Apoptotic cells induce Mer tyrosine kinase-dependent blockade of NF-kappaB activation in dendritic cells

Dendritic cells (DCs) play a key role in immune homeostasis and maintenance of self-tolerance. Tolerogenic DCs can be established by an encounter with apoptotic cells (ACs) and subsequent inhibition of maturation and effector functions. The receptor(s) and signaling pathway(s) involved in AC-induced inhibition of DCs have yet to be defined. We demonstrate that pretreatment with apoptotic but not necrotic cells inhibits activation of IkappaB kinase (IKK) and downstream NF-kappaB. Notably, receptor tyrosine kinase Mer (MerTK) binding of ACs is required for mediating this effect. Monocyte-derived DCs lacking MerTK expression (MerTKKD) or treated with blocking MerTK-specific antibodies (Abs) are resistant to AC-induced inhibition and continue to activate NF-kappaB and secrete proinflammatory cytokines. Blocking MerTK activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway prevents AC-induced inhibition. These results demonstrate an essential role for MerTK-mediated regulation of the PI3K/AKT and NF-kappaB pathways in AC-induced inhibition of monocyte-derived DCs.     

Mature dendritic cells use endocytic receptors to capture and present antigens

In response to inflammatory stimuli, dendritic cells (DCs) trigger the process of maturation, a terminal differentiation program required to initiate T-lymphocyte responses. A hallmark of maturation is down-regulation of endocytosis, which is widely assumed to restrict the ability of mature DCs to capture and present antigens encountered after the initial stimulus. We found that mature DCs continue to accumulate antigens, especially by receptor-mediated endocytosis and phagocytosis. Internalized antigens are transported normally to late endosomes and lysosomes, loaded onto MHC class II molecules (MHCII), and then presented efficiently to T cells. This occurs despite the fact that maturation results in the general depletion of MHCII from late endocytic compartments, with MHCII enrichment being typically thought to be a required feature of antigen processing and peptide loading compartments. Internalized antigens can also be cross-presented on MHC class I molecules, without any reduction in efficiency relative to immature DCs. Thus, although mature DCs markedly down-regulate their capacity for macropinocytosis, they continue to capture, process, and present antigens internalized via endocytic receptors, suggesting that they may continuously initiate responses to newly encountered antigens during the course of an infection.     

Regulation of immune responses to Mycobacterium tuberculosis secretory antigens by dendritic cells

Towards elucidating the immune responses induced by antigens from the Mycobacterium tuberculosis (M. tb) RD-1 region, we have been characterizing their interactions with dendritic cells (DCs) and their precursors. We have shown that incubation of bone marrow DC precursors with M. tb antigens induces the differentiation of DC precursors and also the maturation of various DC subsets. While MTSA differentiated DCs were immature, MTSA matured DCs were terminally mature. However, regardless of their maturation status M. tb secretory antigen-activated DCs down-regulated pro-inflammatory T helper cell responses to a subsequent challenge with M. tb cell extract (CE) while increasing regulatory responses. Investigations into the underlying mechanisms showed that stimulation with M. tb CE changed the polarization of antigen-activated DCs from DC1 to DC2. This resulted in secretion of high levels of IL-10 and TGF-beta together with increased surface expression of CD86. Blocking either IL-10 or TGF-beta or CD86 restored Th1 responses to CE antigens. Conversely, treatment of antigen-activated DCs with IL-12 and/or IFN-gamma fully restored Th1 responses of CE antigens. These results indicate that M. tb strategically secretes antigens from infected macrophages to down-regulate pro-inflammatory immune responses at sites of infection.     

Hepatitis C virus-infected hepatocytes extrinsically modulate dendritic cell maturation to activate T cells and natural killer cells

Dendritic cell maturation critically modulates antiviral immune responses, and facilitates viral clearance. Hepatitis C virus (HCV) is characterized by its high predisposition to persistent infection. Here, we examined the immune response of human monocyte-derived dendritic cells (MoDCs) to the JFH1 strain of HCV, which can efficiently replicate in cell culture. However, neither HCV RNA replication nor antigen production was detected in MoDCs inoculated with JFH1. None of the indicators of HCV interacting with MoDCs we evaluated were affected, including expression of maturation markers (CD80, 83, 86), cytokines (interleukin-6 and interferon-beta), the mixed lymphocyte reaction, and natural killer (NK) cell cytotoxicity. Strikingly, MoDCs matured by phagocytosing extrinsically-infected vesicles containing HCV-derived double-stranded RNA (dsRNA). When MoDCs were cocultured with HCV-infected apoptotic Huh7.5.1 hepatic cells, there was increased CD86 expression and interleukin-6 and interferon-beta production in MoDCs, which were characterized by the potential to activate NK cells and induce CD4+ T cells into the T helper 1 type. Lipid raft-dependent phagocytosis of HCV-infected apoptotic vesicles containing dsRNA was indispensable to MoDC maturation. Colocalization of dsRNA with Toll-like receptor 3 (TLR3) in phagosomes suggested the importance of TLR3 signaling in the MoDC response against HCV. CONCLUSION: The JFH1 strain does not directly stimulate MoDCs to activate T cells and NK cells, but phagocytosing HCV-infected apoptotic cells and their interaction with the TLR3 pathway in MoDCs plays a critical role in MoDC maturation and reciprocal activation of T and NK cells.     

Disease-associated dendritic cells respond to disease-specific antigens through the common heat shock protein receptor

The most abundant intracellular proteins, heat shock proteins (HSPs), serve as molecular chaperones for regulatory and maturation pathways. Diverse families of HSPs have been shown to bind antigenic peptides and to play major roles in innate and adaptive immune responses through the common HSP receptor, CD91. HIV-1+ patients with Kaposi sarcoma (KS) were matched for CD4 count and HIV-1 RNA viral load to HIV-1+ patients without Kaposi sarcoma (and negative for Kaposisarcoma-associated herpesvirus). We then investigated the pathways used by tumor lysates, viral lysates, and viral particles in their activation. In particular, we observed immune responses after HSP depletion using antitumor antibiotics and blockade of the common HSP receptor, CD91. Despite the impaired functional capacity of dendritic cells (DCs) derived from patients with KS, DCs retain the ability to prime the adaptive arm of the immune system through the common HSP receptor, leading to phenotypic activation and stimulation of tetramer-positive CD8+ cytotoxic T cells. We also show that interferon-producing plasmacytoid DCs are selectively depleted in KS-positive compared with matched KS-negative HIV-1-infected patients. Functionally impaired DCs can effectively cross-present immune responses through the common HSP receptor. These results have important implications for the etiopathogenesis of KS and for the development and design of any compounds, including vaccines, derived from cellular lysates.     

Recognition of HBV antigens and HBV DNA by dendritic cells

BACKGROUND:Hepatitis B virus(HBV)is a hepatotropic, noncytopathic,DNA virus which can cause acute and chronic infection.Viral persistence is associated with a weak or absent specific immune responses to HBV,particularly the cellular immune response.Dendritic cells(DCs)are professional antigen-presenting cells with a unique T cell stimulatory aptitude that play a crucial role in the instruction of adaptive immune responses upon infection.An impaired function of DCs was suggested by recent studies to account for the T and B cell hyporesponsiveness in chronic HBV infection.This review summarizes recent insights into the recognition of HBV antigens by DCs. DATA SOURCES:Studies were identified by searching MEDLINE and/or PubMed for articles using the key words"hepatitis B virus (HBV)","dendritic cells","C-type lectins","mannose receptor", "toll-like receptor",and"dendritic cell-specific intercellular-adhesion-molecule-3 grabbing nonintegrin(DC-SIGN)"up to December 2009.Additional papers were identified by a manual search of the references from the key articles. RESULTS:DCs play an important role in the progress of hepatitis B,especially in the recognition of HBV.There are three main ways of recognition of HBV antigens by DCs. First,HBV DNA can be recognized by DCs through toll-like receptor 9(TLR9)which activates the NF-κB signal pathway and p38 MAPK to up-regulate the expression of interferon (IFN)regulatory factor 7(IRF-7)in a manner independent of type I IFN signaling,resulting in secretion of type I IFN and inflammatory cytokines,and induction of DC maturation and the adaptive immune response.Second,HBc/HBeAg cannot be recognized by DCs,but DNA or ssRNA encapsulated within HBcAg can be internalized by DCs through TLRs.Third,HBsAg can be internalized by DCs through the mannose receptor,which lacks the ability to induce DC maturation without the assistance of DC-SIGN.Meanwhile,there is some cross-talk among the three mechanisms,which induces an effective anti-viral response or HBV persistence. CONCLUSIONS:On the basis of these recognition processes, methods have been used to enhance the efficacy of DC-based vaccine against HBV and have been useful in the clinical application of HBV vaccine therapy.But the interactions between HBV antigens/HBV DNA and DCs are not clear, and cross-talk between TLRs and various ligands makes HBV antigen recognition by DCs more complicated.More efforts should be made to define the mechanisms and develop effective vaccines and therapies.     

Vaccination with protein-transduced dendritic cells elicits a sustained response to hepatitis C viral antigens

BACKGROUND & AIMS: Professional antigen-presenting dendritic cells are capable of eliciting a vigorous antiviral response in naive T cells. The administration of antigen-loaded dendritic cells offers a potential approach to induce high-level immunity against hepatitis C virus. METHODS: The dendritic cell population in mice was expanded in vivo by hydrodynamic delivery of naked DNA that encoded the secreted form of human fms-like tyrosine kinase 3 ligand. The CD11c-enriched dendritic cell population obtained from the spleen was transduced in vitro with recombinant hepatitis C virus core and nonstructural 5 proteins by using macromolecular-based protein delivery. Vaccine efficacy was assessed with a cytotoxic T-lymphocyte assay, cytokine enzyme-linked immunosorbent assays, and intracellular cytokine staining in vitro and by a tumor challenge model in vivo. RESULTS: Relative to mice inoculated with nontransduced dendritic cells, splenocytes derived from mice immunized with either hepatitis C virus core-transduced or nonstructural 5-transduced dendritic cells showed 3- to 5-fold greater antigen-specific cytotoxic T lymphocyte activity. The CD4(+) T cells obtained from mice immunized with nonstructural 5-transduced dendritic cells produced interferon gamma, but not interleukin 4, when stimulated with nonstructural 5. In contrast, T cells derived from mice immunized with hepatitis C virus core-transduced dendritic cells produced neither interferon gamma nor interleukin 4 when stimulated with core protein. Mice vaccinated with nonstructural 5-transduced dendritic cells, but not a nonstructural 5-expressing plasmid, showed a sustained antiviral response to nonstructural 5 as evidenced by reduced growth of nonstructural 5-expressing tumor cells inoculated 10 weeks after vaccination. CONCLUSIONS: These findings suggest that vaccination with protein-transduced dendritic cells may constitute an important antiviral strategy for hepatitis C virus.     

Pulmonary dendritic cells in local immunity to inert and pathogenic antigens in the respiratory tract

Subsets of dendritic cells form a series of highly developed networks throughout the respiratory tree, and represent the only professional antigen-presenting cells present within the majority of these tissue microenvironments. Work in noninfectious model systems indicates that they function with high efficiency in immune surveillance for inhaled antigens, responding rapidly to local antigenic challenge via mobilization of resident and precursor populations with kinetics equivalent to neutrophils. Their prime function is to rapidly translocate incoming antigenic signals to the lymph nodes draining the mucosal surfaces of the respiratory tract, and as such they are ideally positioned to orchestrate primary and secondary adaptive immunity to all classes of inhaled antigens, in particular those derived from pathogens such as respiratory viruses.     

Regulation of immune response by Plasmodium-infected red blood cells

During the asexual blood stage infection of the human malaria parasite, Plasmodium falciparum, parasite-derived proteins are inserted onto the surface of the host red blood cell membrane. These proteins are highly variable and were originally thought only to mediate antigenic variation, and sequestration of parasites from peripheral circulation, thus enabling immune evasion. Recent studies have revealed that PfEMP-1 and other molecules on the P. falciparum-infected red blood cell (PfRBC) activate and modulate the immune response. In this review, we discuss how PfRBCs interact with antigen-presenting cells (APCs) and other cells of the immune system, and how such interactions could modulate the host response to Plasmodium infections.     

Cryptosporidium parvum antigens induce mouse and human dendritic cells to generate Th1-enhancing cytokines

Cryptosporidium parvum is an opportunistic intracellular parasite that causes mild to severe diarrhoea, which can be life‐threatening in an immunocompromised host. To increase our understanding of the mechanisms that play a role in host immune responses, we investigated the effects of C. parvum antigens on the phenotype of mouse and human dendritic cells (DCs). Cryptosporidium parvum antigens induced DC activation as indicated by upregulation of the maturation marker CD209, as well as by the production of the cytokines interleukin‐12 p70, IL‐2, IL‐1beta, IL‐6. In particular, significant increases in the expression of IL‐12 p70 were observed from mouse DCs derived from bone marrow in response to solubilized sporozoite antigen and the recombinant cryptosporidial antigens, Cp40 and Cp23. We observed a small but significant increase in IL‐18 expression following the exposure to Cp40. We found that the induction of Th1 cytokines was MyD88 dependent (MyD88 knockout mouse DCs were unresponsive). Additionally, both sporozoite preparations (solubilized and live) significantly induced IL‐12 production by human monocytic dendritic cells (MoDCs). This finding indicates that solubilized as well as recombinant antigens can induce the maturation of DCs and subsequently initiate an innate immune response.     

Targeting hepatitis B virus antigens to dendritic cells by heat shock protein to improve DNA vaccine potency

AIM： To investigate a novel DNA vaccination based upon expression of the HBV e antigen fused to a heat shock protein （HSP） as a strategy to enhance DNA vaccine potency.
METHODS： A pCMV-HBeAg-HSP DNA vaccine and a control DNA vaccine were generated. Mice were immunized with these different construct. Immune responses were measured 2 wk after a second immunization by a T cell response assay, CTL cytotoxicity assay, and an antibody assay in C57BL/6 and BALB/c mice. CT26-HBeAg tumor cell challenge test in vivo was Performed in BALB/c mice to monitor anti-tumor immune responses.
RESULTS： In the mice immunized with pCMV-HBe-HSP DNA, superior CTL activity to target HBV-positive target cells was observed in comparison with mice immunized with pCMV-HBeAg （44% ± 5% vs 30% ± 6% in E： T 〉 50：1, P 〈 0,05）, ELISPOT assays showed a stronger T-cell response from mice immunized with pCMV-HBe- HSP than that from pCMV-HBeAg immunized animals when stimulated either with MHC class I or class Ⅱ epitopes derived from HBeAg （74% ± 9% vs 31% ± 6%, P 〈 0.01）. ELISA assays revealed an enhanced HBeAg antibody response from mice immunized with pCMV- HBe-HSP than from those immunized with pCMV-HBeAg. The lowest tumor incidence and the slowest tumor growth were observed in mice immunized with pCMV- HBe-HSP when challenged with CT26-HBeAg.
CONCLUSION： The results of this study demonstrate a broad enhancement of antigen-specific CD4^＋ helper,CD8^＋ cytotoxic T-cell, and B-cell responses by a novel DNA vaccination strategy. They also proved a stronger antigen-specific immune memory, which may be superior to currently described HBV DNA vaccination strategies for the treatment of chronic HBV infection.     

丝裂霉素诱导凋亡癌细胞致敏树突状细胞后的免疫应答

目的 观察树突状细胞(DC)从丝裂霉素诱导的凋亡胆管癌细胞获取抗原后,抗肿瘤免疫应答及对胆管癌细胞的特异性免疫杀伤效果。 方法 用粒细胞-巨噬细胞集落刺激因子(GM-CSF)加IL-4从人外周血分化、诱导DC,丝裂霉素在体外诱导培养的人胆管癌细胞凋亡,将DC、T淋巴细胞和凋亡胆管癌细胞共培养,同时设计不同类型肿瘤细胞(坏死胆管癌细胞及培养胆管癌细胞)作对照,7d后,分离、富集DC、T淋巴细胞进行免疫应答及肿瘤细胞杀伤试验。 结果 与凋亡胆管癌细胞共培养的DC可以有效提呈胆管癌细胞抗原,有强烈的免疫应答,刺激的细胞毒T淋巴细胞特异性地杀伤胆管癌细胞。 结论 丝裂霉素诱导凋亡癌细胞可以致敏rhGM-CSF加rhIL-4从人外周血单个核细胞诱导、扩增出的DC,并产生显著的杀伤胆管癌细胞的免疫反应,可望成为特异性免疫治疗肿瘤的一条新途径。     

Induction of CD4+/CD25+ regulatory T cells by targeting of antigens to immature dendritic cells

Coupling of ovalbumin (OVA) to anti-DEC-205 monoclonal antibody (mAb) (alphaDEC) induced the proliferation of OVA-specific T cells in vivo. Expansion was short-lived, caused by dendritic cells (DCs), and rendered T cells anergic thereafter. Phenotypic analysis revealed the induction of CD25+/CTLA-4+ T cells suppressing proliferation and interleukin-2 (IL-2) production of effector CD4+ T cells. The findings were supported by 2 disease models: (1) CD4+ T-cell-mediated hypersensitivity reactions were suppressed by the injection of alphaDEC-OVA and (2) the application of hapten-coupled alphaDEC-205 reduced CD8+ T-cell-mediated allergic reactions. Thus, targeting of antigens to immature DCs through alphaDEC antibodies led to the induction of regulatory T cells, providing the basis for novel strategies to induce regulatory T cells in vivo.     

树突状细胞负载肝癌相关抗原后成熟调控的研究

目的 研究树突状细胞（DC）负载肝癌相关抗原后的成熟调控。方法 用SDS－PAGE制备电泳纯化牛结核分枝杆菌热休克蛋白70，用其诱导DC的分化与成熟。结果 DC负载肝癌可溶性抗原后，10％2细胞失去了DC特征，同时其表面CD54（90．0％），CD83（78．0％），CD86（85．0％）分子表达下降；牛分枝杆菌卡介苗－热休克蛋白70（BCG HSP70）的活化有利于负载肝癌可溶性抗原后的DC维持其特异性标志，同时DC表面CD54（92．0％），CD83（90．0％），CD86（91．0％）分子表达增加，DC诱导同种异体淋巴细胞转化的能力增加，淋巴细胞增殖加快，从而促进DC成熟，增加其抗原呈递能力。结论 预示BCG HSP70有可能成为促进DC活化和成熟的另一重要分子。     

CD8alpha-11b+ dendritic cells but not CD8alpha+ dendritic cells mediate cross-tolerance toward intestinal antigens

Cross-presentation is a critical process by which antigen is displayed to CD8 T cells to induce tolerance. It is believed that CD8alpha+ dendritic cells (DCs) are responsible for cross-presentation, suggesting that the CD8alpha+ DC population is capable of inducing both cross-priming and cross-tolerance to antigen. We found that cross-tolerance against intestinal soluble antigen was abrogated in C57BL/6 mice lacking mesenteric lymph nodes (MLNs) and Peyer patches (PPs), whereas mice lacking PPs alone were capable of developing CD8 T-cell tolerance. CD8alpha-CD11b+ DCs but not CD8alpha+ DCs in the MLNs present intestinal antigens to relevant CD8 T cells, while CD8alpha+ DCs but not CD8alpha-CD11b+ DCs in the spleen exclusively cross-present intravenous soluble antigen. Thus, CD8alpha-CD11b+ DCs in the MLNs play a critical role for induction of cross-tolerance to dietary proteins.     

Intrahepatic delivery of alpha-galactosylceramide-pulsed dendritic cells suppresses liver tumor

Alpha-galactosylceramide, a glycosphingolipid, mediates interaction of dendritic cells (DCs) and NKT cells, leading to activation of both innate and acquired immunity. For cancer treatment, conventional DC-based vaccine has been tried, but its clinical efficacy is limited against liver cancer. Intrahepatic injection of alpha-Galactosylceramide-pulsed DCs (alphaGCDC) has not yet been tested in the liver that contains abundant immune cells such as NK, NKT, and T cells. In the present study, we examined the efficacy of alphaGCDC administration in comparison with p53 peptide-pulsed DCs using a well-established murine CMS4 tumor model. Injection of alphaGCDC into CMS4 liver tumors resulted in complete tumor rejection and established long-term survival of the animals, while injection of p53(232-240) peptide-pulsed DCs (pepDC) only partially suppressed tumor growth in the liver. The levels of IFN-gamma in sera of alphaGCDC-treated mice were significantly higher than those of pepDC-treated mice. Hepatic NK cells were efficiently activated by alphaGCDC injection and played a critical role in liver tumor rejection as evidenced by an in vivo antibody-mediated NK cell depletion study. Injection of alphaGCDC into liver tumor led to higher p53(232-240) peptide-specific CD8+ T cell response than that of pepDC. The mice that had been protected from CMS4 liver tumor by alphaGCDC injection became resistant to subcutaneous CMS4 rechallenge, but not to Colon26 rechallenge. CONCLUSION: These results demonstrate that alphaGCDC injection into the liver can efficiently activate NK cells that in turn reject liver tumors to establish potent acquired immunity against the original tumor.     

微载体培养人肝细胞及肝非实质细胞

目的研究用微载体培养人肝细胞及肝非实质细胞的方法．方法采用体外简易两步灌流和差速离心法获取胎肝细胞和肝非实质细胞，在综合限定条件下进行微载体ｃｙｔｏｄｅｘ３粘附培养，并对培养肝细胞的形态及合成葡萄糖和白蛋白的功能进行动态测定．结果分离肝细胞及肝非实质细胞在接种时即呈明显的聚集倾向，将其与微载体混合振荡孵育２０ｍｉｎ后，二者极易相粘附．在被覆聚羟乙基异丁烯酸的培养瓶内，使用激素限定条件培养液培养约４８ｈ，典型的多细胞聚集球形体得以形成．该形态特征以及白蛋白、葡萄糖合成能力可保持１月结论使用微载体培养人肝细胞和肝非实质细胞具有多种优点，有广泛的应用价值．