytes, which could become a potential therapeutic target for treatment of malignant glioma and other immunosuppressive diseases.

Human placental trophoblasts express the immunosuppressive cytokine IL-35

Studies of maternal-fetal tolerance focus on defining mechanisms for establishment of immunological privilege within the uterus during pregnancy. Fetal trophoblasts play a key role in maternal tolerance, in part through cytokines production. As a novel inhibitory cytokine, IL-35 is produced by Foxp3(+) regulatory T cells (Tregs) and mediates maximal suppression of Tregs. The purpose of the study is to analyze the expression of IL-35 in first-trimester human placental trophoblasts. IL-35 expression was detected at both protein and mRNA levels by immunohistochemical staining and quantitative real-time PCR method, respectively and secretion of IL-35 was measured by ELISA assay. Our results demonstrated that human trophoblasts constitutively expressed IL-35. Ebi3 and p35 (two subunits of IL-35) mRNA was shown to be co-expressed in trophoblast cells. Moreover, large amounts of secreted IL-35 were detected in the supernatants of trophoblast cells. But we did not detect the constitutive expression of IL-35 in decidual stromal cells. Our findings confirmed for the first time that first-trimester human trophoblast cells expressed and secreted IL-35, which might contribute to their suppressive capacity to maternal immune cells. Therefore, IL-35 may be an important factor of the cytokine network regulating local immune responses during human pregnancy.

Hypoxia induces T-cell apoptosis by inhibiting chemokine C receptor 7 expression: the role of adenosine receptor A(2).

Hypoxia is a major characteristic of the tumor microenvironment, and its effects on immune cells are proposed to be important factors for the process of tumor immune escape. It has been reported that hypoxia affects the function of dendritic cells and the antitumor function of T cells. Here we discuss the effects of hypoxia on T-cell survival. Our results showed that hypoxia induced apoptosis of T cells. Adenosine and adenosine receptors (AR) are important to the hypoxia-related signaling pathway. Using AR agonists and antagonists, we demonstrated that hypoxia-induced apoptosis of T cells was mediated by A2a and A2b receptors. Furthermore, we are the first, to our knowledge, to report that hypoxia significantly inhibited the expression of chemokine C receptor 7 (CCR7) of T cells via the A2R signal pathway, perhaps representing a mechanism of hypoxia-induced apoptosis of T cells. Collectively, our research demonstrated that hypoxia induces T-cell apoptosis by the A2R signaling pathway partly by suppressing CCR7. Blocking the A2R signaling pathway and/or activation of CCR7 can increase the anti-apoptosis function of T cells and may become a new strategy to improve antitumor potential.

Regulation of Th1/Th2 polarization by tissue inhibitor of metalloproteinase-3 via modulating dendritic cells

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is one of a family of proteins inhibiting matrix metalloproteinases, which has also been identified as a mediator for checking inflammation. Meanwhile, it is well known that inflammation causes the activation of the immune response. However, it is not clear whether TIMP-3 plays a role in the immune system. In the present study, we demonstrated a novel function of TIMP-3 in Th1/Th2 polarization through its influence on the antigen-presenting cells. First, TIMP-3 was found strikingly up-regulated by IL-4 during the differentiation of human dendritic cells via the p38MAPK pathway. Second, the expression of costimulatory molecule-CD86 was repressed by TIMP-3. Besides, the induction of IL-12 in matured dendritic cells was significantly inhibited in a PI3K-dependent manner. Furthermore, dendritic cells matured in the presence of TIMP-3 could stimulate allogeneic naive T helper (Th) cells to display a prominent Th2 polarization. Importantly, in an autoimmune disorder-primary immune thrombocytopenia, TIMP-3 showed a statistically positive correlation with IL-4 and platelet count, but a negative correlation with IFN-γ in patient blood samples. Collectively, these in vitro and in vivo data clearly suggested a novel role of TIMP-3 in Th1/Th2 balance in humans.

Osteopontin splice variants expressed by breast tumors regulate monocyte activation via MCP-1 and TGF-β1

Osteopontin (OPN), a multifunctional glycoprotein, has three transcripts that have distinct roles in tumors in vitro. Whether OPN transcripts have different functions in tumor processes in vivo is unclear. It has been reported that immune cell-derived OPN can promote tumor formation. We propose a hypothesis that tumor-derived OPN may facilitate tumor immune escape by affecting immune cell differentiation and function. In this study, we constructed lentiviral expression vectors of OPN transcripts and transfected them into the MCF-7 cell line. MCF-7 cells transfected with OPN transcripts were injected into the armpit of nude mice, and tumor growth was monitored. The results showed that all OPN transcripts promoted local tumor formation, but that there was no significant difference among transcripts. We also investigated the effect of the OPN expressed by tumor cells on monocyte differentiation by coculturing monocytes with tumor supernatant. We found OPN-c upregulated CD163 levels compared with OPN-a and OPN-b; however, none of the transcripts affected HLA-DR and CD206 levels. All OPN transcripts significantly inhibited TNF-α and enhanced IL-10 production by monocytes. Furthermore, we found that the overexpression of OPN transcripts significantly upregulated TGF-β1 and MCP-1 production by tumor cells. Using neutralizing antibody and recombinant cytokines, we found that OPN overexpressed by tumor cells regulates the production of TNF-α and IL-10 by monocytes partly via MCP-1 and TGF-β1, respectively. Collectively, our results show that OPN transcripts have no distinct role in breast cancer formation in vivo. We also demonstrate that OPN regulates the alternative activation of monocytes via TGF-β1 and MCP-1, which may represent an additional mechanism for tumor immune escape.

RhoBTB2 (DBC2) functions as tumor suppressor via inhibiting proliferation, preventing colony formation and inducing apoptosis in breast cancer cells

RhoBTB2 was isolated recently as a tumor suppressor gene from sporadic breast cancer. Although RhoBTB2 was found to be frequently lost in breast cancer and a variety of cancers, its antitumor effect, however, remains unclear. In this study, we constructed a recombinant expression vector pEGFP-N1-RhoBTB2 and transfected it into RhoBTB2-negative breast tumor cell line T-47D. Stable transformanted cells were identified by fluorescence microscope, RT-PCR and Western blot. Cell viability was measured by MTT assay. Colony forming efficiency of breast tumor cells was detected by colony formation assay. Morphological change of apoptotic cells was observed by hematoxylin-eosin staining. Apoptotic ratio was determined by flow cytometry. Cell invasion and migration ability assay were performed using transwell system. Overexpression of RhoBTB2 in breast tumor cells significantly inhibited the proliferation and colony formation of tumor cells. In addition, RhoBTB2 also elevated the apoptotic ratio and caused typical changes of apoptotic morphology in breast tumor cells of RhoBTB2 overexpression. But RhoBTB2 did not influence the invasion and migration ability of breast tumor cells. Therefore, RhoBTB2 is an important tumor suppressor gene related with breast cancer and may play antitumor roles by inhibiting proliferation, preventing colony formation and promoting the apoptosis of tumor cells. However, the precise mechanism behind the antitumor effects of RhoBTB2 needs to be investigated further.

Aberrant expression of chemokine receptor CCR4 in human gastric cancer contributes to tumor‐induced immunosuppression

The chemokine receptor CCR4 is preferentially expressed on certain immune cells and some hematological tumor cells, which play pivotal roles in suppression of host immune response. However, the reasons for the upmodulation of CCR4 and its immune functions in solid tumors remain unclear. Herein, we aimed to determine the expression profiles of CCR4 in gastric cancer cells and its role in regulating antitumor immunity. CCR4 expression was assessed in 63 cases of gastric carcinomas by immunohistochemistry. We found cancer cells in lymphocyte‐rich carcinomas more frequently showed moderate to strong positive staining for CCR4 than those in conventional carcinomas (P = 0.041), and also found a positive relationship between expression of CCR4 and tumor necrosis factor‐α (P = 0.012). Stimulation of gastric cell lines with various cytokines showed that tumor necrosis factor‐α uniquely upmodulated CCR4 expression through activation of nuclear factor‐κB. Additional coculture experiments showed the forced expression of CCR4 in SGC‐7901 cells caused a significant reduction of γ‐interferon and elevation of interleukin‐10 secretion in the supernatants from cocultured SGC‐7901 cells and PBMCs. In addition, granzyme A production in cancer cell‐cocultured CD56+ natural killer cells was significantly downregulated. Inhibition of the overexpressed CCR4 in cancer cells by an inhibitor of CCR4, compound 39, proved to partly restore the antitumor immunity in respect of the inverse changes in those factors. Our studies suggest that the aberrant expression of CCR4 in human gastric cancer could contribute to tumor‐induced immunosuppression. Conceivably, downmodulation of CCR4 expression could be a promising immunotherapy for human gastric cancer. (Cancer Sci 2011; 102: 1264–1271)