TLR7 Agonist Imiquimod Research Articles

Activation of TLR7 increases CCND3 expression via the downregulation of miR-15b in B cells of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by B-cell hyperreactivity. The Toll-like receptor 7 (TLR7) signaling pathway is abnormally activated in SLE B cells. CyclinD3 (CCND3) plays an important role in B-cell proliferation, development, and differentiation. Although previous studies focused on the B cell-intrinsic role of TLR7 for the development of spontaneous germinal centers, the influence of TLR7 on CCND3 in SLE B cells is still not clear. Here, we used a B-cell profiling chip and found that CCND3 was related to SLE and significantly elevated in SLE B cells. Moreover, we determined that the expression level of CCND3 was higher, while miR-15b was significantly lower in the B cells from SLE patients and B6.MRL-Faslpr/J lupus mice compared to normal subjects. Furthermore, we demonstrated that the activation of TLR7 dramatically increased CCND3 expression but significantly decreased miR-15b in B cells in vitro and we identified that CCND3 is a direct target of miR-15b. To further confirm our results, we established another lupus model by topically treating C57BL/6 (B6) mice with the TLR-7 agonist imiquimod (IMQ) for 8 weeks according to the previously described protocol. Expectedly, topical treatment with IMQ also significantly increased CCND3 and decreased miR-15b in B cells of B6 mice. Taken together, our results identified that the activation of TLR7 increased CCND3 expression via the downregulation of miR-15b in B cells; thus, these findings suggest that extrinsic factor-induced CCND3 expression may contribute to the abnormality of B cell in SLE.

The TLR7 agonist Imiquimod promote the immunogenicity of mesenchymal stem cells.

BackgroundMesenchymal stem cells (MSCs) are considered the best candidate in stem cells therapy due to their multipotent differentiation ability, low expression of co-stimulatory molecules (CD80, CD86, CD34 and HLA-II) and immunosuppression effects on in vivo immune responses. MSCs were now widely used in clinical trials but received no encourage results. The major problem was the fate of engrafted MSCs in vivo could not be defined. Some studies indicated that MSCs could induce immune response and result in the damage and rejection of MSCs. As toll like receptors (TLRs) are important in inducing of immune responses, in this study we study the role of TLR7 in mediating the immune status of MSCs isolated from umbilical cord.ResultsOur results indicated that TLR7 agonist Imiquimod could increase the proliferation of PBMC isolated from healthy human volunteers and release of lactate dehydrogenase (LDH) in supernatant from PBMC-UCMSCs co-culture system. Flow cytometry and quantitative PCR also confirmed the regulated expression of surface co-stimulatory molecules and pro-inflammatory genes (IL-6, IL-8, IL-12, TGF-β and TNF-α). And the down-regulation expression of stem cell markers also confirmed the loss of stemness of UCMSCs. We also found that the osteo-differentiation ability of UCMSCs was enhanced in the presence of Imiquimod.ConclusionTo our knowledge, this is the first report that activation of TLR7 pathway increases the immunogenicity of UCMSCs. Extensive researches have now been conducted to study whether the change of immune status will be help in tumor rejection based on the tumor-tropism of MSCs.

The efficacy of tumor debulking surgery is improved by adjuvant immunotherapy using imiquimod and anti-CD40.

BackgroundTumor debulking surgery followed by adjuvant chemotherapy or radiotherapy is a standard treatment for many solid malignancies. Although this approach can be effective, it often has limited success against recurrent or metastatic cancers and new multimodality approaches are needed. Adjuvant immunotherapy is another potentially effective approach. We therefore tested the efficacy of the TLR7 agonist imiquimod (IMQ) combined with agonistic anti-CD40 in an incomplete debulking model of malignant mesothelioma.MethodsEstablished subcutaneous murine ABA-HA mesothelioma tumors in BALB/c mice were surgically debulked by 75% and treated with either: i) saline; ii) intratumoral IMQ; iii) systemic anti-CD40 antibody, or using a combination of IMQ and anti-CD40. Tumour growth and survival were monitored, and the role of anti-tumor CD4 and CD8 T cells in therapeutic responses was determined.ResultsThe combination therapy of partial debulking surgery, IMQ and anti-CD40 significantly delayed tumor growth in a CD8 T cell dependent manner, and promoted tumor regression in 25% of animals with establishment of immunological memory. This response was associated with an increase in ICOS+ CD8 T cells and tumor-specific CTL activity in tumor draining lymph nodes along with an increase in ICOS+ CD8 T cells in responding tumours.ConclusionsWe show that the post-surgical environment can be significantly altered by the co-administration of adjuvant IMQ and anti-CD40, resulting in strong, systemic anti-tumor activity. Both adjuvants are available for clinical use/trial, hence this treatment regimen has clear translational potential.

Topical rather than intradermal application of the TLR7 ligand imiquimod leads to human dermal dendritic cell maturation and CD8+ T-cell cross-priming.

Toll-like receptor (TLR) ligands are attractive candidate adjuvants for therapeutic cancer vaccines, since TLR signaling stimulates and tunes both humoral and cellular immune responses induced by dendritic cells (DCs). Given that human skin contains a dense network of DCs, which are easily accessible via (intra-)dermal delivery of vaccines, skin is actively explored as an antitumor vaccination site. Here we used a human skin explant model to explore the potential of TLR ligands as adjuvants for DC activation in their complex microenvironment. We show that topical application of Aldara skin cream, 5% of which comprises the TLR7 agonist imiquimod, significantly enhanced DC migration as compared with that resulting from intradermal injection of the TLR7/8 ligand R848 or the soluble form of imiquimod. Moreover, Aldara-treated DCs showed highest levels of the costimulatory molecules CD86, CD83, CD40, and CD70. Topical Aldara induced the highest production of pro-inflammatory cytokines in skin biopsies. When combined with intradermal peptide vaccination, Aldara-stimulated DCs showed enhanced cross-presentation of the melanoma antigen MART-1, which resulted in increased priming and activation of MART-1-specific CD8(+) T cells. These results point to advantageous effects of combining the topical application of Aldara with antitumor peptide vaccination.

Asthma and Allergy Sig 1 – Oral Presentations

Introduction:Some asthmatics display impaired type 1 IFN responses and exaggerated inflammatory responses upon Rhinovirus (RV) infection, however, the molecular basis for these observations remain obscure despite intensive research. Recently, impaired production of antiviral molecules and IFN-induced cytokines have been described in asthmatics when peripheral bloodmononuclear cells (PBMC) were stimulated with the TLR7 agonist Imiquimod but not in response to the TLR3 agonist poly I : C (Roponen et al. ERJ 2009). Interestingly, some asthmatics display variations in their TLR7 genotype that may be related to impaired TLR7 function. Aim:To identify the molecular basis for the aberrant response to RV observed in asthmatics. Method:Bronchial biopsies were taken from well characterized asthmatics and healthy controls. Expression of key pathogen recognition receptors including TLR3 and 7 were enumerated using qPCR. WT and TLR7-/- BALB/c mouse models of RV1B induced exacerbation of house dust mite driven allergic airways disease were employed to determine the relevance of TLR7 signallingin antiviral responses and inflammation. Results:Expression of TLR7 was found to be significantly reduced in bronchial biopsies from eosinophilic but not neutrophilic asthmatics. TLR7-/- mice were found to have a deficient response to RV1B infection with reduced levels of type I and III interferons and elevated viral titre at 24 hr post infection when compared to wild type controls. Treatment with type I or type III interferons limited RV1B replication as well as impaired both neutrophilic and eosinophilic inflammation.Conclusion:Our study suggests deficient TLR7 function as a crucial mechanism underpinning impaired IFN responses to RV1B in allergic airways inflammation. Restoration of IFN not only reduced viral replication but also impaired RV1B-induced inflammation. These studies may indicate that augmentation of TLR7-regulated effector functions could be exploited as a novel therapeutic approach for the treatment of virus-induced asthma exacerbation in asthmatics with deficient IFN responses and/or eosinophilic inflammation.

Toll-like Receptor 7 Rapidly Relaxes Human Airways

Toll-like receptors (TLRs) 7 and 8 detect respiratory virus single-stranded RNA and trigger an innate immune response. We recently described rapid TLR7-mediated bronchodilation in guinea pigs. To characterize TLR7 expression and TLR7-induced airway relaxation in humans and in eosinophilic airway inflammation in guinea pigs. To evaluate the relaxant effects of other TLRs. Human airway smooth muscle strips were contracted with methacholine in vitro, and responses to TLR7 and TLR8 agonists were assessed. TLR7-mediated nitric oxide production was measured using a fluorescent indicator, and TLR7 expression was characterized using immunofluorescence. TLR7 signaling was also evaluated in ovalbumin-challenged guinea pigs. The TLR7 agonist imiquimod (R837) caused rapid dose-dependent relaxation of methacholine-contracted human airways in vitro. This was blocked by the TLR7 antagonist IRS661 and by inhibiting nitric oxide production but not by inhibiting prostaglandin production. TLR7 activation markedly increased fluorescence of a nitric oxide detector. TLR7 was expressed on airway nerves, but not airway smooth muscle, implicating airway nerves as the source of TLR7-induced nitric oxide production. TLR7-mediated relaxation persisted in inflamed guinea pigs airways in vivo. The TLR8 agonists polyuridylic acid and polyadenylic acid also relaxed human airways, and this was not blocked by the TLR7 antagonist or by blocking nitric oxide or prostaglandin production. No other TLRs relaxed the airways. TLR7 is expressed on airway nerves and mediates relaxation of human and animal airways through nitric oxide production. TLR7-mediated bronchodilation may be a new therapeutic strategy in asthma.

Tu2114 Visceral Pain-Related Responses Are Attenuated in Mice With Antibiotic-Induced Intestinal Dysbiosis

BACKGROUND: Toll-like Receptors (TLRs) are effector components in host-bacterial interactions. Within the gut, TLRs participate microbial-related local neuroimmune control, likely including the modulation of epithelial barrier function (EBF). AIMS: To assess changes in colonic EBF associated to the local stimulation of TLR7, with the selective agonist imiquimod, in rats. METHODS: Adult male SD rats were treated intracolonically with the TLR7 agonist imiquimod (100-300 μg/rat, 0.2 ml) or vehicle (0.2 ml/rat). 5 h later colonic samples were obtained to assess EBF in vitro (Ussing chambers). Basal epithelial electrical parameters (short-circuit current; potential difference and conductance) and effects of neural blockade with tetrodotoxin (TTX basolateral, 1 μM) on epithelial secretion were assessed. Paracellular permeability was determined assessing the flux of 4kD fluorescein isothiocyanate-dextran (FD4). Expression of tight-junction-related proteins (occludin, zona occludens-1 [ZO-1], claudin-3, tricellulin and Jam-1), the barrier-related marker proglucagon (precursor of Glucagon-like Peptide-2 [GLP-2]); cytokines (IL-6 and IFN α) and TLR7 was assessed by RTqPCR. Gene expression (RT-qPCR) and distribution (immunohistochemistry) of the poreforming protein claudin-2 were also assessed. RESULTS: Imiquimod (300 μg/rat) showed a tendency to decrease conductance (Table). TTX reduced transepithelial ionic fluxes and conductance, in a similar proportion in either non-treated or imiquimod-treated animals. Imiquimod reduced paracellular flux of FD4 by 17% and 65% at 100 and 300 μg, respectively, vs. vehicle-treated controls (Table). Gene expression of tight-junction-related proteins was not affected by imiquimod. Similarly, expression of proglucagon, the precursor of the barrierenhancer GLP-2, was not modified by imiquimod. Neither gene expression levels nor distribution of the pore-forming protein claudin-2 along the colonic crypts were affected by imiquimod. No inflammatory-like changes associated to imiquimod administration were observed either at the macroscopical, microscopical (histopathology) or molecular (gene expression of cytokines) levels. CONCLUSIONS: In rats, activation of colonic TLR7 enhanced epithelial barrier function, as indicated by the reduction in conductance and permeability to macromolecules. These effects are mediated through yet to defined mechanisms, but were independent of changes in the expression of the main tight-junction-related proteins, the GLP-2 precursor, proglucagon, or the pore-forming protein, claudin-2. Since TLRs are activated by the gut microbiota, increased epithelial tightness associated to their activation might represent a local defensive mechanism generated by dysbiosis and directed towards the prevention of bacterial translocation and the penetration of luminal antigens. Table Effects of activation of TLR7 with imiquimod on colonic EBF

Tu2113 Activation of Colonic Toll-Like Receptor 7 (TLR7) Enhances Epithelial Barrier Function in Rats

BACKGROUND: Toll-like Receptors (TLRs) are effector components in host-bacterial interactions. Within the gut, TLRs participate microbial-related local neuroimmune control, likely including the modulation of epithelial barrier function (EBF). AIMS: To assess changes in colonic EBF associated to the local stimulation of TLR7, with the selective agonist imiquimod, in rats. METHODS: Adult male SD rats were treated intracolonically with the TLR7 agonist imiquimod (100-300 μg/rat, 0.2 ml) or vehicle (0.2 ml/rat). 5 h later colonic samples were obtained to assess EBF in vitro (Ussing chambers). Basal epithelial electrical parameters (short-circuit current; potential difference and conductance) and effects of neural blockade with tetrodotoxin (TTX basolateral, 1 μM) on epithelial secretion were assessed. Paracellular permeability was determined assessing the flux of 4kD fluorescein isothiocyanate-dextran (FD4). Expression of tight-junction-related proteins (occludin, zona occludens-1 [ZO-1], claudin-3, tricellulin and Jam-1), the barrier-related marker proglucagon (precursor of Glucagon-like Peptide-2 [GLP-2]); cytokines (IL-6 and IFN α) and TLR7 was assessed by RTqPCR. Gene expression (RT-qPCR) and distribution (immunohistochemistry) of the poreforming protein claudin-2 were also assessed. RESULTS: Imiquimod (300 μg/rat) showed a tendency to decrease conductance (Table). TTX reduced transepithelial ionic fluxes and conductance, in a similar proportion in either non-treated or imiquimod-treated animals. Imiquimod reduced paracellular flux of FD4 by 17% and 65% at 100 and 300 μg, respectively, vs. vehicle-treated controls (Table). Gene expression of tight-junction-related proteins was not affected by imiquimod. Similarly, expression of proglucagon, the precursor of the barrierenhancer GLP-2, was not modified by imiquimod. Neither gene expression levels nor distribution of the pore-forming protein claudin-2 along the colonic crypts were affected by imiquimod. No inflammatory-like changes associated to imiquimod administration were observed either at the macroscopical, microscopical (histopathology) or molecular (gene expression of cytokines) levels. CONCLUSIONS: In rats, activation of colonic TLR7 enhanced epithelial barrier function, as indicated by the reduction in conductance and permeability to macromolecules. These effects are mediated through yet to defined mechanisms, but were independent of changes in the expression of the main tight-junction-related proteins, the GLP-2 precursor, proglucagon, or the pore-forming protein, claudin-2. Since TLRs are activated by the gut microbiota, increased epithelial tightness associated to their activation might represent a local defensive mechanism generated by dysbiosis and directed towards the prevention of bacterial translocation and the penetration of luminal antigens. Table Effects of activation of TLR7 with imiquimod on colonic EBF

Imiquimod enhances anti-lymphoma response in a tumor cell lysate vaccine and inhibits tumor cells growth.

Event Abstract Back to Event Imiquimod enhances anti-lymphoma response in a tumor cell lysate vaccine and inhibits tumor cells growth. Claudia Mongini1*, Julieta De Toro1, Leticia Hershlik1, María J. Gravisaco2, Claudia Waldner1 and Paula Di Sciullo1 1 National Research Council Of Argentina, Argentina 2 Instituto Nacional de Tecnología Agropecuaria (INTA), Argentina Our aim was to study the immunomodulating and proapoptotic properties of the TLR7 agonist Imiquimod to be used as adjuvant in a tumor vaccine. Imiquimod showed an antiproliferative effect on LBC T-cell lymphoma in vitro at 48 and 72 h with doses between 0.5 and 200 μg/ml, inducing apoptosis in a dose dependent manner. BALB/c mice inoculated intraperitoneally twice with LBC cells lysates with or without 100 μg/mouse of Imiquimod and challenged one week later with LBC cells intraperitoneally, showed a significant increase in survival compared to un-immunized controls (LBC-lysate<0.05, LBC-lysate + IMQ<0.0005) and to mice receiving Imiquimod (p<0.0002; Logrank).Tumor incidence was only 5±5% in LBC-lysate+IMQ group whereas in LBC-lysate group was 52.5±2.5%. Although the percentages of specific cytotoxicity were above normal in both immunized groups (p<0.05, ANOVA), LBC-lysate+IMQ immunization induced the highest levels. A significant enhance in peritoneal cavity CD8+, CD4+ T and B cells (p<0.0001, ANOVA) with an increase IFNγ in supernatant from peritoneal cavity cells and splenocytes was demonstrated in all vaccinated groups compared to normal (p<0.001, ANOVA). Our vaccine also generated circulating antibodies in all immunized mice that rejected the tumor up to 75 days after challenge. Finally, mice that rejected the first tumor challenge were re-challenged with LBC tumor with an increased in survival in all immunized groups over the control (p<0.01, Logrank) with rejection rates close to 100%. We demonstrated that Imiquimod not only modulates immune response efficiently to reject tumor generating a long-lasting memory, but also is capable of inducing tumor apoptosis. Keywords: imiquimod, T-cell lymphoma, TLR7 agonist, Apoptosis, tumor cell lysate vaccine Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Mongini C, De Toro J, Hershlik L, Gravisaco MJ, Waldner C and Di Sciullo P (2013). Imiquimod enhances anti-lymphoma response in a tumor cell lysate vaccine and inhibits tumor cells growth.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01162 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 31 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Claudia Mongini, National Research Council Of Argentina, Buenos Aires, Buenos Aires, 1121, Argentina, cmongini@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Claudia Mongini Julieta De Toro Leticia Hershlik María J Gravisaco Claudia Waldner Paula Di Sciullo Google Claudia Mongini Julieta De Toro Leticia Hershlik María J Gravisaco Claudia Waldner Paula Di Sciullo Google Scholar Claudia Mongini Julieta De Toro Leticia Hershlik María J Gravisaco Claudia Waldner Paula Di Sciullo PubMed Claudia Mongini Julieta De Toro Leticia Hershlik María J Gravisaco Claudia Waldner Paula Di Sciullo Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Topical TLR7 Agonist Imiquimod Can Induce Immune-Mediated Rejection of Skin Metastases in Patients with Breast Cancer

Skin metastases of breast cancer remain a therapeutic challenge. Toll-like receptor 7 agonist imiquimod is an immune response modifier and can induce immune-mediated rejection of primary skin malignancies when topically applied. Here we tested the hypothesis that topical imiquimod stimulates local antitumor immunity and induces the regression of breast cancer skin metastases. A prospective clinical trial was designed to evaluate the local tumor response rate of breast cancer skin metastases treated with topical imiquimod, applied 5 d/wk for 8 weeks. Safety and immunologic correlates were secondary objectives. Ten patients were enrolled and completed the study. Imiquimod treatment was well tolerated, with only grade 1 to 2 transient local and systemic side effects consistent with imiquimod's immunomodulatory effects. Two patients achieved a partial response [20%; 95% confidence interval (CI), 3%-56%]. Responders showed histologic tumor regression with evidence of an immune-mediated response, showed by changes in the tumor lymphocytic infiltrate and locally produced cytokines. Topical imiquimod is a beneficial treatment modality for breast cancer metastatic to skin/chest wall and is well tolerated. Importantly, imiquimod can promote a proimmunogenic tumor microenvironment in breast cancer. Preclinical data generated by our group suggest superior results with a combination of imiquimod and ionizing radiation and we are currently testing in patients whether the combination can further improve antitumor immune and clinical responses.

Skin TLR7 Triggering Promotes Accumulation of Respiratory Dendritic Cells and Natural Killer Cells

The TLR7 agonist imiquimod has been used successfully as adjuvant for skin treatment of virus-associated warts and basal cell carcinoma. The effects of skin TLR7 triggering on respiratory leukocyte populations are unknown. In a placebo-controlled experimental animal study we have used multicolour flow cytometry to systematically analyze the modulation of respiratory leukocyte subsets after skin administration of imiquimod. Compared to placebo, skin administration of imiquimod significantly increased respiratory dendritic cells (DC) and natural killer cells, whereas total respiratory leukocyte, alveolar macrophages, classical CD4+ T helper and CD8+ T killer cell numbers were not or only moderately affected. DC subpopulation analyses revealed that elevation of respiratory DC was caused by an increase of respiratory monocytic DC and CD11bhi DC subsets. Lymphocyte subpopulation analyses indicated a marked elevation of respiratory natural killer cells and a significant reduction of B lymphocytes. Analysis of cytokine responses of respiratory leukocytes after stimulation with Klebsiella pneumonia indicated reduced IFN-γ and TNF-α expression and increased IL-10 and IL-12p70 production after 7 day low dose skin TLR7 triggering. Additionally, respiratory NK cytotoxic activity was increased after 7d skin TLR7 triggering. In contrast, lung histology and bronchoalveolar cell counts were not affected suggesting that skin TLR7 stimulation modulated respiratory leukocyte composition without inducing overt pulmonary inflammation. These data suggest the possibility to modulate respiratory leukocyte composition and respiratory cytokine responses against pathogens like Klebsiella pneumonia through skin administration of a clinically approved TLR7 ligand. Skin administration of synthetic TLR7 ligands may represent a novel, noninvasive means to modulate respiratory immunity.

Skin triggering of TLR7 modulates respiratory dendritic cells and natural killer cells - identification of a novel mode for manipulation of respiratory leukocytes

Objective: Dendritic cell (DC) maturation and activation in situ is a critical element of successful DC vaccination protocols whereas suppression of functional DC activation represents an important step in the development of immunoregulatory cellular therapy protocols. The TLR7 agonist imiquimod has been used successfully as novel topical immune adjuvant but the systemic use has been limited due to significant toxicity. We hypothesized that skin application of a synthetic TLR7 ligand may represent a novel means to modulate respiratory leukocytes. Methods: In a placebo-controlled experimental animal study we have used 7-colour flow cytometry to systematically analyze the modulation of murine respiratory leukocyte subsets after skin administration of the synthetic TLR7 ligand imiquimod. Results: Compared to placebo, skin administration of imiquimod markedly increased respiratory dendritic cells and innate lymphocytes within 24h, whereas total respiratory leukocytes, alveolar macrophages, T helper and killer lymphocyte numbers were not affected or reduced. DC subpopulation analyses revealed that elevation of respiratory DC was related to an increase of monocytic DC (CD11c+CD11b+MHC-classIIdimSiglecFneg,CD45+). Lymphocyte subpopulation analyses indicated a marked elevation of respiratory natural killer cells and a reduction of B cells. In contrast, bronchoalveolar cell counts and histopathology were not affected suggesting that skin TLR7 stimulation markedly modulated respiratory leukocyte composition without inducing a respiratory inflammatory response. Conclusion: These data suggest for the first time the possibility to modulate respiratory leukocyte subsets after skin administration of a clinically approved TLR7 ligand. Skin administration of synthetic TLR7 ligands may represent a novel and safe means to modulate respiratory immunity. DC vaccination trials targeting the respiratory system may be modulated by skin TLR7 triggering.

Sustained viral load reduction in treatment-naive HCV genotype 1 infected patients after therapeutic peptide vaccination

BackgroundNovel antivirals augment treatment efficacy in chronic HCV infection, to overcome limitations on safety profile alternative approaches are warranted. The effect of a therapeutic peptide vaccine on HCV viral load was investigated in treatment-naïve genotype 1 HCV patients. MethodsFifty patients received 8 intradermal IC41 vaccinations biweekly with topical application of the TLR7 agonist imiquimod (Group A). In Group B, 21 patients received a condensed schedule of 16 subcutaneous vaccinations weekly without imiquimod. ResultsAt Week 16 Group A (n=44) showed a statistically significant (p=0.0013) HCV viral load decline of 0.21log. 24 weeks after the last vaccination the viral load decreased by 0.47log (p<0.0001) in 34 subjects. This effect was more pronounced in 17 patients with high baseline HCV (>2×106IU/ml) with a 0.61log decline, which was statistically significant (p<0.02) starting two weeks after the third vaccination. No apparent effect on HCV viral load was observed in Group B (n=21). In Group A eight patients (24%) showed a viral load response defined as a decline of >0.8log. Overall, about 30–55% of patients showed T cell responses during the vaccination series and up to six months in both groups. No significant correlations between the HCV viral load decrease and T cell immune response were detected. ConclusionsThis is the first report on a significant antiviral effect of a peptide vaccine in HCV infected patients. Response kinetics with increased HCV RNA decline 24 weeks after the last IC41 vaccination is encouraging.

Skin Langerin+ Dendritic Cells Transport Intradermally Injected Anti–DEC-205 Antibodies but Are Not Essential for Subsequent Cytotoxic CD8+ T Cell Responses

Incorporation of Ags by dendritic cells (DCs) increases when Ags are targeted to endocytic receptors by mAbs. We have previously demonstrated in the mouse that mAbs against C-type lectins administered intradermally are taken up by epidermal Langerhans cells (LCs), dermal Langerin(neg) DCs, and dermal Langerin(+) DCs in situ. However, the relative contribution of these skin DC subsets to the induction of immune responses after Ag targeting has not been addressed in vivo. We show in this study that murine epidermal LCs and dermal DCs transport intradermally injected mAbs against the lectin receptor DEC-205/CD205 in vivo. Skin DCs targeted in situ with mAbs migrated through lymphatic vessels in steady state and inflammation. In the skin-draining lymph nodes, targeting mAbs were found in resident CD8α(+) DCs and in migrating skin DCs. More than 70% of targeted DCs expressed Langerin, including dermal Langerin(+) DCs and LCs. Numbers of targeted skin DCs in the nodes increased 2-3-fold when skin was topically inflamed by the TLR7 agonist imiquimod. Complete removal of the site where OVA-coupled anti-DEC-205 had been injected decreased endogenous cytotoxic responses against OVA peptide-loaded target cells by 40-50%. Surprisingly, selective ablation of all Langerin(+) skin DCs in Langerin-DTR knock-in mice did not affect such responses independently of the adjuvant chosen. Thus, in cutaneous immunization strategies where Ag is targeted to DCs, Langerin(+) skin DCs play a major role in transport of anti-DEC-205 mAb, although Langerin(neg) dermal DCs and CD8α(+) DCs are sufficient to subsequent CD8(+) T cell responses.

Menstrual Cycle Influences Toll-Like Receptor Responses

Background: Toll-like receptors (TLRs) are pattern recognition receptors that play an important role as mediators of innate immunity. Human studies have shown changes in endometrial TLR expression during the menstrual cycle and during pregnancy. Our objective was to measure peripheral TLR activity over the course of the menstrual cycle. Methods: We recruited 11 healthy females, and using ELISA we measured sex hormone levels and IL-1β, IL-6, IL-8 and TNF-α following stimulation of whole blood with different TLR agonists during follicular, and early and late luteal phases of the menstrual cycle. Results: During the follicular phase, we observed lower levels of IL-6 and TNF-α following stimulation with the TLR2 agonist HKLM when compared with the early luteal phase; lower levels of IL-1β, IL-6 and TNF-α following stimulation with the TLR4 agonist LPS, and lower levels of IL-1β and TNF-α following stimulation with the TLR5 agonist flagellin. Decreased IL-6 levels in the late compared to the early luteal phase were also observed following stimulation with the TLR5 agonist flagellin. Compared with the follicular phase, the late luteal phase of the cycle resulted in decreased levels of IL-1β and TNF-α following stimulation with the TLR1/TLR2 agonist Pam3CSK and the TLR6/TLR2 agonist FSL1, as well as decreased levels of TNF-α following stimulation with the TLR8 agonist ssRNA40. There were no differences in cytokine release across the menstrual cycle following stimulation with the TLR3 agonist polyriboinosinic polyribocytidylic acid, or the TLR7 agonist Imiquimod. Conclusion: This study is the first to demonstrate that TLR responsivity in peripheral blood fluctuates throughout the menstrual cycle.

Regulatory T Cells and IL-10 Independently Counterregulate Cytotoxic T Lymphocyte Responses Induced by Transcutaneous Immunization

BackgroundThe imidazoquinoline derivate imiquimod induces inflammatory responses and protection against transplanted tumors when applied to the skin in combination with a cognate peptide epitope (transcutaneous immunization, TCI). Here we investigated the role of regulatory T cells (Treg) and the suppressive cytokine IL-10 in restricting TCI-induced cytotoxic T lymphocyte (CTL) responses.Methodology/Principal FindingsTCI was performed with an ointment containing the TLR7 agonist imiquimod and a CTL epitope was applied to the depilated back skin of C57BL/6 mice. Using specific antibodies and FoxP3-diphteria toxin receptor transgenic (DEREG) mice, we interrogated inhibiting factors after TCI: by depleting FoxP3+ regulatory T cells we found that specific CTL-responses were greatly enhanced. Beyond this, in IL-10 deficient (IL-10-/-) mice or after blocking of IL-10 signalling with an IL-10 receptor specific antibody, the TCI induced CTL response is greatly enhanced indicating an important role for this cytokine in TCI. However, by transfer of Treg in IL-10-/- mice and the use of B cell deficient JHT-/- mice, we can exclude Treg and B cells as source of IL-10 in the setting of TCI.Conclusion/SignificanceWe identify Treg and IL-10 as two important and independently acting suppressors of CTL-responses induced by transcutaneous immunization. Advanced vaccination strategies inhibiting Treg function and IL-10 release may lead the development of effective vaccination protocols aiming at the induction of T cell responses suitable for the prophylaxis or treatment of persistent infections or tumors.

Glucocorticoids inhibit dendritic cell maturation induced by Toll-like receptor 7 and Toll-like receptor 8

GCs are widely prescribed to treat inflammatory disorders and autoimmune and allergic diseases. Their anti-inflammatory and immunosuppressive effects may be related, in part, to their ability to control the maturation and functions of DCs. Here, we report that GCs inhibit the maturation of human CD34-DCs induced by the TLR7 agonist imiquimod and the TLR8 agonist 3M-002. GCs down-regulate the expression of CD86, CD40, CD83, CCR7, and HLA-DR on DCs and inhibit IL-6 and IL-12p40 production by DCs following TLR7 and TLR8 stimulation. This inhibitory effect is abolished by RU486, suggesting a role for GR transcriptional activity. Our results also show that GCs do not affect TLR-mediated DNA-binding activity of NF-κBp65. We observe that GCs control the activation of JNK induced by TLR agonists, without affecting its upstream MKK4. However, p38MAPK activation is not affected by GCs. Concomitantly to JNK inhibition, we observe the induction of the DUSP MKP-1 but not of other DUSPs by GCs. However, although silencing of MKP-1 in DCs reverses GC-mediated JNK inhibition, no significant effect on GC-induced inhibition of DC maturation was evidenced. Our results show that GCs alter DC maturation in response to TLR7 or TLR8 through a mechanism involving GR transcriptional activity.

TLR7 and TLR9 Trigger Distinct Neuroinflammatory Responses in the CNS

Toll-like receptors (TLRs) 7 and 9 recognize nucleic acid determinants from viruses and bacteria and elicit the production of type I interferons and proinflammatory cytokines. TLR7 and TLR9 are similar regarding localization and signal transduction mechanisms. However, stimulation of these receptors has differing effects in modulating viral pathogenesis and in direct toxicity in the central nervous system (CNS). In the present study, we examined the potential of the TLR7 agonist imiquimod and the TLR9 agonist cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN) to induce neuroinflammation after intracerebroventricular inoculation. CpG-ODN induced a more robust inflammatory response than did imiquimod after inoculation into the CNS, with higher levels of several proinflammatory cytokines and chemokines. The increase in cytokines and chemokines correlated with breakdown of the blood-cerebrospinal fluid barrier and recruitment of peripheral cells to the CNS in CpG-ODN-inoculated mice. In contrast, TLR7 agonists induced a strong interferon β response in the CNS but only low levels of other cytokines. The difference in response to these agonists was not due to differences in distribution or longevity of the agonists but rather was correlated with cytokine production by choroid plexus cells. These results indicate that despite the high similarity of TLR7 and TLR9 in binding nucleic acids and inducing similar downstream signaling, the neuroinflammation response induced by these receptors differs dramatically due, at least in part, to activation of cells in the choroid plexus.

Abstract 747: Tumor vaccine adjuvant effect of TLR7 agonist Imiquimod is limited by inducing immusuppressive cells

Abstract Toll-like receptor (TLR) agonists are immune stimulators and have been studied as potential vaccine adjuvants with great interest. We evaluated the adjuvant effects of the TLR7 agonist imiquimod as compared to GM-CSF, which is widely used as a vaccine adjuvant in clinical trials. We found that both topical imiquimod and intradermal GM-CSF effectively induced the accumulation and activation of dentritic cells in draining lymph nodes and equally enhanced OVA specific CD4+ and CD8+ T cell responses in an OVA-tg mouse model. We further evaluated the efficacy of tumor prevention of imiquimod and GM-CSF in a neu-transgenic (neu-tg) mouse model of human breast cancer which over-expresses both neu and IGFBP2 tumor associated antigens. The neu-tg mice were immunized with a vaccine composed of three immunogenic IGFBP2 peptides prior to being inoculated with syngeneic MMC tumor cells. The tumor growth was significantly inhibited in the groups which received GM-CSF as an adjuvant, but not in imiquimod-treated groups. We questioned whether imiquimod induces immune suppression in response to tumor associated antigens and assessed the frequencies of myeloid-derived suppressive cells (MDSC) and T regulatory cells (Treg) in the splenocytes after three immunizations. We found that both CD11c+GR1+ MDSC and CD4+FOXP3+Treg cells were significantly increased in imiquimod-treated mice, but did not increase in GM-CSF-treated group. Serum levels of IL-10 were also significantly increased in imiquimod-treated, but not in GM-CSF-treated group. IFN-gamma secretion in response to IGFBP2 antigen, on the contrary, was remarkably enhanced in the GM-CSF-treated mice, but not in the imiquimod-treated mice as tested by an IFN-gamma ELISPOT assay. Thus, the TLR7 agonist imiquimod, as a vaccine adjuvant did not achieve a protective effect on breast cancer growth. The limited anti-tumor effect observed is associated with elevated levels of immunosuppressive MDSC and Treg cells. These results may have significant implications in the future development of imiquimod as a vaccine adjuvant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 747. doi:10.1158/1538-7445.AM2011-747

Abstract 748: TLR3 agonist Ampligen® as an adjuvant inhibits the growth of breast cancer

Abstract A potent adjuvant is the key for a successful vaccine against tumor antigens. We evaluated the adjuvant effect of a TLR3 agonist, Ampligen® on the growth of breast cancer in a transgenic mouse model. Ampligen® [rintatolimod; poly(I)·poly(C12,U)] is a synthetic mismatched double strand RNA that specifically stimulates TLR3 (Hemispherx Biopharma, Philadelphia, PA). A neu-tg mice [strain name, FVB/N-TgN (MMTVneu) 202Mul] over-expressing both neu and IGFBP-2 tumor associated antigens (Park KH et al CR 2008) were used for this study. Three IGFBP2 peptides were used as immunogen. The mice were vaccinated with the peptides and Ampligen® three times, 2 weeks apart, with PBS, peptides alone, and Ampligen® alone as controls. Syngeneic MMC tumor cells were injected into the mice two weeks after the last vaccine, and the growth of tumors was monitored afterwards. Three other immune modulators (GM-CSF, TLR7 agonist Imiquimod and TLR9 agonist CpG ODN 2395) were also used to evaluate the efficacy of Ampligen® and combination adjuvant approaches. Our results indicate that peptides alone did not mediate an anti-tumor effect. Although Ampligen® alone at high concentration (100ug) had a weak anti-tumor effect as compared to the control with 23% tumor inhibition, vaccination with IGFBP2 antigen and Ampligen® remarkably inhibited tumor growth by 60%. A dose response of Ampligen® was observed with 20ug as the optimal dose. The analysis of the levels of multiplex cytokines (IL-12p70, IL-1a, IL-1b, IL-2, IL-5, IL-10, IL-17, IFNg and TNFa) in serum two days after last vaccine showed that the level of IL-12p70 was increased in peptides/Ampligen® treated mice as compared to control (5.9±0.9 vs. 3.1±0.8 pg/ml;n=5; p=0.041) or other adjuvants. The serum levels of other cytokines were not statistically different. We compared the adjuvant effect of Ampligen® with GM-CSF, Imiquimod and ODN 2395. Besides Imiquimod, all these adjuvants significantly inhibited tumor growth as compared with peptides alone (p&amp;lt;0.002). Ampligen® was more effective than ODN2394 (p=0.02). The combination of Ampligen® with these other immunostimulators didn't further enhance the antitumor effect. In conclusion, Ampligen® as an adjuvant inhibited the growth of breast cancer with an increased level of Th1 cytokines and is a potent tumor vaccine adjuvant with the potential for clinical use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 748. doi:10.1158/1538-7445.AM2011-748