Human C1q Regulates Influenza A Virus Infection and Inflammatory Response via Its Globular Domain

The Influenza A virus (IAV) is a severe respiratory pathogen. C1q is the first subcomponent of the complement system’s classical pathway. C1q is composed of 18 polypeptide chains. Each of these chains contains a collagen-like region located at the N terminus, and a C-terminal globular head region organized as a heterotrimeric structure (ghA, ghB and ghC). This study was aimed at investigating the complement activation-independent modulation by C1q and its individual recombinant globular heads against IAV infection. The interaction of C1q and its recombinant globular heads with IAV and its purified glycoproteins was examined using direct ELISA and far-Western blotting analysis. The effect of the complement proteins on IAV replication kinetics and immune modulation was assessed by qPCR. The IAV entry inhibitory properties of C1q and its recombinant globular heads were confirmed using cell binding and luciferase reporter assays. C1q bound IAV virions via HA, NA and M1 IAV proteins, and suppressed replication in H1N1, while promoting replication in H3N2-infected A549 cells. C1q treatment further triggered an anti-inflammatory response in H1N1 and pro-inflammatory response in H3N2-infected cells as evident from differential expression of TNF-α, NF-κB, IFN-α, IFN-β, IL-6, IL-12 and RANTES. Furthermore, C1q treatment was found to reduce luciferase reporter activity of MDCK cells transfected with H1N1 pseudotyped lentiviral particles, indicative of an entry inhibitory role of C1q against infectivity of IAV. These data appear to demonstrate the complement-independent subtype specific modulation of IAV infection by locally produced C1q.     

Initiators of Classical and Lectin Complement Pathways Are Differently Engaged after Traumatic Brain Injury—Time-Dependent Changes in the Cortex,Striatum, Thalamus and Hippocampus in a Mouse Model

The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein—GFAP), microglia/macrophages (allograft inflammatory factor 1—IBA-1), and microglia (transmembrane protein 119—TMEM119); moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.     

CFH Loss in Human RPE Cells Leads to Inflammation and Complement System Dysregulation via the NF-κB Pathway

Age-related macular degeneration (AMD), the leading cause of vision loss in the elderly, is a degenerative disease of the macula, where retinal pigment epithelium (RPE) cells are damaged in the early stages of the disease, and chronic inflammatory processes may be involved. Besides aging and lifestyle factors as drivers of AMD, a strong genetic association to AMD is found in genes of the complement system, with a single polymorphism in the complement factor H gene (CFH), accounting for the majority of AMD risk. However, the exact mechanism of CFH dysregulation confers such a great risk for AMD and its role in RPE cell homeostasis is unclear. To explore the role of endogenous CFH locally in RPE cells, we silenced CFH in human hTERT-RPE1 cells. We demonstrate that endogenously expressed CFH in RPE cells modulates inflammatory cytokine production and complement regulation, independent of external complement sources, or stressors. We show that loss of the factor H protein (FH) results in increased levels of inflammatory mediators (e.g., IL-6, IL-8, GM-CSF) and altered levels of complement proteins (e.g., C3, CFB upregulation, and C5 downregulation) that are known to play a role in AMD. Moreover, our results identify the NF-κB pathway as the major pathway involved in regulating these inflammatory and complement factors. Our findings suggest that in RPE cells, FH and the NF-κB pathway work in synergy to maintain inflammatory and complement balance, and in case either one of them is dysregulated, the RPE microenvironment changes towards a proinflammatory AMD-like phenotype.     

Identification of Lectins from Metastatic Cancer Cells through Magnetic Glyconanoparticles

Cancer cells can have characteristic carbohydrate binding properties. Previously, it was shown that a highly metastatic melanoma cell line B16F10 bound to galactoside-functionalized nanoparticles much stronger than the corresponding less metastatic B16F1 cells. To better understand the carbohydrate binding properties of cancer cells, herein, we report the isolation and characterization of endogenous galactose binding proteins from B16F10 cells using magnetic glyconanoparticles. The galactose-coated magnetic glyconanoparticles could bind with lectins present in the cells and be isolated through magnet-mediated separation. Through Western blot and mass spectrometry, the arginine/serine rich splicing factor Sfrs1 was identified as a galactose-selective endogenous lectin, overexpressed in B16F10 cells, compared with B16F1 cells. In addition, galactin-3 was found in higher amounts in B16F10 cells. Finally, the glyconanoparticles exhibited a superior efficiency in lectin isolation, from both protein mixtures and live cells, than the corresponding more traditional microparticles functionalized with carbohydrates. Thus, the magnetic glyconanoparticles present a useful tool for discovery of endogenous lectins, as well as binding partners of lectins, without prior knowledge of protein identities.     

Cinnamtannin B-1 Regulates Cell Proliferation of Spinal Cord Astrocytes and Protects the Cell from Oxygen-Glucose-Serum Deprivation/Reoxygenation-Induced Apoptosis

Astrocytes are important for protecting neurons in the central nervous system. It has been reported that some antioxidants could protect astrocytes from ischemia/reperfusion-induced dysfunction. Cinnamtannin B-1 is a naturally occurring A-type proanthocyanidin that exhibits anti-oxidant properties. In this study, we investigated the effects of cinnamtannin B-1 on spinal cord astrocytes. Astrocytes were subjected to oxygen-glucose-serum deprivation for eight hours followed by reoxygenation with or without cinnamtannin B-1. We found that cinnamtannin B-1 protected astrocytes from oxygen-glucose-serum deprivation and reoxygenation-induced apoptosis. Concurrently, cinnamtannin B-1 promoted the proliferation of astrocytes whereas the extracellular regulated protein kinase (ERK) inhibitor reversed this effect. The results indicated that cinnamtannin B-1 protects astrocytes from oxygen-glucose-serum deprivation/reoxygenation-induced apoptosis by promoting astrocyte proliferation via an ERK pathway. Therefore, as an anti-oxidant, cinnamtannin B-1 might provide extra benefit for astrocyte protection during ischemia/reperfusion in the central nervous system.     

Insight into Cisplatin-Resistance Signaling of W1 Ovarian Cancer Cells Emerges mTOR and HSP27 as Targets for Sensitization Strategies

The microenvironment possesses a strong impact on the tumor chemoresistance when cells bind to components of the extracellular matrix. Here we elucidate the signaling pathways of cisplatin resistance in W1 ovarian cancer cells binding to collagen type 1 (COL1) and signaling interference with constitutive cisplatin resistance in W1CR cells to discover the targets for sensitization. Proteome kinase arrays and Western blots were used to identify the signaling components, their impact on cisplatin resistance was evaluated by inhibitory or knockdown approaches. W1 cell binding to COL1 upregulates integrin-associated signals via FAK/PRAS40/mTOR, confirmed by β1-integrin (ITGB1) knockdown. mTOR appears as key for resistance, its blockade reversed COL1 effects on W1 cell resistance completely. W1CR cells compensate ITGB1-knockdown by upregulation of discoidin domain receptor 1 (DDR1) as alternative COL1 sensor. COL1 binding via DDR1 activates the MAPK pathway, of which JNK1/2 appears critical for COL1-mediated resistance. JNK1/2 inhibition inverts COL1 effects in W1CR cells, whereas intrinsic cisplatin resistance remained unaffected. Remarkably, knockdown of HSP27, another downstream MAPK pathway component overcomes intrinsic resistance completely sensitizing W1CR cells to the level of W1 cells for cisplatin cytotoxicity. Our data confirm the independent regulation of matrix-induced and intrinsic chemoresistance in W1 ovarian cancer cells and offer novel targets for sensitization.     

Insulin-like Growth Factor 1 Promotes Cell Proliferation by Downregulation of G-Protein-Coupled Receptor 17 Expression via PI3K/Akt/FoxO1 Signaling in SK-N-SH Cells

Insulin-like growth factor 1 (IGF-1) not only regulates neuronal function and development but also is neuroprotective in the setting of acute ischemic stroke. G-protein-coupled receptor 17 (GPR17) expression in brain tissue serves as an indicator of brain damage. As whether IGF-1 regulates GPR17 expression remains unknown, the aim of this study is to investigate how IGF-1 regulates GPR17 expression in vitro. Human neuroblastoma SK-N-SH cells were used. Lentivirus-mediated short hairpin RNA (shRNA) was constructed to mediate the silencing of FoxO1, while adenoviral vectors were used for its overexpression. Verification of the relevant signaling cascade was performed using a FoxO1 inhibitor (AS1842856), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), and a GPR17 antagonist (cangrelor). Cell proliferation was analyzed using EdU staining; immunofluorescence staining was used to detect the expression and subcellular localization of FoxO1. Chromatin immunoprecipitation was used to analyze the binding of FoxO1 to the GPR17 promoter in SK-N-SH cells. The expression of FoxO1, GPR17, and protein kinase B (also known as Akt) mRNA and protein as well as the levels of FoxO1 and Akt phosphorylation were investigated in this study. IGF-1 was found to downregulate FoxO1 and GPR17 expression in SK-N-SH cells while promoting cell viability and proliferation. Inhibition of FoxO1 and antagonism of GPR17 were found to play a role similar to that of IGF-1. Silencing of FoxO1 by lentivirus-mediated shRNA resulted in the downregulation of FoxO1 and GPR17 expression. The overexpression of FoxO1 via adenoviral vectors resulted in the upregulation of FoxO1 and GPR17 expression. Blocking of PI3K signaling by LY294002 inhibited the effect of IGF-1 on GPR17 suppression. Results from chromatin immunoprecipitation revealed that IGF-1 promotes FoxO1 nuclear export and reduces FoxO1 binding to the GPR17 promoter in SK-N-SH cells. Here, we conclude that IGF-1 enhances cell viability and proliferation in SK-N-SH cells via the promotion of FoxO1 nuclear export and reduction of FoxO1 binding to the GPR17 promoter via PI3K/Akt signaling. Our findings suggest that the enhancement of IGF-1 signaling to antagonize GPR17 serves as a potential therapeutic strategy in the management of acute ischemic stroke.     

Molecular Basis of Complement C1q Collagen-Like Region Interaction with the Immunoglobulin-Like Receptor LAIR-1

The immune system homeostasis relies on a tight equilibrium of interconnected stimulatory and inhibitory signals. Disruption of this balance is characteristic of autoimmune diseases such as systemic lupus erythematosus (SLE). Aside from activating the classical complement pathway and enhancing pathogens and apoptotic cells phagocytosis, C1q has been recently shown to play an important role in immune modulation and tolerance by interacting with several inhibitory and stimulatory immune receptors. Due to its functional organization into collagen-like (CLR) and globular (GR) regions and its multimeric nature, C1q is able to interact simultaneously with several of these receptors and locally congregate pro- and anti-inflammatory signals, thus modulating the immune response. Leukocyte associated immunoglobulin-like (Ig-like) receptor 1 (LAIR-1), a ubiquitous collagen receptor expressed in many immune cell types, has been reported to interact with the CLR of C1q. In this study, we provide new insights into the molecular and structural determinants underlying C1q/LAIR-1 interaction. Recombinant LAIR-1 extracellular Ig-like domain was produced and tested for its interaction with C1q. A molecular dissection of C1q combined with competition assays reveals that LAIR-1 interacts with C1q’s CLR through a binding site close but different from the one of its associated C1r2s2 proteases tetramer. On the other side, we identified LAIR-1 residues involved in C1q interaction by site-directed mutational analysis. All together, these results lead to propose a possible model for C1q interaction with LAIR-1 and will contribute to the fundamental understanding of C1q-mediated immune tolerance.     

A Role of Complement in the Pathogenic Sequelae of Mouse Neonatal Germinal Matrix Hemorrhage

Germinal matrix hemorrhage (GMH) is a devastating disease of infancy that results in intraventricular hemorrhage, post-hemorrhagic hydrocephalus (PHH), periventricular leukomalacia, and neurocognitive deficits. There are no curative treatments and limited surgical options. We developed and characterized a mouse model of GMH based on the injection of collagenase into the subventricular zone of post-natal pups and utilized the model to investigate the role of complement in PHH development. The site-targeted complement inhibitor CR2Crry, which binds deposited C3 complement activation products, localized specifically in the brain following its systemic administration after GMH. Compared to vehicle, CR2Crry treatment reduced PHH and lesion size, which was accompanied by decreased perilesional complement deposition, decreased astrocytosis and microgliosis, and the preservation of dendritic and neuronal density. Complement inhibition also improved survival and weight gain, and it improved motor performance and cognitive outcomes measured in adolescence. The progression to PHH, neuronal loss, and associated behavioral deficits was linked to the microglial phagocytosis of complement opsonized neurons, which was reversed with CR2Crry treatment. Thus, complement plays an important role in the pathological sequelae of GMH, and complement inhibition represents a novel therapeutic approach to reduce the disease progression of a condition for which there is currently no treatment outside of surgical intervention.     

Activation of the Complement System on Human Endothelial Cells by Urban Particulate Matter Triggers Inflammation-Related Protein Production

Exposure to particulate matter (PM) is becoming a major global health issue. The amount and time of exposure to PM are known to be closely associated with cardiovascular diseases. However, the mechanism through which PM affects the vascular system is still not clear. Endothelial cells line the interior surface of blood vessels and actively interact with plasma proteins, including the complement system. Unregulated complement activation caused by invaders, such as pollutants, may promote endothelial inflammation. In the present study, we sought to investigate whether urban PM (UPM) acts on the endothelial environment via the complement system. UPM-treated human endothelial cells with normal human serum showed the deposition of membrane attack complexes (MACs) on the cell surface via the alternative pathway of the complement system. Despite the formation of MACs, cell death was not observed, and cell proliferation was increased in UPM-mediated complement activation. Furthermore, complement activation on endothelial cells stimulated the production of inflammation-related proteins. Our results revealed that UPM could activate the complement system in human endothelial cells and that complement activation regulated inflammatory reaction in microenvironment. These findings provide clues with regard to the role of the complement system in pathophysiologic events of vascular disease elicited by air pollution.     

重组B7-H1IgV融合蛋白的表达、纯化及活性鉴定

目的以B7-H1为靶点,研制肿瘤免疫治疗蛋白疫苗。方法将人B7-H1胞外片段IgV区基因插入pQE-30原核表达载体,转化大肠杆菌,经IPTG诱导表达。Ni2+-NTA亲和层析纯化蛋白,Westernblot鉴定。用纯化的rhB7-H1IgV蛋白免疫昆明小鼠,经ELISA、流式细胞术、免疫组化技术和CDC试验测定融合蛋白及其抗血清的生物学活性。结果所构建的pQE-30-TT-B7-H1IgV表达载体,能稳定表达rhB7-H1IgV蛋白,经纯化后免疫昆明小鼠,可获得高滴度抗B7-H1抗血清。经流式细胞术和免疫组化检测显示,其抗血清可与HT-29/B7-H1+及SP2/0肿瘤细胞结合,且在CDC试验中,可依赖补体杀伤HT-29/B7-H1+及SP2/0肿瘤细胞。结论rhB7-H1IgV融合蛋白不仅可引发小鼠体液免疫应答,而且其抗体还能与表达B7-H1的肿瘤细胞相结合,并介导补体依赖的体外杀伤作用。     

Characterization and Differentiation of the Tumor Microenvironment (TME) of Orthotopic and Subcutaneously Grown Head and Neck Squamous Cell Carcinoma (HNSCC) in Immunocompetent Mice

For the development and evaluation of new head and neck squamous cell carcinoma (HNSCC) therapeutics, suitable, well-characterized animal models are needed. Thus, by analyzing orthotopic versus subcutaneous models of HNSCC in immunocompetent mice, we evaluated the existence of adenosine-related immunosuppressive B- and T lymphocyte populations within the tumor microenvironment (TME). Applying the SCC VII model for the induction of HNSCC in immunocompetent C3H/HeN mice, the cellular TME was characterized after tumor initiation over time by flow cytometry. The TME in orthotopic grown tumors revealed a larger population of tumor-infiltrating lymphocytes (TIL) with more B cells and CD4⁺ T cells than the subcutaneously grown tumors. Immune cell populations in the blood and bone marrow showed a rather distinct reaction toward tumor induction and tumor location compared to the spleen, lymph nodes, or thymus. In addition, large numbers of immunosuppressive B- and T cells were identified within the TME but also in secondary lymphoid organs, independently of the tumor initiation site. The altered immunogenic TME may influence the response to any treatment attempt. Moreover, when analyzing the TME and other lymphoid organs of tumor-bearing mice, we observed conditions reflecting largely those of patients suffering from HNSCC suggesting the C3H/HeN mouse model as a suitable tool for studies aiming to target immunosuppression to improve anti-cancer therapies.     

Angulin-1 (LSR) Affects Paracellular Water Transport,However Only in Tight Epithelial Cells

Water transport in epithelia occurs transcellularly (aquaporins) and paracellularly (claudin-2, claudin-15). Recently, we showed that downregulated tricellulin, a protein of the tricellular tight junction (tTJ, the site where three epithelial cells meet), increased transepithelial water flux. We now check the hypothesis that another tTJ-associated protein, angulin-1 (alias lipolysis-stimulated lipoprotein receptor, LSR) is a direct negative actuator of tTJ water permeability depending on the tightness of the epithelium. For this, a tight and an intermediate-tight epithelial cell line, MDCK C7 and HT-29/B6, were stably transfected with CRISPR/Cas9 and single-guide RNA targeting angulin-1 and morphologically and functionally characterized. Water flux induced by an osmotic gradient using 4-kDa dextran caused water flux to increase in angulin-1 KO clones in MDCK C7 cells, but not in HT-29/B6 cells. In addition, we found that water permeability in HT-29/B6 cells was not modified after either angulin-1 knockout or tricellulin knockdown, which may be related to the presence of other pathways, which reduce the impact of the tTJ pathway. In conclusion, modulation of the tTJ by knockout or knockdown of tTJ proteins affects ion and macromolecule permeability in tight and intermediate-tight epithelial cell lines, while the transepithelial water permeability was affected only in tight cell lines.     

天然产物Combretastatin B-1的合成

该文基于Perkin反应合成了具有血管阻断及抗肿瘤活性的天然产物Combretastatin B-1(CB1)。以异香兰素为起始原料,用二溴海因对其进行选择性溴代得到2-溴-3-羟基-4-甲氧基苯甲醛(Ⅱ),该化合物与3,4,5-三甲氧基苯乙酸(Ⅲ)发生Perkin反应得到(Z/E)-2-(3,4,5-三甲氧基苯基)-3-(2'-溴-3'-乙酰氧基-4'-甲氧基)丙烯酸(Z-4、E-4),再经羟化反应及脱羧-异构化反应制备得到E-Combretastatin A-1(E-CA1),最后经催化氢化反应得到CB1,总收率为53.4%。     

JAK2-Mediated Phosphorylation of Stress-Induced Phosphoprotein-1 (STIP1) in Human Cells

Stress-induced phosphoprotein-1 (STIP1)—a heat shock protein (HSP)70/HSP90 adaptor protein—is commonly overexpressed in malignant cells, where it controls proliferation via multiple signaling pathways, including JAK2/STAT3. We have previously shown that STIP1 stabilizes the protein tyrosine kinase JAK2 in cancer cells via HSP90 binding. In this study, we demonstrate that STIP1 may act as a substrate for JAK2 and that phosphorylation of tyrosine residues 134 and 152 promoted STIP1 protein stability, induced its nuclear-cytoplasmic shuttling, and promoted its secretion into the extracellular space. We also found that JAK2-mediated STIP1 phosphorylation enhanced cell viability and increased resistance to cisplatin-induced cell death. Conversely, interference STIP1 with JAK2 interaction—attained either through site-directed mutagenesis or the use of cell-penetrating peptides—decreased JAK2 protein levels, ultimately leading to cell death. On analyzing human ovarian cancer specimens, JAK2 and STIP1 expression levels were found to be positively correlated with each other. Collectively, these results indicate that JAK2-mediated phosphorylation of STIP-1 is critical for sustaining the JAK2/STAT3 signaling pathway in cancer cells.     

ROS-Mediated Anti-Tumor Effect of Coptidis Rhizoma against Human Hepatocellular Carcinoma Hep3B Cells and Xenografts

Coptidis Rhizoma is the dried rhizome from the Coptis chinensis Franch. that has been shown to have a number of beneficial pharmacological properties including antioxidant, anti-inflammatory, and anti-cancer effects. However, the anti-cancer effects of Coptidis Rhizoma on hepatocellular carcinoma (HCC) remain unclear. In this study, we investigated the anti-cancer properties of Coptidis Rhizoma ethanol extract (CR) in HCC Hep3B cells and in a xenograft mouse model. Our results showed that the CR significantly inhibited cell growth and induced apoptosis in Hep3B cells through increased expression of Bcl-2 associated x-protein (Bax) and cleavage of poly-ADP ribose polymerase (PARP), reduced expression of Bcl-2, and activated caspases. CR also increased the generation of intracellular reactive oxygen species (ROS), which caused a loss of mitochondrial membrane potential (MMP, ΔΨm) and activation of the mitochondria-mediated intrinsic apoptosis pathway. Moreover, N-acetylcysteine (NAC), a ROS inhibitor, markedly blocked the effects of CR on apoptotic pathways. CR also induced the expression of light chain 3 (LC3)-I/II, a key autophagy regulator, whereas CR-mediated autophagy was significantly suppressed by NAC. In addition, pre-treatment with NAC perfectly attenuated the inhibition of cell invasion and migration of CR-stimulated Hep3B cells. Furthermore, oral administration of CR suppressed Hep3B tumor growth in xenograft mice without toxicity, alterations to body weight, or changes in hematological and biochemical profiles. Taken together, our findings suggest that CR has anti-tumor effects that result from ROS generation, and may be a potential pharmacological intervention for HCC.     

A Chimeric IL-15/IL-15Rα Molecule Expressed on NFκB-Activated Dendritic Cells Supports Their Capability to Activate Natural Killer Cells

Natural killer (NK) cells, members of the innate immune system, play an important role in the rejection of HLA class I negative tumor cells. Hence, a therapeutic vaccine, which can activate NK cells in addition to cells of the adaptive immune system might induce a more comprehensive cellular response, which could lead to increased tumor elimination. Dendritic cells (DCs) are capable of activating and expanding NK cells, especially when the NFκB pathway is activated in the DCs thereby leading to the secretion of the cytokine IL-12. Another prominent NK cell activator is IL-15, which can be bound by the IL-15 receptor alpha-chain (IL-15Rα) to be transpresented to the NK cells. However, monocyte-derived DCs do neither secrete IL-15, nor express the IL-15Rα. Hence, we designed a chimeric protein consisting of IL-15 and the IL-15Rα. Upon mRNA electroporation, the fusion protein was detectable on the surface of the DCs, and increased the potential of NFκB-activated, IL-12-producing DC to activate NK cells in an autologous cell culture system with ex vivo-generated cells from healthy donors. These data show that a chimeric IL-15/IL-15Rα molecule can be expressed by monocyte-derived DCs, is trafficked to the cell surface, and is functional regarding the activation of NK cells. These data represent an initial proof-of-concept for an additional possibility of further improving cellular DC-based immunotherapies of cancer.