To Toll-like Receptor 3 Research Articles

Design of a novel multi-epitope vaccine against Marburg virus using immunoinformatics studies

Marburg virus (MARV) is a highly contagious and virulent agent belonging to Filoviridae family. MARV causes severe hemorrhagic fever in humans and non-human primates. Owing to its highly virulent nature, preventive approaches are promising for its control. There is currently no approved drug or vaccine against MARV, and management mainly involves supportive care to treat symptoms and prevent complications. Our aim was to design a novel multi-epitope vaccine (MEV) against MARV using immunoinformatics studies. In this study, various proteins (VP35, VP40 and glycoprotein precursor) were used and potential epitopes were selected. CTL and HTL epitopes covered 79.44% and 70.55% of the global population, respectively. The designed MEV construct was stable and expressed in Escherichia coli (E. coli) host. The physicochemical properties were also acceptable. MARV MEV candidate could predict comprehensive immune responses such as those of humoral and cellular in silico. Additionally, efficient interaction to toll-like receptor 3 (TLR3) and its agonist (β-defensin) was predicted. There is a need for validation of these results using further in vitro and in vivo studies.

Double-Stranded RNA Enhances Matrix Metalloproteinase-1 and -13 Expressions through TLR3-Dependent Activation of Transglutaminase 2 in Dermal Fibroblasts.

UV-irradiation induces the secretion of double-stranded RNA (dsRNA) derived from damaged noncoding RNAs in keratinocytes, which enhance the expression of matrix metalloproteinases (MMP) in non-irradiated dermal fibroblasts, leading to dysregulation of extracellular matrix homeostasis. However, the signaling pathway responsible for dsRNA-induced MMP expression has not been fully understood. Transglutaminase 2 (TG2) is an enzyme that modifies substrate proteins by incorporating polyamine or crosslinking of proteins, thereby regulating their functions. In this study, we showed that TG2 mediates dsRNA-induced MMP-1 expression through NF-κB activation. Treatment of poly(I:C), a synthetic dsRNA analogue binding to toll-like receptor 3 (TLR3), generates ROS, which in turn activates TG2 in dermal fibroblast. Subsequently, TG2 activity enhances translocation of p65 into the nucleus, where it augments transcription of MMP. We confirmed these results by assessing the level of MMP expression in Tlr3−/−, TG2-knockdowned and Tgm2−/− dermal fibroblasts after poly(I:C)-treatment. Moreover, treatment with quercetin showed dose-dependent suppression of poly(I:C)-induced MMP expression. Furthermore, ex vivo cultured skin from Tgm2−/− mice exhibited a significantly reduced level of MMP mRNA compared with those from wild-type mice. Our results indicate that TG2 is a critical regulator in dsRNA-induced MMP expression, providing a new target and molecular basis for antioxidant therapy in preventing collagen degradation.

Immunoinformatics prediction of overlapping CD8+ T-cell, IFN-γ and IL-4 inducer CD4+ T-cell and linear B-cell epitopes based vaccines against COVID-19 (SARS-CoV-2)

At the beginning of the year 2020, the world was struck with a global pandemic virus referred to as SARS-CoV-2 (COVID-19) which has left hundreds of thousands of people dead. To control this virus, vaccine design becomes imperative. In this study, potential epitopes-based vaccine candidates were explored. Six hundred (600) genomes of SARS-CoV-2 were retrieved from the viPR database to generate CD8+ T-cell, CD4+ T-cell and linear B-cell epitopes which were screened for antigenicity, immunogenicity and non-allergenicity. The results of this study provide 19 promising candidate CD8+ T-cell epitopes that strongly overlap with 8 promising B-cells epitopes. Another 19 CD4+ T-cell epitopes were also identified that can induce IFN-γ and IL-4 cytokines. The most conserved MHC-I and MHC-II for both CD8+ and CD4+ T-cell epitopes are HLA-A*02:06 and HLA-DRB1*01:01 respectively. These epitopes also bound to Toll-like receptor 3 (TLR3). The population coverage of the conserved Major Histocompatibility Complex Human Leukocyte Antigen (HLA) for both CD8+ T-cell and CD4+ T-cell ranged from 65.6% to 100%. The detailed analysis of the potential epitope-based vaccine and their mapping to the complete COVID-19 genome reveals that they are predominantly found in the location of the surface (S) and membrane (M) glycoproteins suggesting the potential involvement of these structural proteins in the immunogenic response and antigenicity of the virus. Since the majority of the potential epitopes are located on M protein, the design of multi-epitope vaccine with the structural protein is highly promising though the whole M protein could also serve as a viable epitope for the development of an attenuated vaccine. Our findings provide a baseline for the experimental design of a suitable vaccine against SARS-CoV-2.

Towards a novel peptide vaccine for Middle East respiratory syndrome coronavirus and its possible use against pandemic COVID-19

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging health concern due to its high mortality rate of 35%. At present, no vaccine is available to protect against MERS-CoV infections. Therefore, an in silico search for potential antigenic epitopes in the non-redundant proteome of MERS-CoV was performed herein. First, a subtractive proteome-based approach was employed to look for the surface exposed and host non-homologous proteins. Following, immunoinformatics analysis was performed to predict antigenic B and T cell epitopes that were used in the design of a multi-epitopes peptide. Molecular docking study was carried out to predict vaccine construct affinity of binding to Toll-like receptor 3 (TLR3) and understand its binding conformation to extract ideas about its processing by the host immune system. We identified membrane protein, envelope small membrane protein, non-structural protein ORF3, non-structural protein ORF5, and spike glycoprotein as potential candidates for subunit vaccine designing. The designed multi-epitope peptide then linked to β-defensin adjuvant is showing high antigenicity. Further, the sequence of the designed vaccine construct is optimized for maximum expression in the Escherichia coli expression system. A rich pattern of hydrogen and hydrophobic interactions of the construct was observed with the TLR3 allowing stable binding of the construct at the docked site as predicted by the molecular dynamics simulation and MM-PBSA binding energies. We expect that the panel of subunit vaccine candidates and the designed vaccine construct could be highly effective in immunizing populations from infections caused by MERS-CoV and could possible applied on the current pandemic COVID-19.

Heterozygous TLR3 Mutation in Patients with Hantavirus Encephalitis

Puumala hantavirus (PUUV) hemorrhagic fever with renal syndrome (HFRS) is common in Northern Europe; this infection is usually self-limited and severe complications are uncommon. PUUV and other hantaviruses, however, can rarely cause encephalitis. The pathogenesis of these rare and severe events is unknown. In this study, we explored the possibility that genetic defects in innate anti-viral immunity, as analogous to Toll-like receptor 3 (TLR3) mutations seen in HSV-1 encephalitis, may explain PUUV encephalitis. We completed exome sequencing of seven adult patients with encephalitis or encephalomyelitis during acute PUUV infection. We found heterozygosity for the TLR3 p.L742F novel variant in two of the seven unrelated patients (29%, p = 0.0195). TLR3-deficient P2.1 fibrosarcoma cell line and SV40-immortalized fibroblasts (SV40-fibroblasts) from patient skin expressing mutant or wild-type TLR3 were tested functionally. The TLR3 p.L742F allele displayed low poly(I:C)-stimulated cytokine induction when expressed in P2.1 cells. SV40-fibroblasts from three healthy controls produced increasing levels of IFN-λ and IL-6 after 24 h of stimulation with increasing concentrations of poly(I:C), whereas the production of the cytokines was impaired in TLR3 L742F/WT patient SV40-fibroblasts. Heterozygous TLR3 mutation may underlie not only HSV-1 encephalitis but also PUUV hantavirus encephalitis. Such possibility should be further explored in encephalitis caused by these and other hantaviruses.

Yansuanmalingua inhibits replication of type 2 porcine reproductive and respiratory syndrome virus via activating the caspase-8 apoptosis pathway.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the pork industry. The present study showed that Yansuanmalingua (YASML) can inhibit type 2 PRRSV replication using plaque assay, quantitative reverse transcriptase-polymerase chain reaction, and immunofluorescence assay. Furthermore, inhibition of PRRSV replication was shown to be related to Toll-like receptor 3 (TLR3)-dependent apoptosis-induction by YASML in the PRRSV-infected MARC-145, and TLR3-dependent apoptosis-induction by YASML was found to suppress PRRSV replication via the activation of caspase-8 and caspase-3 pathways, respectively. Meanwhile, activation of the caspase-3 pathway seemed to be related to thedownregulation of myeloid cell leukemia 1 (Mcl-1) expression. Our results showed that YASML-induced TLR3-dependent apoptosis could be blocked by a pan-caspase inhibitor and small interfering RNA against TLR3. In conclusion, the present study demonstrates that YASML exerts its anti-PRRSV effect by activating the caspase-8/caspase-3 signaling pathway and by negatively regulating Mcl-1 expression. These findings not only provide new insights into the molecular mechanism of YASML inhibition of PRRSV replication via the TLR3-dependent apoptosis pathway but also suggest potential, new antiviral drugs by expressing caspase-3 or down expressing Mcl-1.

Phase I study of a new concept cancer vaccine composed artificial intelligence (AI)-designed shared-antigen peptides plus combined synergistically activating antigen-specific CTL reaction (CYT001) in patients with advanced hepatocellular carcinoma (CRESCENT 1).

TPS595 Background: CYT001 (CYTLIMIC Inc.) is a novel cancer vaccine involving artificial intelligence (AI)-designed shared-antigen peptides and optimal combined adjuvants that boost the cancer-immunity cycle. The two multi-HLA reactive peptides heat shock protein 70 (HSP70) and glypican 3 (GPC3) were screened by an AI-based prediction system according to the proteome, mRNA, and histopathology data fromhuman samples. These immunogenic peptides were confirmed to show cross-reactivity to HLA-A 24:02, 02:01, and 02:06. Poly-ICLC (Oncovir Inc.) binds to Toll-like receptor 3 (TLR3) and melanoma differentiation antigen 5 (MDA5) on antigen-presenting cells (APCs)and activates APCs. LAG-3Ig (Immutep Inc.) binds to the major histocompatibility complex (MHC) class II molecules of APCs and activates APCs. Both poly-ICLC and LAG-3Ig synergistically activate antigen-specific CTL reactions as effective combination adjuvants. The present study aims to evaluate the safety and tolerability of CYT001 (mixture of HSP70 peptide [2.0 mg], GPC3 peptide [2.0 mg], poly-ICLC [1.0 mg], and LAG-3Ig [1.4 mg]) in patients with advanced hepatocellular carcinoma (HCC). Methods: This is a single-center, phase 1, open-label, single-arm, investigator-initiated clinical trial of CYT001 for advanced HCC patients with no eligible standard systemic therapy, Child–Pugh A liver disease, and HLA-A 24:02, 02:01, or 02:06. Enrolled patients will receive CYT001 as a subcutaneous injection on days 1, 8, 15, and 21 in the 1st and 2nd cycles, days 1 and 15 in the 3rd and 4th cycles, and day 1 in the 5th cycle or later of 28-day cycles. The primary endpoint is dose-limiting toxicity, and the secondary endpoints are safety and the response rate. The transition of the CTL reactions of both the HSP70 and GPC3 peptides will be evaluated using blood samples of the subjects. Exploratory analyses include investigation of candidate biomarkers for treatment efficacy using liver tumor biopsy samples (baseline and after the 1st cycle) and blood samples (baseline and every days of administration). Clinical trial information: jRCT2031190072.

A marine-sourced fucoidan solution inhibits Toll-like-receptor-3-induced cytokine release by human bronchial epithelial cells.

Fucoidans are sulfated polysaccharides from brown algae, known to have immunomodulatory activity. Their effects on the response of airway epithelial cells to Toll-like receptor 3 (TLR3) stimulation have not been characterized. Our objective was to evaluate the effects of a marine-sourced fucoidan solution (MFS) on the TLR3-induced expression and/or production of cytokines and prostaglandin by human primary bronchial epithelial cells as a model of the airway epithelium. The cells were incubated with MFS in the presence or absence of Poly(I:C) (a TLR3 agonist that mimics viral RNA). Cytokine expression and production were assessed using RT-qPCR and ELISA. The expression of cyclooxygenase-2 and the production of prostaglandin E2 were also measured. Relative to control, exposure to MFS was associated with lower Poly(I:C)-induced mRNA expression of various cytokines and chemokines, and lower COX-2 production. The MFS inhibited the production of some cytokines (IL-1α, IL-1β, TNFα, and IL-6), chemokines (CCL5, CCL22, CXCL1, CXCL5 and CXCL8) and prostaglandin E2 but did not alter the production of IL-12/25, CCL2 and CCL20. At clinically relevant concentrations, the MFS inhibited the TLR3-mediated production of inflammatory mediators by human primary bronchial epithelial cells - suggesting that locally applied MFS might help to reduce airway inflammation in viral infections.

Physiological responses to central and peripheral injection of polyinosinic-polycytidylic acid in chicks

ABSTRACT1. The purpose of the present study was to determine if intracerebroventricular (ICV) and intraperitoneal (IP) injection of polyinosinic-polycytidylic acid (poly I:C), a viral mimetic that binds to toll-like receptor-3 (TLR3), affects food intake, voluntary activity, cloacal temperature, plasma corticosterone (CORT) and glucose concentrations, and crop emptying rate in chicks (Gallus gallus).2. Both ICV and IP injection of poly I:C significantly decreased food intake.3. IP but not ICV injection of poly I:C significantly suppressed voluntary activity, whereas ICV injection decreased time spent sitting. Both ICV and IP injection of poly I:C significantly increased plasma CORT and glucose concentration. Neither ICV nor IP injection of poly I:C significantly affected cloacal temperature.4. In addition, ICV injection of poly I:C significantly reduced crop emptying rate, whereas IP injection had no effect.5. These results suggested that central TLR3 is related to anorexia, stress response and retardation of crop emptying while peripheral TLR3 is related to anorexia, change in behaviour and stress responses during viral infection in chicks.

Molecular characterization and expression of a disintegrin and metalloproteinase with thrombospondin motifs 8 in chicken.

ObjectiveA disintegrin and metallopeptidase with thrombospondin motifs type 8 (ADAMTS8) is crucial for diverse physiological processes, such as inflammation, tissue morphogenesis, and tumorigenesis. The chicken ADAMTS8 (chADAMTS8) gene was differentially expressed in the kidney following exposure to different calcium concentrations, suggesting a pathological role of this protein in metabolic diseases. We aimed to examine the molecular characteristics of chADAMTS8 and analyze the gene-expression differences in response to toll-like receptor 3 (TLR3) stimulation.MethodsThe ADAMTS8 mRNA and amino acid sequences of various species (chicken, duck, cow, mouse, rat, human, chimpanzee, pig, and horse) were retrieved from the Ensembl database and subjected to bioinformatics analyses. Reverse-transcription polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) experiments were performed with various chicken tissues and the chicken fibroblast DF-1 cell line, which was stimulated with polyinosinic-polycytidylic acid (poly[I:C]; a TLR3 ligand).ResultsThe chADAMTS8 gene was predicted to contain three thrombospondin type 1 (TSP1) domains, whose amino acid sequences shared homology among the different species, whereas sequences outside the TSP1 domains (especially the amino-terminal region) were very different. Phylogenetic analysis revealed that chADAMTS8 is evolutionarily clustered in the same clade with that of the duck. chADAMTS8 mRNA was broadly expressed in chicken tissues, and the expression was significantly up-regulated in the DF-1 cells in response to poly(I:C) stimulation (p<0.05). These results showed that chADAMTS8 may be a target gene for TLR3 signaling.ConclusionIn this report, the genetic information of chADAMTS8 gene, its expression in chicken tissues, and chicken DF-1 cells under the stimulation of TLR3 were shown. The result suggests that chADAMTS8 expression may be induced by viral infection and correlated with TLR3-mediated signaling pathway. Further study of the function of chADAMTS8 during TLR3-dependent inflammation (which represents RNA viral infection) is needed and it will also be important to examine the molecular mechanisms during different regulation, depending on innate immune receptor activation.

Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

Apoptosis and programmed necrosis (necroptosis) determine cell fate, and antagonize infection. Execution of these complementary death pathways involves the formation of receptor-interacting protein kinase 1 (RIPK1) containing complexes. RIPK1 binds to adaptor proteins, such as TRIF (Toll-IL-1 receptor-domain-containing-adaptor-inducing interferon-beta factor), FADD (Fas-associated-protein with death domain), NEMO (NF-κB regulatory subunit IKKγ), SQSTM1 (sequestosome 1/p62), or RIPK3 (receptor-interacting protein kinase 3), which are involved in RNA sensing, NF-κB signaling, autophagosome formation, apoptosis, and necroptosis. We report that a range of rhinoviruses impair apoptosis and necroptosis in epithelial cells late in infection. Unlike the double-strand (ds) RNA mimetic poly I:C (polyinosinic:polycytidylic acid), the exposure of dsRNA to toll-like receptor 3 (TLR3) in rhinovirus-infected cells did not lead to apoptosis execution. Accordingly, necroptosis and the production of ROS (reactive oxygen species) were not observed late in infection, when RIPK3 was absent. Instead, a virus-induced alternative necrotic cell death pathway proceeded, which led to membrane rupture, indicated by propidium iodide staining. The impairment of dsRNA-induced apoptosis late in infection was controlled by the viral 3C-protease (3Cpro), which disrupted RIPK1-TRIF/FADD /SQSTM1 immune-complexes. 3Cpro and 3C precursors were found to coimmuno-precipitate with RIPK1, cleaving the RIPK1 death-domain, and generating N-terminal RIPK1 fragments. The depletion of RIPK1 or chemical inhibition of its kinase at the N-terminus did not interfere with virus progeny formation or cell fate. The data show that rhinoviruses suppress apoptosis and necroptosis, and release progeny by an alternative cell death pathway, which is controlled by viral proteases modifying innate immune complexes.

Hepatitis B virus genome replication triggers toll-like receptor 3-dependent interferon responses in the absence of hepatitis B surface antigen.

The hepatitis B virus (HBV) has been described as stealth virus subverting immune responses initially upon infection. Impaired toll-like receptor signaling by the HBV surface antigen (HBsAg) attenuates immune responses to facilitate chronic infection. This implies that HBV replication may trigger host innate immune responses in the absence of HBsAg. Here we tested this hypothesis, using highly replicative transgenic mouse models. An HBV replication-dependent expression of antiviral genes was exclusively induced in HBsAg-deficient mice. These interferon responses attributed to toll-like receptor 3 (TLR3)-activated Kupffer and liver sinusoidal endothelial cells and further controlled the HBV genome replication. However, activation of TLR3 with exogenous ligands indicated additional HBs-independent immune evasion events. Our data demonstrate that in the absence of HBsAg, hepatic HBV replication leads to Tlr3-dependent interferon responses in non-parenchymal liver cells. We hypothesize that HBsAg is a major HBV-mediated evasion mechanism controlling endogenous antiviral responses in the liver. Eradication of HBsAg as a therapeutic goal might facilitate the induction of endogenous antiviral immune responses in patients chronically infected with HBV.

Interleukin-24 inhibits influenza A virus replication in vitro through induction of toll-like receptor 3 dependent apoptosis

New anti-viral agents and strategies are urgently needed to fight rapidly mutating viruses, as vaccine programs cannot react fast enough to prevent pandemics. Recently, we have shown that interleukin-24 (IL-24) sensitizes tumor cells to toll-like receptor 3 (TLR3) mediated apoptosis. As influenza A virus stimulates the TLR3 receptor, we hypothesized that IL-24 might also exert an anti-viral effect. This study demonstrates that IL-24 reduces the titer of different influenza A virus subtypes independently of type I interferon in an apoptosis dependent manner. The anti-viral effect of IL-24 correlated with caspase-3 activation and could be blocked by a pan-caspase inhibitor and by small interfering RNA (siRNA) directed towards TLR3. Surprisingly, caspase-3 activation in influenza A virus/IL-24-stimulated cells correlated with the down-regulation of the B-cell lymphoma 2 (Bcl-2) family member myeloid cell leukemia 1 (Mcl-1). Correspondingly, knockdown of Mcl-1 by siRNA enhanced caspase activation in influenza A virus infected cells and was furthermore linked to a reduction of viral titers. We conclude that IL-24 exerts an anti-viral role selectively purging virally infected cells by leading to a down-regulation of Mcl-1. Our findings might therefore represent the first step towards a new rational concept in the development of anti-viral strategies based on the induction of apoptosis.

Medroxyprogesterone acetate impairs human dendritic cell activation and function.

Does medroxyprogesterone acetate (MPA) impair human dendritic cell (DC) activation and function? In vitro MPA treatment suppressed expression of CD40 and CD80 by human primary DCs responding to Toll-like receptor 3 (TLR3) agonist stimulation (i.e. DC activation). Moreover, this MPA-mediated decrease in CD40 expression impaired DC capacity to stimulate T cell proliferation (i.e. DC function). MPA is the active molecule in Depo-Provera(®) (DMPA), a commonly used injectable hormonal contraceptive (HC). Although DMPA treatment of mice prior to viral mucosal tissue infection impaired the capacity of DCs to up-regulate CD40 and CD80 and prime virus-specific T cell proliferation, neither DC activation marker expression nor the ability of DCs to promote T cell proliferation were affected by in vitro progesterone treatment of human DCs generated from peripheral blood monocytes. This cross-sectional study examined MPA-mediated effects on the activation and function of human primary untouched peripheral blood DCs. Human DCs isolated from peripheral blood mononuclear cells by negative immunomagnetic selection were incubated for 24 h with various concentrations of MPA. After an additional 24 h incubation with the TLR3 agonist polyinosinic:polycytidylic acid (poly I:C), flow cytometry was used to evaluate DC phenotype (i.e. expression of CD40, CD80, CD86, and HLA-DR). In separate experiments, primary untouched human DCs were sequentially MPA-treated, poly I:C-activated, and incubated for 7 days with fluorescently labeled naïve allogeneic T cells. Flow cytometry was then used to quantify allogeneic T cell proliferation. Several pharmacologically relevant concentrations of MPA dramatically reduced CD40 and CD80 expression in human primary DCs responding to the immunostimulant poly I:C. In addition, MPA-treated DCs displayed a reduced capacity to promote allogeneic CD4(+) and CD8(+) T cell proliferation. In other DC: T cell co-cultures, the addition of antibody blocking the CD40-CD154 (CD40L) interaction mirrored the decreased T cell proliferation produced by MPA treatment, while addition of recombinant soluble CD154 restored the capacity of MPA-treated DCs to induce T cell proliferation to levels produced by non-MPA-treated controls. While our results newly reveal that pharmacologically relevant MPA concentrations suppress human DC function in vitro, additional research is needed to learn if DMPA similarly inhibits DC maturation and function in the human female genital tract. Identification of a mechanism by which MPA impairs human DC activation and function increases the biological plausibility for the relationships currently suspected between DMPA use and enhanced susceptibility to genital tract infection. Funding provided by the NIH (grant R01HD072663) and The Ohio State University College of Medicine. The authors have no conflicts of interest to declare.

Abstract A65: Toll-like receptor 3 signaling in ovarian cancer.

Abstract The purpose of this study is to examine the cytokine secretion (in vitro) and leukocyte infiltration profiles (in vivo) in response to Toll-Like Receptor 3 (TLR3) activation in murine epithelial ovarian carcinoma. Ovarian cancer is the most deadly gynecological neoplasm, typically treated with surgery and chemotherapy, and has a less than a 50% 5-year survival rate. Drug resistance develops frequently, and it is necessary to investigate more targeted therapies. Chronic inflammation, which is seen in many cancers, can render an immunosuppressive tumor microenvironment characterized by cytokines that promote leukocyte infiltration, which can potentiate angiogenesis, and subsequent tumor growth and metastasis. Pathways that can generate such inflammation are important to characterize as they may be targeted in the development of novel cancer therapeutics. One regulator of inflammation in some types of epithelial ovarian cancer is TLR3, a protein that senses dsRNA and activates NF-κB, a potent initiator of cytokine secretion. Although typically expressed in immune cells, TLR3 is also present in cancer cells, and may function to direct inflammation at the tumor site towards an immunosuppressive state capable of recruiting pro-angiogenic leukocytes. Using PCR, ELISA, western blot, and microarray experiments on siRNA-generated TLR3-knock-downs and control samples, we are characterizing the cytokine secretion profiles following TLR3 activation with synthetic dsRNA ligands. Furthermore, we will employ this ovarian cancer cell line (ID8) with C57BL/6 mice to generate transplantation tumors and determine the effects of TLR3 signaling on the leukocyte population and presence of angiogenic molecules at the tumor site using flow cytometry and immunohistochemistry. We have determined that treatment of ID8 epithelial ovarian carcinoma cells with TLR3 ligands poly (I:C) or poly (A:U) results in increased secretion of pleiotropic cytokines, such as CCL5 and IL6, that may potentiate leukocyte migration to the tumor site. In vivo studies with C57BL/6 mice will determine whether this TLR3-induced cytokine secretion in the tumor cells translates to the physiological environment and promotes leukocyte infiltration and angiogenesis. By elucidating the contribution of TLR3 to the recruitment of pro-angiogenic leukocytes in ovarian epithelial carcinoma, the results of this project may be useful in the development of anti-angiogenic therapies for certain ovarian tumors. Citation Format: Maria Muccioli, Michelle Pate, Fabian Benencia. Toll-like receptor 3 signaling in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A65.

Selected Toll-like Receptor Ligands and Viruses Promote Helper-Independent Cytotoxic T Cell Priming by Upregulating CD40L on Dendritic Cells

CD40L (CD154) on CD4(+) T cells has been shown to license dendritic cells (DCs) via CD40 to prime cytotoxic T lymphocyte (CTL) responses. We found that the converse (CD40L on DCs) was also important. Anti-CD40L treatment decreased endogenous CTL responses to both ovalbumin and influenza infection even in the absence of CD4(+) T cells. DCs expressed CD40L upon stimulation with agonists to Toll-like receptor 3 (TLR3) and TLR9. Moreover, influenza infection, which stimulates CTLs without help, upregulated CD40L on DCs, but herpes simplex infection, which elicits CTLs through help, did not. CD40L-deficient (Cd40lg(-/-)) DCs are suboptimal both in vivo in bone marrow chimera experiments and in vitro in mixed lymphocyte reactions. In contrast, Cd40lg(-/-) CD8(+) T cells killed as effectively as wild-type cells. Thus, CD40L upregulation on DCs promoted optimal priming of CD8(+) T cells without CD4(+) T cells, providing a mechanism by which pathogens may elicit helper-independent CTL immunity.