Particulate formulations for the delivery of poly(I:C) as vaccine adjuvant.

Current research and development of antigens for vaccination often center on purified recombinant proteins, viral subunits, synthetic oligopeptides or oligosaccharides, most of them suffering from being poorly immunogenic and subject to degradation. Hence, they call for efficient delivery systems and potent immunostimulants, jointly denoted as adjuvants. Particulate delivery systems like emulsions, liposomes, nanoparticles and microspheres may provide protection from degradation and facilitate the co-formulation of both the antigen and the immunostimulant. Synthetic double-stranded (ds) RNA, such as polyriboinosinic acid-polyribocytidylic acid, poly(I:C), is a mimic of viral dsRNA and, as such, a promising immunostimulant candidate for vaccines directed against intracellular pathogens. Poly(I:C) signaling is primarily dependent on Toll-like receptor 3 (TLR3), and on melanoma differentiation-associated gene-5 (MDA-5), and strongly drives cell-mediated immunity and a potent type I interferon response. However, stability and toxicity issues so far prevented the clinical application of dsRNAs as they undergo rapid enzymatic degradation and bear the potential to trigger undue immune stimulation as well as autoimmune disorders. This review addresses these concerns and suggests strategies to improve the safety and efficacy of immunostimulatory dsRNA formulations. The focus is on technological means required to lower the necessary dosage of poly(I:C), to target surface-modified microspheres passively or actively to antigen-presenting cells (APCs), to control their interaction with non-professional phagocytes and to modulate the resulting cytokine secretion profile.

Tuning the immune response of dendritic cells to surface-assembled poly(I:C) on microspheres through synergistic interactions between phagocytic and TLR3 signaling.

The artificial dsRNA polyriboinosinic acid-polyribocytidylic acid, poly(I:C), is a potent adjuvant candidate for vaccination, as it strongly drives cell-mediated immunity. However, because of its effects on non-immune bystander cells, poly(I:C) administration may bear danger for the development of autoimmune diseases. Thus poly(I:C) should be applied in the lowest dose possible. We investigated microspheres carrying surface-assembled poly(I:C) as a two-in-one adjuvant formulation to stimulate maturation of monocyte-derived dendritic cells (MoDCs). Negatively charged polystyrene microspheres were equipped with a poly(ethylene glycol) corona through electrostatically driven surface assembly of a library of polycationic poly(l-lysine)-graft-poly(ethylene glycol) copolymers, PLL-g-PEG. Stable surface assembly of poly(I:C) was achieved by incubation of polymer-coated microspheres in an aqueous poly(I:C) solution. Surface-assembled poly(I:C) exhibited a strongly enhanced efficacy to stimulate maturation of MoDCs by up to two orders of magnitude, as compared to free poly(I:C). Multiple phagocytosis events were the key factor to enhance the efficacy. The cytokine secretion pattern of MoDCs after exposure to surface-assembled poly(I:C) differed from that of free poly(I:C), while their ability to stimulate T cell proliferation was similar. Overall, phagocytic signaling plays an important role in defining the resulting immune response to such two-in-one adjuvant formulations.

Surface assembly of poly(I:C) on PEGylated microspheres to shield from adverse interactions with fibroblasts.

By expressing an array of pattern recognition receptors (PRRs), fibroblasts play an important role in stimulating and modulating the response of the innate immune system. The TLR3 ligand polyriboinosinic acid-polyribocytidylic acid, poly(I:C), a mimic of viral dsRNA, is a vaccine adjuvant candidate to activate professional antigen presenting cells (APCs). However, owing to its ligation with extracellular TLR3 on fibroblasts, subcutaneously administered poly(I:C) bears danger towards autoimmunity. It is thus in the interest of its clinical safety to deliver poly(I:C) in such a way that its activation of professional APCs is as efficacious as possible, whereas its interference with non-immune cells such as fibroblasts is controlled or even avoided. Complementary to our previous work with monocyte-derived dendritic cells (MoDCs), here we sought to control the delivery of poly(I:C) surface-assembled on microspheres to human foreskin fibroblasts (HFFs). Negatively charged polystyrene (PS) microspheres were equipped with a poly(ethylene glycol) (PEG) corona through electrostatically driven coatings with a series of polycationic poly(L-lysine)-graft-poly(ethylene glycol) copolymers, PLL-g-PEG, of varying grafting ratios g from 2.2 up to 22.7. Stable surface assembly of poly(I:C) was achieved by incubation of polymer-coated microspheres with aqueous poly(I:C) solutions. Notably, recognition of both surface-assembled and free poly(I:C) by extracellular TLR3 on HFFs halted their phagocytic activity. Ligation of surface-assembled poly(I:C) with extracellular TLR3 on HFFs could be controlled by tuning the grafting ratio g and thus the chain density of the PEG corona. When assembled on PLL-5.7-PEG-coated microspheres, poly(I:C) was blocked from triggering class I MHC molecule expression on HFFs. Secretion of interleukin (IL)-6 by HFFs after exposure to surface-assembled poly(I:C) was distinctly lower as compared to free poly(I:C). Overall, surface assembly of poly(I:C) may have potential to contribute to the clinical safety of this vaccine adjuvant candidate.

Nuclear factor I-C links platelet-derived growth factor and transforming growth factor beta1 signaling to skin wound healing progression.

Transforming growth factor beta (TGF-beta) and platelet-derived growth factor A (PDGFAlpha) play a central role in tissue morphogenesis and repair, but their interplay remain poorly understood. The nuclear factor I C (NFI-C) transcription factor has been implicated in TGF-beta signaling, extracellular matrix deposition, and skin appendage pathologies, but a potential role in skin morphogenesis or healing had not been assessed. To evaluate this possibility, we performed a global gene expression analysis in NFI-C(-/-) and wild-type embryonic primary murine fibroblasts. This indicated that NFI-C acts mostly to repress gene expression in response to TGF-beta1. Misregulated genes were prominently overrepresented by regulators of connective tissue inflammation and repair. In vivo skin healing revealed a faster inflammatory stage and wound closure in NFI-C(-/-) mice. Expression of PDGFA and PDGF-receptor alpha were increased in wounds of NFI-C(-/-) mice, explaining the early recruitment of macrophages and fibroblasts. Differentiation of fibroblasts to contractile myofibroblasts was also elevated, providing a rationale for faster wound closure. Taken together with the role of TGF-beta in myofibroblast differentiation, our results imply a central role of NFI-C in the interplay of the two signaling pathways and in regulation of the progression of tissue regeneration.

Genetic analysis of c-Myc in mouse bone marrow and liver

1. Summary The transcription factor and proto-oncogene c-myc plays an important role in integrating many mitogenic signals within the cell. The consequences are both broad and varied and include the regulation of apoptosis, cellular differentiation, cellular growth and cell cycle progression. It is found to be mis-regulated in over 70% of all cancers, however, our knowledge about c-Myc remains limited and very little is known about its physiological role in mammalian development and in adulthood. We have addressed the physiological role of c-Myc in both the bone marrow and the liver of mice by generating adult c-myc flox/flox mice that lacked c-myc in either the bone marrow or the liver after conversion of the c-myc flox alleles into null alleles by the inducible Mx¬Cre transgene with polyI-polyC. In investigating the role of c-Myc in the haematopoietic system, we concentrated on the aspects of cellular proliferation, cellular differentiation and apoptosis. Mice lacking c-Myc develop anaemia between 3-8 weeks and all more differentiated cell types are severely depleted leading to death. However in addition to its role in driving proliferation in transient amplifying cells, we unexpectedly discovered a new role for c-Myc in controlling haematopoietic stem cell (HSC) differentiation. c-Myc deficient HSCs are able to proliferate normally in vivo. In addition, their differentiation into more committed progenitors is blocked. These cells expressed increased adhesion molecules, which possibly prevent HSCs from being released from the special stem cell supporting stromal niche cells with which they closely associate. Secondly we used the liver as a model system to address the role of c-Myc in cellular growth, meaning the increase in cell size, and also cellular proliferation. Our results revealed c-Myc to play no role in metabolic cellular growth following a period of fasting. Following treatment with the xenobiotic TCPOBOP, c-Myc deficient hepatocytes increased in cell size as control hepatocytes and could surprisingly proliferate albeit at a reduced rate demonstrating a c-Myc independent proliferation pathway to exist in parenchymal cells. However, following partial hepatectomy, in which two-thirds of the liver was removed, mutant livers were severely restricted in their regeneration capacity compared to control livers demonstrating that c-Myc is essential for liver regeneration. Résumé Le facteur de transcription et proto-oncogène c-myc joue un rôle important dans l'intégration de nombreux signaux mitogéniques dans la cellule. Les conséquences de son activation sont étendues et variées et incluent la régulation de l'apoptose, de la différenciation, de la croissance et de la progression du cycle cellulaire. Même si plus de 20% des cancers montrent une dérégulation de c-myc, les connaissances sur ce facteur de transcription restent limitées et ses rôles physiologiques au cours du développement et chez l'adulte sont très peu connus. Nous avons étudié le rôle physiologique de c-Myc dans la molle osseuse et le foie murin en générant des souris adultes c-myc flox/flox. Dans ces souris, les allèles c-myc flox sont convertis en allèles nuls par le transgène Mx-Cre après induction avec du Poly-I.C. Pour notre étude du rôle de c-Myc dans le système hématopoiétique, nous nous sommes concentrés sur les aspects de la prolifération et de la différenciation cellulaire, ainsi que sur l'apoptose. Les souris déficientes pour c-Myc développent une anémie 3 à 8 semaines après la délétion du gène; tous les différents types cellulaires matures sont progressivement épuisés ce qui entraîne la mort des animaux. Néanmoins, outre sa capacité à induire la prolifération des cellules transitoires de la molle osseuse, nous avons inopinément découvert un nouveau rôle pour c-Myc dans le contrôle de la différenciation des cellules souches hématopoiétiques (HSC). Les HSC déficientes pour c-Myc prolifèrent normalement in vivo mais leur différenciation en progéniteurs plus engagés dans une voie de différenciation est bloquée. Ces cellules surexpriment certaines molécules d'adhésion ce qui empêcherait les HSC d'être relachées du stroma spécialisé, ou niche, auquel elles sont étroitement associées. D'autre part, nous avons utilisé le foie comme système modèle pour étudier le rôle de c-Myc dans la prolifération et dans la croissance cellulaire, c'est à dire l'augmentation de taille des cellules. Nos résultats ont révélé que c-Myc ne joue pas de rôle dans le métabolisme cellulaire qui suit une période de jeûne. L'augmentation de la taille cellulaire des hépatocytes déficients pour c-Myc suite au traitement avec l'agent xénobiotique TCPOBOP est identique à celle observée pour les cellules de contrôle. Le taux de prolifération des hépatocytes mutants est par contre réduit, indiquant qu'une voie de différenciation indépendante de c-Myc existe dans les cellules parenchymales. Néanmoins, après hépatectomie partielle, où deux-tiers du foie sont éliminés chirurgicalement, les foies mutants sont sévèrement limités dans leur capacité de régénération par rapport aux foies de contrôle, montrant ainsi que c-Myc est essentiel pour la régénération hépatique.

Complex organization of CTF/NF-I, C/EBP, and HNF3 binding sites within the promoter of the liver-specific vitellogenin gene.

Vitellogenin genes are expressed specifically in the liver of female oviparous vertebrates under the strict control of estrogen. To explain this tissue-specific expression, we performed a detailed analysis of the Xenopus laevis vitellogenin gene B1 promoter by DNase I footprinting and gel mobility-shift assays. We characterized five binding sites for the ubiquitous factor CTF/NF-I. Two of these sites are close to the TATA-box, whereas the others are located on both sides of the estrogen responsive unit formed by two imperfect estrogen response elements. Moreover two liver-enriched factors, C/EBP and HNF3, were found to interact with multiple closely spaced proximal promoter elements in the first 100 base pairs upstream of the TATA-box. To confirm the physiological significance of this in vitro analysis, in vivo DNase I footprinting experiments were carried out using the ligation-mediated polymerase chain reaction technique. The various cis-elements characterized in vitro as binding sites for known transcription factors and more particularly for liver-enriched transcription factors are efficiently recognized in vivo as well, suggesting that they play an important role in the control of the liver-specific vitellogenin gene B1 expression.

Nuclear factor I-C regulates TGF-{beta}-dependent hair follicle cycling.

Skin appendages such as teeth and hair share several common signaling pathways. The nuclear factor I C (NFI-C) transcription factor has been implicated in tooth development, but a potential role in hair growth had not been assessed. In this study we found that NFI-C regulates the onset of the hair growth cycle. NFI-C(-/-) mice were delayed in the transition from the telogen to anagen phase of the hair follicle cycle after either experimental depilation or spontaneous hair loss. Lack of NFI-C resulted in delayed induction of the sonic hedgehog, Wnt5a, and Lef1 gene expression, which are key regulators of the hair follicle growth initiation. NFI-C(-/-) mice also showed elevated levels of transforming growth factor β1 (TGF-β1), an inhibitor of keratinocyte proliferation, and of the cell cycle inhibitor p21 at telogen. Reduced expression of Ki67, a marker of cell proliferation, was noted at the onset of anagen, indicating impaired activation of the hair progenitor cells. These findings implicate NFI-C in the repression of TGF-β1 signaling during telogen stage, resulting in the delay of progenitor cell proliferation and hair follicle regeneration in NFI-C-deficient mice. Taken together with prior observations, these findings also designate NFI-C as a regulator of adult progenitor cell proliferation and of postnatal tissue growth or regeneration.

Type I interferon inhibits interleukin-1 production and inflammasome activation.

Type I interferon (IFN) is a common therapy for autoimmune and inflammatory disorders, yet the mechanisms of action are largely unknown. Here we showed that type I IFN inhibited interleukin-1 (IL-1) production through two distinct mechanisms. Type I IFN signaling, via the STAT1 transcription factor, repressed the activity of the NLRP1 and NLRP3 inflammasomes, thereby suppressing caspase-1-dependent IL-1β maturation. In addition, type I IFN induced IL-10 in a STAT1-dependent manner; autocrine IL-10 then signaled via STAT3 to reduce the abundance of pro-IL-1α and pro-IL-1β. In vivo, poly(I:C)-induced type I IFN diminished IL-1β production in response to alum and Candida albicans, thus increasing susceptibility to this fungal pathogen. Importantly, monocytes from multiple sclerosis patients undergoing IFN-β treatment produced substantially less IL-1β than monocytes derived from healthy donors. Our findings may thus explain the effectiveness of type I IFN in the treatment of inflammatory diseases but also the observed "weakening" of the immune system after viral infection.

Nuclear Factor I-C acts as a regulator of hepatocyte proliferation at the onset of liver regeneration.

BACKGROUND & AIMS: Knockout studies of the murine Nuclear Factor I-C (NFI-C) transcription factor revealed abnormal skin wound healing and growth of its appendages, suggesting a role in controlling cell proliferation in adult regenerative processes. Liver regeneration following partial hepatectomy (PH) is a well-established regenerative model whereby changes elicited in hepatocytes lead to their rapid and phased proliferation. Although NFI-C is highly expressed in the liver, no hepatic function was yet established for this transcription factor. This study aimed to determine whether NFI-C may play a role in hepatocyte proliferation and liver regeneration. METHODS: Liver regeneration and cell proliferation pathways following two-thirds PH were investigated in NFI-C knockout (ko) and wild-type (wt) mice. RESULTS: We show that the absence of NFI-C impaired hepatocyte proliferation because of plasminogen activator I (PAI-1) overexpression and the subsequent suppression of urokinase plasminogen activator (uPA) activity and hepatocyte growth factor (HGF) signalling, a potent hepatocyte mitogen. This indicated that NFI-C first acts to promote hepatocyte proliferation at the onset of liver regeneration in wt mice. The subsequent transient down regulation of NFI-C, as can be explained by a self-regulatory feedback loop with transforming growth factor beta 1 (TGF-ß1), may limit the number of hepatocytes entering the first wave of cell division and/or prevent late initiations of mitosis. CONCLUSION: NFI-C acts as a regulator of the phased hepatocyte proliferation during liver regeneration.

Function of TRADD in tumor necrosis factor receptor 1 signaling and in TRIF-dependent inflammatory responses.

Tumor necrosis factor receptor 1 (TNFR1) and Toll-like receptors (TLRs) regulate immune and inflammatory responses. Here we show that the TNFR1-associated death domain protein (TRADD) is critical in TNFR1, TLR3 and TLR4 signaling. TRADD deficiency abrogated TNF-induced apoptosis, prevented recruitment of the ubiquitin ligase TRAF2 and ubiquitination of the adaptor RIP1 in the TNFR1 signaling complex, and considerably inhibited but did not completely abolish activation of the transcription factor NF-kappaB and mitogen-activated protein kinases 'downstream' of TNFR1. TRIF-dependent cytokine production induced by the synthetic double-stranded RNA poly(I:C) and lipopolysaccharide was lower in TRADD-deficient mice than in wild-type mice. Moreover, TRADD deficiency inhibited poly(I:C)-mediated RIP1 ubiquitination and activation of NF-kappaB and mitogen-activated protein kinase signaling in fibroblasts but not in bone marrow macrophages. Thus, TRADD is an essential component of TNFR1 signaling and has a critical but apparently cell type-specific function in TRIF-dependent TLR responses.

Intravaginal TLR agonists increase local vaccine-specific CD8 T cells and human papillomavirus-associated genital-tumor regression in mice.

Human papillomaviruses (HPV)-related cervical cancer is the second leading cause of cancer death in women worldwide. Despite active development, HPV E6/E7 oncogene-specific therapeutic vaccines have had limited clinical efficacy to date. Here, we report that intravaginal (IVAG) instillation of CpG-ODN (TLR9 agonist) or poly-(I:C) (TLR3 agonist) after subcutaneous E7 vaccination increased ∼fivefold the number of vaccine-specific interferon-γ-secreting CD8 T cells in the genital mucosa (GM) of mice, without affecting the E7-specific systemic response. The IVAG treatment locally increased both E7-specific and total CD8 T cells, but not CD4 T cells. This previously unreported selective recruitment of CD8 T cells from the periphery by IVAG CpG-ODN or poly-(I:C) was mediated by TLR9 and TLR3/melanoma differentiation-associated gene 5 signaling pathways, respectively. For CpG, this recruitment was associated with a higher proportion of GM-localized CD8 T cells expressing both CCR5 and CXCR3 chemokine receptors and E-selectin ligands. Most interestingly, IVAG CpG-ODN following vaccination led to complete regression of large genital HPV tumors in 75% of mice, instead of 20% with vaccination alone. These findings suggest that mucosal application of immunostimulatory molecules might substantially increase the effectiveness of parenterally administered vaccines.Mucosal Immunology advance online publication 12 September 2012; doi:10.1038/mi.2012.83.

Adjuvants that improve the ratio of antigen-specific effector to regulatory T cells enhance tumor immunity.

Antitumor immunity is strongly influenced by the balance of tumor antigen-specific effector and regulatory T cells. However, the impact that vaccine adjuvants have in regulating the balance of antigen-specific T cell populations is not well understood. We found that antigen-specific T regulatory cells (Treg) were induced following subcutaneous vaccination with either OVA or melanoma-derived peptides, with a restricted expansion of effector T cells. Addition of the adjuvants CpG-ODN or Poly(I:C) preferentially amplified effector T cells over Tregs, dramatically increasing the antigen-specific T effector:Treg ratios and inducing polyfunctional effector cells. In contrast, two other adjuvants, imiquimod and Quil A saponin, favored an expansion of antigen-specific Tregs and failed to increase effector T cell:Treg ratios. Following therapeutic vaccination of tumor-bearing mice, high ratios of tumor-specific effector T cells:Tregs in draining lymph nodes were associated with enhanced CD8+ T cell infiltration at the tumor site and a durable rejection of tumors. Vaccine formulations of peptide+CpG-ODN or Poly(I:C) induced selective production of pro-inflammatory Type I cytokines early after vaccination. This environment promoted CD8+ and CD4+ effector T cell expansion over that of antigen-specific Tregs, tipping the effector T cell to Treg balance to favor effector cells. Our findings advance understanding of the influence of different adjuvants on T cell populations, facilitating the rational design of more effective cancer vaccines.

Characterization of cytoplasmic antiviral signaling pathways

Summary Multicellular organisms have evolved the immune system to protect from pathogen such as viruses, bacteria, fungi or parasites. Detection of invading pathogens by the host innate immune system is crucial for mounting protective responses and depends on the recognition of microbial components by specific receptors. The results presented in this manuscript focus on the signaling pathways involved in the detection of viral infection by the sensing of viral nucleic acids. First, we describe a new regulatory mechanism controlling RNA-sensing antiviral pathways. Our results indicate that TRIF and Cardif, the crucial adaptor proteins for endosomal and cytoplasmic RNA detection signaling pathway, are processed and inactivated by caspases. The second aspect investigated here involves a signaling pathway triggered upon cytosolic DNA sensing. The interferon inducible protein DAI was recently described as a DNA sensor able to induce the activation of IRFs and NF-κΒ transcription factors leading to type I interferon production. Here we identify two RIP homotypic interaction motifs (RHIMs) in DAI and demonstrate that they mediate the recruitment of RIP1 and RIP3 and the subsequent NF-κΒ activation. Moreover, we observed that the mouse cytomegalovirus RHIM- containing protein M45 has the potential to block this signaling cascade by interfering with the formation of the DAI-RIP1/3 signaling complex. Finally, we report the generation and the initial characterization of NLRX1-deficient mice. NLRX1 is a member of the NOD-like receptor family localized to the mitochondria. The function of NLRX1 is still controversial: one study proposed that NLRX1 acts as an inhibitor of the RIG-like receptor (RLR) antiviral pathway by binding the adaptor protein Cardif, whereas another report implicated NLRX1 in the generation of reactive oxygen species (ROS) and the amplification of NF-κΒ and JNK triggered by TNF-α, poly(I:C) or Shigella infection. Collectively, our results indicate that NLRX1-deficiency does not affect RLR signaling nor TNF-α induced responses. Proteomics analysis identified UQCRC2, a subunit of the complex III of the mitochondrial respiratory chain, as a NLRX1 binding partner. This observation might reveal a possible functional link between NLRX1 and mitochondrial respiration and/or ROS generation. Résumé Au cours de l'évolution, les organismes multicellulaires ont développé le système immunitaire afin de se protéger contre les pathogènes. Une étape cruciale pour le déclenchement des réponses protectrices est la reconnaissance par les cellules du système immunitaire de molécules propres aux microbes grâce à des récepteurs spécifiques. Les résultats présentés dans cette thèse décrivent des nouveaux aspects concernant les voies de signalisation impliquées dans la détection des virus. Le premier projet décrit un mécanisme de régulation des voies activées par la détection d'ARN virale. Nos résultats montrent que TRIF et Cardif, des protéines adaptatrices des voies déclenchées par la reconnaissance de ces acides nucléiques au niveau des endosomes et du cytoplasme, sont clivés et inactivés par les caspases. Le projet suivant de notre recherche concerne une voie de signalisation activée par la détection d'ADN au niveau du cytoplasme. La protéine DAI a été récemment décrite comme un senseur pour cet ADN capable d'activer les facteurs de transcription IRF et NF-κΒ et d'induire ainsi la production des interférons de type I. Ici on démontre que DAI interagit avec RIP1 et RIP3 par le biais de domaines appelés RHIM et que ce complexe est responsable de l'activation de NF-κΒ. On a aussi identifié une protéine du cytomégalovirus de la souris, M45, qui contient ce même domaine et on a pu démontrer qu'elle a la capacité d'interférer avec la formation du complexe entre DAI et RIP1/RIP3 bloquant ainsi l'activation de NF-κΒ. Enfin on décrit ici la génération de souris déficientes pour le gène qui code pour la protéine NLRX1. Cette protéine fait partie de la famille des récepteurs NOD et est localisée dans la mitochondrie. Une étude a suggéré que NLRX1 agit comme un inhibiteur des voies antivirales activées par les récepteurs du type RIG-I (RLR) en interagissant avec la protéine adaptatrice Cardif. Une autre étude propose par contre que NLRX1 participe à la production des dérivés réactifs de l'oxygène et contribue ainsi à augmenter l'activation de NF- κΒ et JNK induite par le TNF-α ou le poly(I:C). Nos résultats montrent que l'absence de NLRX1 ne modifie ni la voie de signalisation RLR ni les réponses induites par le TNF-α. Des analyses ultérieures ont permis d'identifier comme partenaire d'interaction de NLRX1 la protéine UQCRC2, une des sous-unités qui composent le complexe III de la chaîne respiratoire mitochondriale. Cette observation pourrait indiquer un lien fonctionnel entre NLRX1 et la respiration mitochondriale ou la production des dérivés réactifs de l'oxygène au niveau de cette organelle.

Toll-like receptor 3 expressed by melanoma cells as a target for therapy?

PURPOSE: The immunomodulatory properties of Toll-like receptors (TLR) agonists have inspired their use as experimental adjuvants for vaccination of cancer patients. However, it is now well recognized that TLR expression is not restricted to immune cells but can also be found in many cell types, including those giving rise to tumors. It is therefore mandatory to explore the potential effects of TLR triggering directly on tumor cells. EXPERIMENTAL DESIGN: In the present work, we have investigated TLR3 protein expression in melanoma cell lines derived from patients, and analyzed the effects of TLR3 agonists on tumor cell survival. Moreover, we used RNA interference to stably knock down TLR3 expression and study the involvement of this receptor in dsRNA-induced effects on melanoma cells viability. RESULTS: Human melanoma cells can express functional TLR3 protein. Interestingly, the engagement of the receptor by TLR3 agonists can directly inhibit cell proliferation and induce tumor cell death when combined to treatment with either type I IFN or protein synthesis inhibitors. These effects were shown by RNA interference to be largely dependent on TLR3. Moreover, TLR3-mediated cell death involves the activation of caspases and engages both extrinsic and intrinsic apoptotic pathways. CONCLUSION: TLR3 protein can be expressed in human melanoma cells, where it can deliver proapoptotic and antiproliferative signaling. Altogether, these results suggest that TLR3 agonists represent very promising adjuvants for cancer vaccines not only based on their well-described immunostimulatory properties, but also due to their newly identified cytostatic and cytotoxic effects directly on tumor cells.