Ligand R848 Research Articles

Expression of TLR7/8 in canine sperm and evaluation of the effect of ligand R848 on the sorting of canine X/Y sperm

The aim of this study was to analyze the expression pattern of Toll receptor 7/8 (TLR7/8) in canine sperm, and explore the feasibility of using TLR7/8 ligand resiquimod(R848)to separate canine X and Y sperm. In this study, cellular immunofluorescence was used to analyze the expression of TLR7/8 in canine sperm, real-time fluorescence quantitative PCR was used to calculate the proportion of X sperm in the lower layer of the incubation solution with R848 to evaluate the sorting effect of R848 on canine X/Y sperm, and sperm quality detection system was used to analyze the effect of R848 on the motility of canine sperm. The mechanism of effect of R848 on canine sperm motility was analyzed by Western blot. The results showed that TLR8 was not expressed in all canine sperm, while TLR7 was expressed in all canine sperm and was localized in the head and tail of sperm. When 0.4 μM R848 was incubated with canine sperm for 1 h, the total motility, average path velocity (VAP), average straight-line velocity (VSL), and average curved-line velocity (VCL) of canine sperm were significantly decreased(P<0.05). There was no significant difference between the lower and upper layers of the R848 treatment group and the control group(P>0.05), and the proportion of X sperm was nearly half. The levels of NF-κB and GSK3α/β phosphorylation of sperm in R848 treatment group were significantly increased compared with control group(P<0.05). The above results showed that TLR7/8 was not differentially expressed in canine X and Y sperm. R848 could decrease the motility of canine spermatozoa and inhibit sperm motility by the GSK3α/ β-hexokinase pathway through the phosphorylation of NFκB and GSK3α/β, while could not separate X and Y spermatozoa. The method of sorting X/Y sperm based on TLR7/8 is not feasible for dogs.

In vitro characteristics of X- and Y-bearing ram spermatozoa sorted by bovine serum albumin (BSA) column and TLR7/8 ligand R848

The quality of the separated fractions in sex-sorted semen is very important for the success of the artificial insemination. This study aimed to evaluate some in vitro characteristics (DNA quantity, kinematic parameters and enzymes activity) of X- and Y-bearing ram spermatozoa sorted by bovine serum albumin (BSA) column and toll-like receptors (TLR)7/8 ligand R848. The ejaculates from six rams were collected by artificial vagina and subjected to a computer-assisted semen analysis (CASA). Total motility and percentage of the sperms with rapid and medium progressivity or non-progressivity in whole ejaculates and in X and Y fractions were analyzed. Activity of the enzymes ALP, GGT, CK, LDH and accumulation of lactate in the seminal plasma of ejaculates and in the environmental fluid of sexed spermatozoa were measured by biochemical analyzer. DNA was isolated from precipitated spermatozoa, and its quantity was measured. For both protocols the DNA mass from X-bearing fractions was higher, than from Y-bearing fractions. The high total motility of X- and Y-bearing spermatozoa as well as greater percent sperms with progressive motility were observed after use of BSA protocol. The application of TLR7/8 ligand R848 protocol led to reducing of Y-sperm motility and enhancement of non-progressivity in both fractions, which corresponded to the determined high amount of the extracellular lactate. For both methods, the significantly reduced activity of enzymes in the X and Y spermatozoa environmental fluids was established. Both protocols produce X- and Y-sperm fractions with satisfactory quality (over 80% total motility and over 50% rapid and medium progressive spermatozoa in each fraction).

Long-term in vivo vitamin D3 supplementation modulates bovine IL-1 and chemokine responses

Vitamin D deficiency at birth, followed by prolonged insufficiency in early life may predispose bovine calves to infection and disease. However, the effects of vitamin D levels on innate immunity are unclear due to the lack of long-term supplementation trials in vivo and reliable approaches for reproducibly assessing immune function. Here, a standardized whole blood immunophenotyping assay was used to compare innate immune responses to infection relevant ligands (LPS, Pam3CSK4 and R848) between Holstein–Friesian calves supplemented with vitamin D (n = 12) from birth until 7 months of age and control calves (n = 10) raised on an industry standard diet. Transcriptomic analysis in unstimulated whole blood cells revealed increased expression of type I interferons and chemokines in vitamin D supplemented calves, while IL-1 and inflammasome gene expression was decreased. In response to stimulation with the bacterial ligand LPS, supplemented calves had significantly increased expression of CASP1, CX3CR1, CAT, whereas STAT1 was decreased. Stimulation with the bacterial ligand Pam3CSK4 revealed increased expression of IL1A, IL1B and CAT genes; and decreased C5AR1 expression. In response to the viral ligand R848, STAT1 and S100A8 expression was significantly decreased. An increased IL-1 and inflammasome gene expression signature in vitamin D supplemented calves in response to LPS and Pam3CSK4 was also found, with ELISA confirming increased IL-1β protein production. In contrast, a decreased chemokine gene expression signature was found in response to R848 in supplemented animals, with decreased IL-8 protein expression exhibited in response to all PAMPs also found. These results demonstrated expression of several cytokine, chemokine and inflammasome genes were impacted by vitamin D supplementation in the first 7 months of life, with IL-8 expression particularly responsive to vitamin D. Overall, vitamin D supplementation induced differential innate immune responses of blood immune cells that could have important implications for disease susceptibility in cattle.

Impact of Selected Bacterial and Viral Toll-like Receptor Agonists on the Phenotype and Function of Camel Blood Neutrophils.

Innate recognition of pathogens depends on the interaction between microbial structures known as pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) in host cells. Toll-like receptors (TLR) are among the most important PRRs being expressed on and in a wide range of immune cell types. Studies on the interaction mechanisms between different pathogen species and the immune system of the dromedary camel are still scarce. The present study aimed to investigate the immunomodulatory effect of synthetic bacterial and viral TLR ligands on some phenotypic properties and selected functions of neutrophils purified from dromedary camel blood. Neutrophils were separated from camel blood (n = five animals) and were stimulated in vitro with the TLR ligands LPS, Pam3CSK4, R848 (Resiquimod), and Poly IC or were left without stimulation. Stimulation with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) was used as a positive control stimulation. Shape change, phagocytosis activity, ROS production, the expression of cell surface markers, and cell vitality were compared between stimulated and non-stimulated cells. With exception of the TLR3 agonist Poly IC, all TLR ligands used showed the potential to stimulate camel neutrophils resulting in increased cell size and the upregulation of CD18 and CD14 on their surface. Similarly, the phagocytosis activity of camel neutrophils was significantly improved after priming with all TLR ligands, except Poly IC, which, in contrast, resulted in a reduced percentage of phagocytosis-positive cells. In contrast to stimulation with PMA, which induced a significant ROS production in camel neutrophils, none of the TLR ligands used stimulated ROS generation in neutrophils. Only stimulation with Pam3CSK4 increased the expression of MHCII molecules on camel neutrophils, resulting in an expanded MHCIIhigh fraction within camel neutrophils. Our study indicates selective immunomodulating effects of TLR agonists on purified camel neutrophils without affecting their vitality.

Co-stimulation by TLR7/8 ligand R848 modulates IFN-γ production of porcine γδ T cells in a microenvironment-dependent manner

Gamma-Delta (γδ) T cells represent a prominent lymphocyte subset in pigs. Their role and function, however, remains largely unknown. Toll-like receptors (TLR) are key receptors for the recognition of pathogens, but so far, it is unknown if porcine γδ T cells express TLRs and therefore have the innate ability to recognize pathogens through pattern recognition receptors. In this study, we compared γδ T cells in different age groups of pigs and investigated the functional relevance of TLR7/8 expression. We found that the major γδ T cell phenotype shifts from CD2−CD8α−/dimCD27+ in young pigs to CD2+CD8αhighCD27− in 3-year-old pigs impacting their ability to produce IFN-γ upon cytokine and TLR stimulation. Furthermore, we report that stimulation with TLR7/8 ligand R848 increased IFN-γ production in purified γδ T cells upon co-stimulation with IL-2 and IL-12. However, the effect of R848 as a co-activator of γδ T cells was abrogated by the addition of monocytes or within PBMCs, suggesting that γδ T cells respond to multiple direct and indirect stimulations. Thus, our results indicate that γδ T cells express TLRs, are modulated by TLR7/8 ligand R848 and have subset-specific responses.

Anti - Inflammatory Activity of Quercetin and Kaempferol Is Limited to Tlr4 Stimulation in Monocytes

Flavonoids are known to exert anti-inflammatory activity but their mechanism at receptor level and their comparative potencies are less studied. Objective of the study is to evaluate, rank order few flavonoids for their cytokine inhibitory activity in monocytes (THP-1) and explore the inhibitory signature among toll-like receptors (TLRs). We tested flavonoids for their potential to inhibit LPS induced cytokines IL-6, TNF alpha in human monocyte cell line THP-1 and mouse macrophage cell line using Enzyme linked immunosorbent assay (ELISA) assays. Quercetin and Kaempferol reduced LPS induced cytokine secretion in monocyte and macrophage cell lines. Quercetin and Kaempferol demonstrated dose dependent inhibition of cytokines IL-6 and TNF alpha. Quercetin demonstrated IC50 of 6.9µM whereas Kaempferol demonstrated IC50 of 14.3µM in cytokine reduction. In THP-1, NF-kB activity is measured using secreted embryonic alkaline phosphatase (SEAP) reporter with different toll-like receptor activation. TLR4 stimulated NF-kB activity was inhibited by Quercetin and Kaempferol in THP-1 cell line, while these flavonoids did not impact signaling of other TLR ligands Pam3CSK4 (TLR1/2) FLS (TLR2/6), Flagellin (TLR5) and R848 (TLR7/8). I. Quercetin and Kaempferol modulate NF-kB activity when stimulated with TLR4, but not with TLR1/2, TLR2/6, TLR5 and TLR7/8 in Monocytes. Understanding the inhibition profile of flavonoids against Toll receptors will help in design better inhibitor which can alleviate inflammatory diseases

Genetic alteration of heparan sulfate in CD11c + immune cells inhibits inflammation and facilitates pathogen clearance during influenza A virus infection

Survival from influenza A virus (IAV) infection largely depends on an intricate balance between pathogen clearance and immunomodulation in the lung. We demonstrate that genetic alteration of the glycan heparan sulfate (HS) in CD11c + cells via Ndst1f/f CD11cCre + mutation, which inhibits HS sulfation in a major antigen presenting cell population, reduces lung inflammation by A/Puerto Rico/8/1934(H1N1) influenza in mice. Mutation was also characterized by a reduction in lung infiltration by CD4+ regulatory T (Treg) cells in the late infection/effector phase, 9 days post inoculation (p.i.), without significant differences in lung CD8 + T cells, or Treg cells at an earlier point (day 5) following infection. Induction of under-sulfated HS via Ndst1 silencing in a model dendritic cell line (DC2.4) resulted in up-regulated basal expression of the antiviral cytokine interferon β (IFN-β) relative to control. Stimulating cells with the TLR9 ligand CpG resulted in greater nuclear factor-κB (NFκB) phosphorylation in Ndst1 silenced DC2.4 cells. While stimulating cells with CpG also modestly increased IFN-β expression, this did not lead to significant increases in IFN-β protein production. In further IFN-β protein response studies using primary bone marrow DCs from Ndst1f/f CD11cCre + mutant and Cre− control mice, while trace IFN-β protein was detected in response to CpG, stimulation with the TLR7 ligand R848 resulted in robust IFN-β production, with significantly higher levels associated with DC Ndst1 mutation. In vivo, improved pathogen clearance in Ndst1f/f CD11cCre + mutant mice was suggested by reduced IAV AA5H nucleoprotein in lung examined in the late/effector phase. Earlier in the course of infection (day 5 p.i.), mean viral load, as measured by viral RNA, was not significantly different among genotypes. These findings point to novel regulatory roles for DC HS in innate and adaptive immunity during viral infection. This may have therapeutic potential and guide DC targeted HS engineering platforms in the setting of IAV or other respiratory viruses.

Sustained IL-4 priming of macrophages enhances the inflammatory response to TLR7/8 ligand R848.

Macrophages (Mϕ) are highly plastic, and can acquire a variety of functional phenotypes depending on the presence of different stimuli in their local environment. Mφ stimulated by interleukin (IL)-4 induce an alternative activation state and function as anti-inflammatory cells and promote tissue repair. However, there is overwhelming evidence that IL-4 can play a role in promoting inflammation. In asthma and allergic inflammation, IL-4 mediates proinflammatory responses that lead to tissue damage. Thus the effect of IL-4 on the outcome of the immune responses is greatly influenced by other cofactors and cytokines present in the microenvironment. R848 (resiquimod), a TLR7/8 agonist is a novel vaccine adjuvant, triggering a strong Th1-skewed response but its efficacy as a vaccine adjuvant shows variable results. It is not currently known whether the presence of IL-4 can dampen or enhance immunity in response to TLR7 agonists. In the present study, we sought to investigate the impact of IL-4-induced Mφ polarization on the outcome of R848 stimulation. The activation marker expression and production of cytokines were measured in murine spleen-derived Mφ. Protein expression levels of innate recognition molecules and transcription factors involved, including retinoic-acid inducible gene I, mitochondrial antiviral signaling protein, stimulator of interferon genes (STING), and IFN regulatory factors were evaluated in activated Mφ. These play a crucial role in the control of viral replication and optimal CD8+ T cell priming. We report that sustained priming with IL-4 alone promotes an antiviral response in Mφ, and enhances proinflammatory responses to R848 treatment. This highlights the need for better understanding of IL-4 proinflammatory functions and its potential use as a broad-acting antiviral in combination with R848 may be used in combination with other therapies to target the innate arm of immunity against emerging infections.

Treatment with a Toll-like Receptor 7 ligand evokes protective immunity against atherosclerosis in hypercholesterolaemic mice.

The interplay between innate and adaptive immunity is central in life-threatening clinical complications of atherosclerosis such as myocardial infarction and stroke. The specific mechanisms involved and their protective versus detrimental effects in the disease process remain poorly understood. We have previously shown that higher levels of Toll-like receptor 7 (TLR7) expression in human atherosclerotic lesions are correlated with better patient outcome. In this study, we explored whether TLR7 activation can ameliorate disease in experimental atherosclerosis in mice. Apolipoprotein E deficient mice (Apoe-/- ) with established disease were injected for five weeks intraperitoneally with the TLR7 ligand R848. Local effects were evaluated by characterization of the lesion. Systemic effects of the treatment were investigated by immune composition analysis in the spleen and plasma measurements. The in vivo treatment arrested lesion progression in the aorta. We also detected expansion of marginal zone B cells and Treg in the spleen together with increased plasma IgM antibodies against oxidized low-density lipoprotein (oxLDL) and reduced plasma cholesterol levels. These changes were accompanied by increased accumulation of IgM antibodies, decreased necrosis and fewer apoptotic cells in atherosclerotic lesions. Our findings show that TLR7 stimulation could ameliorate atherosclerotic lesion burden and reduce plasma cholesterol in Apoe-/- mice. TLR7 stimulation was associated with an atheroprotective B-cell and Treg response, which may have systemic and local effects within lesions that could prevent arterial lipid accumulation and inflammation.

Elotuzumab Enhances the Th2-Mediated Immune Response of Dendritic Cell Induced By Immunomodulatory Drugs (IMiDs)

Background: Elotuzumab, a humanized IgG1 monoclonal antibody targeting SLAMF7, is useful for the treatment of Relapsed or Refractory multiple myeloma (RRMM) in combination with Lenalidomide (LEN). However, cellular and molecular mechanisms underlying the immunomodulatory effects of elotuzumab still remain largely unclear. We have previously reported that LEN displays immunopotentiating activity that enhances Th2-mediated response at dendritic cell (DC) phase as upstream immune cascade associated with humoral immunity. DCs are pivotal cells in the sense of orchestrating both cell-mediated (linking with Th1) and humoral (linking with Th2) immunity as masters of the immune system. Series of analyses have clarified myeloid DCs (mDCs) play an important role in allergic immune response by the induction of Th2 response. Here, we focused on the effects of elotuzumab in combination with LEN on the function of human mDCs. Methods: Purified blood human CD11+ mDCs from healthy adult volunteers using cell sorting were cultured and analyzed by flow cytometry and ELISA. Serum were obtained from 16 MM patients with before and after elotuzumab therapy. This study was approved by the Institutional Review Board of Kansai Medical University. Results: We found that surface expression of SLAMF7 on mDCs was upregulated in response to Th2-inducing cytokine, thymic stromal lymphopoietin (TSLP) and the expression level was higher in response to TSLP than in response to toll-like receptor ligand R848. Elotuzumab at clinical in vivo plasma concentration of 30 to 300 µg/ml did not affect mDC survival and their CD86 and OX40-ligand expression when stimulated with 0.3 µM LEN and/or TSLP for 24 h. LEN enhanced TSLP-mediated Th2-recruiting chemokine CCL17/TARC from mDCs which functions as chemoattractant for memory Th2 cells and contribute to allergy and humoral immune responses, and elotuzumab significantly enhanced the LEN-mediated production of CCL17/TARC (TSLP+LEN as control vs. TSLP+LEN+100 µg/ml elotuzumab; 1.23 fold increase: p=0.003, and control vs. TSLP+LEN+300 µg/ml elotuzumab; 1.38 fold increase: p=0.038). This finding suggest elotuzumab enhances Th2-mediated immune profile at upstream phase of humoral immunity. In addition, serum CCL17 levels were analyzed in RRMM patients before and after 3 cycle elotuzumab administration (n=16). We found, serum CCL17 levels after elotuzumab administration were significantly higher compared with those before elotuzumab treatment (after; 1512 ± 459 pg/ml vs. before; 402.2 ± 87.4 pg/ml: p = 0.013). Conclusion: MM involves an element of humoral immune dysfunction. Immune status is important for the prognosis of MM, and clinical outcome can be improved by the recovery of immune status. In this context, our data showing the enhancement of Th2-mediated response by elotuzumab provide a plausible explanation for the observed clinical benefit of this antibody-drug in MM. This function of elotuzumab seems to be relevant to the treatment of MM patients under humoral immune dysfunction. Based on our data in focusing on DCs in the immune system, elotuzumab and IMiDs could function as immunostimulators of humoral immunity via mDCs, and this finding elucidated an additional cellular target of elotuzumab. Disclosures Ito: Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding.

Eicosanoid Metabolites Associated with Type-1 Interferon Production from Plasmacytoid Dendritic Cells Stimulated with Toll-like Receptors (P19-012-19)

ObjectivesCommon among several autoimmune diseases is a prominent interferon (IFN) signature caused by aberrant IFN-α production from plasmacytoid dendritic cells (pDC). This uncontrolled response occurs when pDCs are activated through endosomal toll-like receptors (TLR). Within the endosomal compartment, TLR 7/8 and 9 are stimulated through nuclear immune complexes. The objective of this work was to determine the eicosanoids associated with IFN-α production upon TLR stimulation of murine bone marrow derived dendritic cells. MethodsFemale C57BL/6Ncrl mouse bone marrow was cultured in FLT3-ligand (FMS-like tyrosine kinase 3) for 8 days to generate dendritic cell subsets. On day 8, TLR ligands were added. Supernatant was analyzed for eicosanoids by liquid chromatography- mass spectrometry and cell pellets were analyzed for phospholipid fatty acid content by gas chromatography- mass spectrometry and IFN-α by ELISA. ResultsMurine pDCs stimulated with TLR 7 ligand Poly U (GU-rich ssRNA40) and TLR 3 ligand Poly I: C (polyinosinic-polycytidylic acid) produced no observable IFN-α production. Upon stimulation with TLR 9 ligand ODN1585 CpG and TLR 8 ligand R848 (resiquimod), IFN-α was significantly increased with both TLR agonists as compared to unstimulated no target samples. Targeted lipidomics analysis of eicosanoids showed that in cultures stimulated with TLR8 ligand and TLR9 ligand, pro-inflammatory eicosanoid production of prostaglandin E2 and leukotriene B4 were increased. Phospholipid analysis revealed high levels of arachidonic acid and low eicosapentaenoic acid levels within the phospholipid membranes. ConclusionsUnderstanding the role of fatty acids associated with IFN-α production in pDCs may provide therapeutic and mechanistic targets for autoimmune diseases with an IFN signature. Funding SourcesFunding for this project was provided by university internal funds.

Lupus-Associated Immune Complexes Activate Human Neutrophils in an FcγRIIA-Dependent but TLR-Independent Response.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of autoantibodies against nucleic acids and nucleoproteins. Anti-dsDNA Abs are considered a hallmark of SLE, and previous studies have indicated that nucleic acid-containing immune complexes (ICs) induce B cell and dendritic cell activation in a TLR-dependent process. How ICs containing nucleic acids affect neutrophil function has not been well investigated. In this study, we report that nucleic acid-containing ICs derived from the sera of SLE patients induce human and mouse neutrophil activation through TLR-independent mechanisms. Soluble ICs containing Sm/RNP, an RNA Ag, activate human neutrophils to produce reactive oxygen species (ROS) and IL-8. In contrast, ICs containing DNA have to be immobilized to efficiently activate neutrophils. We found that deleting TLR7 or TLR9, the receptors for RNA and DNA, had no effect on mouse neutrophil activation induced by RNA-containing and immobilized DNA-containing ICs. Binding of ICs are mediated through FcγRIIA and FcγRIIIB. However, neutrophil activation induced by RNA- and DNA-containing ICs requires FcγRIIA, as blocking FcγRIIA inhibited ROS release from neutrophils. RNA-containing ICs induce calcium flux, whereas TLR7/8 ligand R848 do not. Surprisingly, chloroquine inhibits calcium flux induced by RNA-containing ICs, suggesting that this lesser known function of chloroquine is involved in the neutrophil activation induced by ICs. These data indicate the SLE-derived ICs activate neutrophils to release ROS and chemokines in an FcγRIIA-dependent and TLR7- and TLR9-independent manner that likely contributes to local tissue inflammation and damage.

Aberrant Cytokine Production in Myelofibrosis Is Not Rectified By Ruxolitinib and Is Differentially Sensitive to Inhibition of JAK/STAT, MAP Kinase, and NFκB Signaling

Inflammatory cytokine production is characteristic of myeloproliferative neoplasms including myelofibrosis (MF). Clonal dominance and bone marrow fibrosis have been attributed in part to cytokine overproduction. Elevated plasma levels of several cytokines have been associated with decreased survival in MF patients; notably IL-8/CXCL8, which has been associated with transformation to secondary acute myeloid leukemia (sAML; Tefferi et al., 2011, J. Clin. Oncol.).In MF patients, JAK2 inhibition with ruxolitinib ameliorates constitutional symptoms and splenomegaly; but improvements in anemia, fibrosis, and malignant clonal burden are infrequent, and survival benefit is modest. We have measured blood plasma cytokines in MF patients prior to and on treatment with ruxolitinib, versus healthy controls and sAML patients. Cytokines significantly elevated in MF patients versus controls (VEGF, TNF, IL-6, IL-10, IL-16) showed minimal differences in pre-versus-on ruxolitinib levels. Indeed, both pre- and on ruxolitinib cytokine levels in MF were similar to those observed in sAML. Therefore, overall cytokine-producing activity in MF is of similar magnitude to that in leukemic hematopoiesis, and persists despite ruxolitinib treatment.To identify cell populations responsible for cytokine production, MF blood cells were studied using mass cytometry (CyTOF). In MF versus healthy control samples, 14/15 cytokines measured were found to be constitutively overproduced in myeloid cell populations. IL-8 was highly expressed in CD34+ hematopoietic stem and progenitor cells (HSPC), which are prevalent in MF blood. The prognostic impact of IL-8 may therefore reflect overproduction of phenotypic HSPC, which could indicate incipient transformation to sAML. For the majority of MF overproduced cytokines, however, the largest source population was monocytes. This indicates that cytokines produced by monocytes may impact disease-propagating HSPC in a non-cell-autonomous fashion.To evaluate mechanisms driving cytokine overproduction, MF patient and control samples were exposed to 4-hour incubations with TPO, TNF, and/or TLR ligands PAM3CSK4 and R848. These compounds activate either JAK/STAT or NFκB signaling. The majority of cytokines elevated in MF exhibited hypersensitivity to these agents in MF versus normal monocytes. TPO induced cytokines in monocytes via its receptor MPL, which was expressed in a subset of both normal and MF monocytes. Cytokine induction by TPO was observed in normal and MF monocytes isolated by flow sorting (CD14+CD34-CD61-), confirming that MPL and JAK2 can induce cytokine overproduction in monocytes cell-autonomously. TPO induced cytokine production in monocytes could be suppressed either by ruxolitinib or pevonedistat, a NEDD8-activating enzyme inhibitor that blocks NFκB activation. This implies that both JAK2 and NFκB signaling pathways must be intact for maximal cytokine induction downstream of MPL.In contrast to TPO induced cytokine production, basal constitutively elevated cytokine production in MF monocytes was not consistently reduced by ruxolitinib. Among the TPO/TNF/TLR inducible cytokines, a subset (including TNF, IL-6, IL-8, IL-10) was minimally sensitive to ruxolitinib. All TPO/TNF/TLR-sensitive cytokines, however, were at least partially sensitive to pharmacologic inhibition of NFκB and/or MAP kinase signaling, as observed from incubation with pevonedistat, trametinib (MEK inhibitor), VX-745 (p38 MAPK inhibitor), and/or JNKi8 (JNK inhibitor). In contrast, these inhibitors did not suppress basal production of non-TPO-inducible cytokines (e.g. TGF-β, IFNγ), which are also overproduced in MF.These results indicate that overproduction of one set of cytokines in MF is driven by a combination of JAK/STAT, NFκB, and MAP kinase signaling; while that of another set is not directly driven by any of these pathways. Cytokines whose production is refractory to ruxolitinib may underlie features of MF that resist ruxolitinib therapy. A combined inhibitory approach may provide therapeutic benefit beyond that observed with ruxolitinib. Based on this hypothesis, our group has initiated a Phase 1 clinical trial combining pevonedistat with ruxolitinib for MF treatment (NCT03386214). Further results will indicate whether inhibition of NFκB confers an additional benefit to that observed from ruxolitinib alone. DisclosuresZhou:Incyte: Speakers Bureau. Oh:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Takeda: Research Funding; CTI Biopharma: Research Funding; Janssen: Research Funding.

The TLR7 ligand R848 prevents mouse graft-versus-host disease and cooperates with anti-interleukin-27 antibody for maximal protection and regulatory T-cell upregulation.

In spite of considerable therapeutic progress, acute graft-versus-host disease still limits allogeneic hematopoietic cell transplantation. We recently reported that mouse infection with nidovirus lactate dehydrogenase elevating virus impairs disease in non-conditioned B6D2F1 recipients of parental B6 spleen cells. As this virus activates TLR7, we tested a pharmacological TLR7 ligand, R848, in this model and observed complete survival if donor and recipients were treated before transplantation. Mixed lymphocyte culture performed 48 h after R848-treatment of normal mice demonstrated that both T-cell allo-responsiveness and antigen presentation by CD11b+ and CD8α+ dendritic cells were inhibited. These inhibitions were dependent on IFNAR-1 signaling. In the B6 to B6D2F1 transplantation model, R848 decelerated, but did not abrogate, donor T-cell implantation and activation. However, it decreased interferon-gamma, tumor necrosis factor-alpha and interleukin-27 while upregulating active transforming growth factor-beta 1 plasma levels. In addition, donor and recipient Foxp3+ regulatory T-cell numbers were increased in recipient mice and their elimination compromised disease prevention. R848 also strongly improved survival of lethally irradiated BALB/c recipients of B6 hematopoietic cells and this also correlated with an upregulation of CD4 and CD8 Foxp3+ regulatory T cells that could be further increased by inhibition of interleukin-27. The combination of anti-interleukin-27p28 mono -clonal antibody and R848 showed strong synergy in preventing disease in the B6 to B6D2F1 transplantation model when recipients were sublethally irradiated and this also correlated with upregulation of regulatory T cells. We conclude that R848 modulates multiple aspects of graft-versus-host disease and offers potential for safe allogeneic bone marrow transplantation that can be further optimized by inhibition of interleukin-27.

Hsa-miR-99b/let-7e/miR-125a Cluster Regulates Pathogen Recognition Receptor-Stimulated Suppressive Antigen-Presenting Cells.

Antigen-presenting cells (APCs) regulate the balance of our immune response toward microbes. Whereas immunogenic APCs boost inflammation and activate lymphocytes, the highly plastic cells can switch into a tolerogenic/suppressive phenotype that dampens and resolves the response. Thereby the initially mediated inflammation seems to prime the switch of APCs while the strength of activation determines the grade of the suppressive phenotype. Recently, we showed that pathogen recognition receptor-mediated pro-inflammatory cytokines reprogram differentiating human blood monocytes in vitro toward an immunosuppressive phenotype through prolonged activation of signal transducer and activator of transcription (STAT) 3. The TLR7/8 ligand R848 (Resiquimod) triggers the high release of cytokines from GM-CSF/IL-4-treated monocytes. These cytokines subsequently upregulate T cell suppressive factors, such as programmed death-ligand 1 (PD-L1) and indolamin-2,3-dioxygenase (IDO) through cytokine receptor-mediated STAT3 activation. Here, we reveal an essential role for the microRNA (miR, miRNA) hsa-miR-99b/let-7e/miR-125a cluster in stabilizing the suppressive phenotype of R848-stimulated APCs on different levels. On the one hand, the miR cluster boosts R848-stimulated cytokine production through regulation of MAPkinase inhibitor Tribbles pseudokinase 2, thereby enhancing cytokine-stimulated activation of STAT3. One the other hand, the STAT3 inhibitor suppressor of cytokine signaling-1 is targeted by the miR cluster, stabilizing the STAT3-induced expression of immunosuppressive factors PD-L1 and IDO. Finally, hsa-miR-99b/let-7e/miR-125a cluster regulates generation of the suppressive tryptophan (Trp) metabolite kynurenine by targeting the tryptophanyl-tRNA synthetase WARS, the direct competitor of IDO in terms of availability of Trp. In summary, our results reveal the hsa-miR-99b/let-7e/miR-125a cluster as an important player in the concerted combination of mechanisms that stabilizes STAT3 activity and thus regulate R848-stimulated suppressive APCs.

Quantitative and functional alterations of 6-sulfo LacNac dendritic cells in multiple myeloma

ABSTRACTMultiple myeloma (MM) results from expansion of abnormal plasma cells in the bone marrow (BM). Previous studies have shown that monocytes play a crucial role in MM pathophysiology. A 6-sulfo LacNAc-expressing population of dendritic cells (Slan-DCs) that overlaps with intermediate and non-classical monocytes in terms of phenotype has been described. Slan-DCs represent a circulating and tissue proinflammatory myeloid population which has been shown to play a role in different cancer contexts, and which exhibits a remarkable plasticity. Herein, we studied Slan-DCs from the BM and blood of MM patients. We performed quantitative and functional analyses of these cells from 54 patients with newly diagnosed, symptomatic MM, 21 patients with MGUS and 24 responding MM patients. We found that circulating Slan-DCs were significantly decreased in MM patients as compared to those of healthy donors or patients with MGUS, while CD14+CD16+ intermediate monocytes accumulate in the BM. Moreover, after activation with TLR7/8 ligand R848, IL-12-producing Slan-DCs from the BM or peripheral blood from MM patients were decreased as compared with healthy donors. We show that MM cell lines or MM cells isolated from patients at diagnosis were able to inhibit the production of IL-12 by Slan-DCs, as well as to shift the phenotype of Slan-DCs towards an intermediate monocyte-like phenotype. Finally, Slan-DCs that have been cultured with MM cells reduced their capacity to induce T cell proliferation and Th1 polarization. We conclude that Slan-DCs represent previously unrecognized players in MM development and may represent a therapeutic target.

Human CD141+ Dendritic Cell and CD1c+ Dendritic Cell Undergo Concordant Early Genetic Programming after Activation in Humanized Mice In Vivo.

Human immune cell subsets develop in immunodeficient mice following reconstitution with human CD34+ hematopoietic stem cells. These “humanized” mice are useful models to study human immunology and human-tropic infections, autoimmunity, and cancer. However, some human immune cell subsets are unable to fully develop or acquire full functional capacity due to a lack of cross-reactivity of many growth factors and cytokines between species. Conventional dendritic cells (cDCs) in mice are categorized into cDC1, which mediate T helper (Th)1 and CD8+ T cell responses, and cDC2, which mediate Th2 and Th17 responses. The likely human equivalents are CD141+ DC and CD1c+ DC subsets for mouse cDC1 and cDC2, respectively, but the extent of any interspecies differences is poorly characterized. Here, we exploit the fact that human CD141+ DC and CD1c+ DC develop in humanized mice, to further explore their equivalency in vivo. Global transcriptome analysis of CD141+ DC and CD1c+ DC isolated from humanized mice demonstrated that they closely resemble those in human blood. Activation of DC subsets in vivo, with the TLR3 ligand poly I:C, and the TLR7/8 ligand R848 revealed that a core panel of genes consistent with DC maturation status were upregulated by both subsets. R848 specifically upregulated genes associated with Th17 responses by CD1c+ DC, while poly I:C upregulated IFN-λ genes specifically by CD141+ DC. MYCL expression, known to be essential for CD8+ T cell priming by mouse DC, was specifically induced in CD141+ DC after activation. Concomitantly, CD141+ DC were superior to CD1c+ DC in their ability to prime naïve antigen-specific CD8+ T cells. Thus, CD141+ DC and CD1c+ DC share a similar activation profiles in vivo but also have induce unique signatures that support specialized roles in CD8+ T cell priming and Th17 responses, respectively. In combination, these data demonstrate that humanized mice provide an attractive and tractable model to study human DC in vitro and in vivo.

Abstract 3659: WT1 and PRAME mRNA transfected TLR 7/8-polarized fast DC vaccines in AML patients mount specific immune responses and impact progression free survival

Abstract Patients diagnosed with acute myeloid leukemia (AML) may not be eligible for curative intensive treatment due to co-morbidity factors or age. Here we report results of 5 AML patients in morphological remission after incomplete induction/consolidation chemotherapy treated with dendritic cells (DCs) targeting WT-1 and PRAME. DCs were produced as described previously (Subklewe et al 2014), using a maturation cocktail containing the TLR 7/8 ligand R848. These DCs show a polarized release of IL-12p70 combined with low IL-10 upon stimulation. 2.5 or 5E+6 DCs per antigen were injected intradermally once weekly for 4 weeks (wks), in wk 6 and thereafter at monthly intervals. Blood and bone marrow (BM) samples were collected at regular intervals. Minimal residual disease (MRD) was measured in BM by quantitative PCR of WT-1 and PRAME expression and by morphology. Immune responses were assessed by analysis of intracellular interferon gamma expression or proliferation following stimulation with peptides spanning WT-1, PRAME, hTERT and survivin or after stimulation with autologous WT-1 and PRAME DCs. A 57 year old woman with intermediate risk M4 AML was vaccinated over 22 months after chemotherapy. Five weeks after start of vaccination she mounted strong CD8 responses against PRAME combined with an increase in hTERT response, suggesting epitope spreading. WT-1 signals in BM showed low positive levels throughout vaccination, but she remains in morphological remission 33 months after end of chemotherapy (EoC). A 50 year old man with M2 intermediate risk AML, initially not eligible for BM transplantation, showed specific immune responses against WT-1 during 10 months of vaccination. Due to Bell’s Palsy he was treated with cortisone which immediately reduced the vaccine effect, accompanied by increase of blasts in the bone marrow. Following new induction therapy he received BM transplantation and is currently in CR. A 68 year old woman with M1 intermediate risk AML is under vaccination for 24 months. WT-1 signals in BM continue to be slightly elevated without any sign of morphological relapse. CD4 responses and low CD8 responses are detected against WT-1, PRAME and hTERT. A 73 year old woman with M1 good risk AML relapsed after 6 months of DC vaccination without mounting specific immune responses. DC treatment was combined with 5-Azacytidine without any immunological and clinical effects. A 59 year old woman with good risk AML has been vaccinated for 14 months and is in remission for 21 months since EoC. WT-1 in BM remains slightly elevated whilst the initially positive PRAME signal is negative. Our results show that in 4 out of 5 AML patients fast TLR-polarized DC vaccination induced or supported specific T cell responses. 3 patients continue to be in CR after 21, 25 and 33 months respectively, following suboptimal primary chemotherapy. Immune responses are under investigation and will be presented. Citation Format: Iris Bigalke, Guri Solum, Dag Josefsen, Yngvar Fløisand, Kirsti Hønnåshagen, Lisbeth Skoge, Signe Spetalen, Stein Sæbøe-Larssen, Dolores J. Schendel, Gunnar Kvalheim. WT1 and PRAME mRNA transfected TLR 7/8-polarized fast DC vaccines in AML patients mount specific immune responses and impact progression free survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3659. doi:10.1158/1538-7445.AM2017-3659

Enhanced stimulation of human tumor-specific T cells by dendritic cells matured in the presence of interferon-\u03b3 and multiple toll-like receptor agonists

Dendritic cell (DC) vaccines have been demonstrated to elicit immunological responses in numerous cancer immunotherapy trials. However, long-lasting clinical effects are infrequent. We therefore sought to establish a protocol to generate DC with greater immunostimulatory capacity. Immature DC were generated from healthy donor monocytes by culturing in the presence of IL-4 and GM-CSF and were further differentiated into mature DC by the addition of cocktails containing different cytokines and toll-like receptor (TLR) agonists. Overall, addition of IFNγ and the TLR7/8 agonist R848 during maturation was essential for the production of high levels of IL-12p70 which was further augmented by adding the TLR3 agonist poly I:C. In addition, the DC matured with IFNγ, R848, and poly I:C also induced upregulation of several other pro-inflammatory and Th1-skewing cytokines/chemokines, co-stimulatory receptors, and the chemokine receptor CCR7. For most cytokines and chemokines the production was even further potentiated by addition of the TLR4 agonist LPS. Concurrently, upregulation of the anti-inflammatory cytokine IL-10 was modest. Most importantly, DC matured with IFNγ, R848, and poly I:C had the ability to activate IFNγ production in allogeneic T cells and this was further enhanced by adding LPS to the cocktail. Furthermore, epitope-specific stimulation of TCR-transduced T cells by peptide- or whole tumor lysate-loaded DC was efficiently stimulated only by DC matured in the full maturation cocktail containing IFNγ and the three TLR ligands R848, poly I:C, and LPS. We suggest that this cocktail is used for future clinical trials of anti-cancer DC vaccines.

Modulation of the energy metabolism inhibits plasmacytoid dendritic cell activation and delays onset of autoantibody production in murine models of systemic lupus erythematosus

Abstract Despite the heterogeneity of systemic lupus erythematosus (SLE) clinical manifestations, an increased expression of type I interferon (IFN) regulated genes, termed the interferon signature, has been reported in most SLE patients. Long recognized to facilitate the progression of SLE, Type I IFNs can be produced in small amounts by most cell types, but the plasmacytoid dendritic cells (pDCs) are the primary producers. Since murine models of SLE have shown that depletion of pDCs stop the onset and progression of autoimmunity, we speculated that it is important to suppress pDC activities that go beyond the production of type I IFNs. We have confirmed that changes in fatty acid metabolism are essential for pDC activation. Using inhibitors of fatty acid oxidation, oxidative phosphorylation, and glycolysis, we have found that metabolic modulation prevents pDC activation upon stimulation with TLR ligands CpG and R848. This metabolic inhibition did not affect pDC viability. Moreover, in vivo treatment of mice with metabolic inhibitors delayed the onset of autoantibody production in murine models of SLE. Indeed, the inhibition of energy metabolism strongly reduced the production of anti-dsDNA and antichromatin autoanti-bodies in i) a model of lupus induced by Graft versus Host Disease (GvHD) and ii) in lupus-prone mice accelerated by TLR stimulation. Therefore, our findings suggest that the in vivo suppression of the major producers of type I IFN through inhibition of energy metabolism may therapeutically downregulate the production of autoantibodies in murine lupus. Our data raises the possibility of manipulating cellular metabolic pathways to regulate the detrimental type I IFN effects in SLE.