In-silico formulation of a next-generation polyvalent vaccine against multiple strains of monkeypox virus and other related poxviruses.

Mpox (formerly known as monkeypox) virus and some related poxviruses including smallpox virus pose a significant threat to public health, and effective prevention and treatment strategies are needed. This study utilized a reverse vaccinology approach to retrieve conserved epitopes for monkeypox virus and construct a vaccine that could provide cross-protection against related viruses with similar antigenic properties. The selected virulent proteins of monkeypox virus, MPXVgp165, and Virion core protein P4a, were subjected to epitope mapping for vaccine construction. Two vaccines were constructed using selected T cell epitopes and B cell epitopes with PADRE and human beta-defensins adjuvants conjugated in the vaccine sequence. Both constructs were found to be highly antigenic, non-allergenic, nontoxic, and soluble, suggesting their potential to generate an adequate immune response and be safe for humans. Vaccine construct 1 was selected for molecular dynamic simulation studies. The simulation studies revealed that the TLR8-vaccine complex was more stable than the TLR3-vaccine complex. The lower RMSD and RMSF values of the TLR8 bound vaccine compared to the TLR3 bound vaccine suggested better stability and consistency of hydrogen bonds. The Rg values of the vaccine chain bound to TLR8 indicated overall stability, whereas the vaccine chain bound to TLR3 showed deviations throughout the simulation. These results suggest that the constructed vaccine could be a potential preventive measure against monkeypox and related viruses however, further experimental validation is required to confirm these findings.

CXCL4 synergizes with TLR8 for TBK1-IRF5 activation, epigenomic remodeling and inflammatory response in human monocytes.

Regulation of endosomal Toll-like receptor (TLR) responses by the chemokine CXCL4 is implicated in inflammatory and fibrotic diseases, with CXCL4 proposed to potentiate TLR responses by binding to nucleic acid TLR ligands and facilitating their endosomal delivery. Here we report that in human monocytes/macrophages, CXCL4 initiates signaling cascades and downstream epigenomic reprogramming that change the profile of the TLR8 response by selectively amplifying inflammatory gene transcription and interleukin (IL)-1ß production, while partially attenuating the interferon response. Mechanistically, costimulation by CXCL4 and TLR8 synergistically activates TBK1 and IKKε, repurposes these kinases towards an inflammatory response via coupling with IRF5, and activates the NLRP3 inflammasome. CXCL4 signaling, in a cooperative and synergistic manner with TLR8, induces chromatin remodeling and activates de novo enhancers associated with inflammatory genes. Our findings thus identify new regulatory mechanisms of TLR responses relevant for cytokine storm, and suggest targeting the TBK1-IKKε-IRF5 axis may be beneficial in inflammatory diseases.

TLR8/TLR7 dysregulation due to a novel TLR8 mutation causes severe autoimmune hemolytic anemia and autoinflammation in identical twins.

Our study presents a novel germline c.1715G>T (p.G572V) mutation in the gene encoding Toll-like receptor 8 (TLR8) causing an autoimmune and autoinflammatory disorder in a family with monozygotic male twins, who suffer from severe autoimmune hemolytic anemia worsening with infections, and autoinflammation presenting as fevers, enteritis, arthritis, and CNS vasculitis. The pathogenicity of the mutation was confirmed by in vitro assays on transfected cell lines and primary cells. The p.G572V mutation causes impaired stability of the TLR8 protein, cross-reactivity to TLR7 ligands and reduced ability of TLR8 to attenuate TLR7 signaling. This imbalance toward TLR7-dependent signaling leads to increased pro-inflammatory responses, such as nuclear factor-κB (NF-κB) activation and production of pro-inflammatory cytokines IL-1ß, IL-6, and TNFα. This unique TLR8 mutation with partial TLR8 protein loss and hyperinflammatory phenotype mediated by TLR7 ligands represents a novel inborn error of immunity with childhood-onset and a good response to TLR7 inhibition.

Photoactivation of Innate Immunity Receptor TLR8 in Live Mammalian Cells by Genetic Encoding of Photocaged Tyrosine.

The effectiveness of innate immune responses relies on an intricate balance between activation and regulation. TLR8, a member of the Toll-like receptor (TLR) family, plays a fundamental role in host defense by sensing viral single-stranded RNAs (ssRNAs). However, the molecular recognition and regulatory mechanism of TLR8 is not fully understood, especially in a whole-cell environment. Here, we engineer the first light-controllable TLR8 model by genetically encoding a photocaged tyrosine, NBY, into specific sites of TLR8. In the caged forms, the activity of TLR8 is masked but can be restored upon decaging by exposure to UV light. To explain the mechanism clearly, we divide the sites with light responsiveness into three groups. They can separately block the ligands that bind to the pockets of TLR8, change the interaction modes between two TLR8 protomers, and interfere with the interactions between TLR8 cytosolic domains with its downstream adaptor. Specifically, we use this chemical caging strategy to probe and evaluate the function of several tyrosine sites located at the interface of TLR8 homodimers with a previously unknown regulatory mode, which may provide a new strategy for TLR8 modulator development. Effects on downstream signaling pathways are monitored at the transcriptional and translational levels in various cell lines. By photoactivating specific cells within a larger population, this powerful tool can provide novel mechanistic insights, with potential in biotechnological and pharmaceutical applications.

TLR activation, immune response and viral protection elicited in cattle by a commercial vaccine against Bovine Herpesvirus-1.

The innate and acquired immune response induced by a commercial inactivated vaccine against Bovine Herpesvirus-1 (BoHV-1) and protection conferred against the virus were analyzed in cattle. Vaccination induced high levels of BoHV-1 antibodies at 30, 60, and 90 days post-vaccination (dpv). IgG1 and IgG2 isotypes were detected at 90 dpv, as well as virus-neutralizing antibodies. An increase of anti-BoHV-1 IgG1 in nasal swabs was detected 6 days post-challenge in vaccinated animals. After viral challenge, lower virus excretion and lower clinical score were observed in vaccinated as compared to unvaccinated animals, as well as BoHV-1-specific proliferation of lymphocytes and production of IFNγ, TNFα, and IL-4. Downregulation of the expression of endosome Toll-like receptors 8-9 was detected after booster vaccination. This is the first thorough study of the immunity generated by a commercial vaccine against BoHV-1 in cattle.

Oral Selective TLR8 Agonist Selgantolimod Induces Multiple Immune Cell Responses in Humans.

TLR8 agonists have the potential for use as immunomodulatory components in therapeutic modalities for viral infections such as chronic HBV (CHB) and HIV. In this study, using peripheral blood samples from a phase 1a clinical trial, we examined the acute effects of a single oral administration of a selective TLR8 agonist on immune cell phenotypes. Administration of the TLR8 agonist selgantolimod (SLGN) in healthy individuals resulted in alteration in frequencies of peripheral blood monocytes, pDCs, mDCs and MAIT cells. Frequencies of mDCs and lymphoid cells significantly reduced after 8 h of SLGN administration, whereas pDC frequencies significantly increased, with changes possibly reflecting migration of different cell types between peripheral and tissue compartments in response to the agonist. Myeloid cell activation was evident by an upregulated expression of co-stimulatory molecules CD40 and CD86 accompanied by the production of IL-6 and IL-18 from these cells. Concomitantly, there was induction of the early activation marker CD69 on innate and adaptive lymphoid cells, including MAIT and NK cell subsets. Further, these activated lymphoid cells had enhanced expression of the effector molecules granzyme B and perforin. Microarray analysis of isolated lymphocytes and monocytes from baseline and post-SLGN treatment revealed changes in expression of genes involved in cellular response to cytokine stimulus, innate immune response, myeloid cell differentiation and antigen receptor-mediated signaling pathway. In a preliminary analysis of samples from CHB patients treated with selgantolimod, activation of innate and adaptive lymphocytes was evident. In conclusion, this first in-human study shows that selgantolimod administration in humans results in activation of multiple immune cell responses with antiviral potential.

Further hit optimization of 6-(trifluoromethyl)pyrimidin-2-amine based TLR8 modulators: Synthesis, biological evaluation and structure-activity relationships.

Toll-like receptor 8 (TLR8) is an endosomal TLR that has an important role in the innate human immune system, which is involved in numerous pathological conditions. Excessive activation of TLR8 can lead to inflammatory and autoimmune diseases, which highlights the need for development of TLR8 modulators. However, only a few small-molecule modulators that selectively target TLR8 have been developed. Here, we report the synthesis and systematic investigation of the structure-activity relationships of a series of novel TLR8 negative modulators based on previously reported 6-(trifluoromethyl)pyrimidin-2-amine derivatives. Four compounds showed low-micromolar concentration-dependent inhibition of TLR8-mediated signaling in HEK293 cells. These data confirm that the 6-trifluoromethyl group and two other substituents on positions 2 and 4 are important structural elements of pyrimidine-based TLR8 modulators. Substitution of the main scaffold at position 2 with a methylsulfonyl group or para hydroxy/hydroxymethyl substituted benzylamine is essential for potent negative modulation of TLR8. Our best-in-class TLR8-selective modulator 53 with IC50 value of 6.2 µM represents a promising small-molecule chemical probe for further optimization to a lead compound with potent immunomodulatory properties.

In-silico immunoinformatic analysis of SARS-CoV-2 virus for the development of putative vaccine construct.

COVID-19 (CoronaVirus disease 2019) is caused by the SARS-CoV-2 virus (severe acute respiratory syndrome corona virus 2). SARS-CoV-2 virus is highly contagious and affects the human respiratory tract resulting in symptoms such as high fever, body ache, cough, dysfunctions of tastebuds and smelling sense of body. The objective of the present study involves immunoinformatic analysis to predict COVID-19 protein for vaccine construct based on the genomic information SARS-CoV-2 virus. At present, as per WHO estimates, around 133 COVID-19 novel vaccines under development. Three amino acid sequences of SARS-CoV-2 were retrieved from the NCBI database for the analysis of vaccine construct. This study involves computational and immunoinformatic methods. The Immunoinformatic tools used in the present study are NetCTL server, IFN epitope server, Toxin PRED, BCPred, CTL + HTL + ADJUVANTS + LINKERS, AlgPredserver, VaxiJenserver, ProtParam to predict vaccine construct. The secondary and tertiary structure prediction is done by PSIPRED, I-TASSER, Galaxy refine, prosA + Ramachandran. Finally, docking of the vaccine constructs and ligand was done with the help of Cluspro 2.0. C-ImmSimm webserver to simulate the potential vaccine construct. The present study demonstrated three potential Vaccine constructs for the SARS-CoV-2 virus, which were docked with TLR8 (Toll-likereceptor8). Interestingly from these, all constructs one having a high potential for the inhibition effect of the SARS-CoV-2virus. Immunological simulation data shows significant elevated amount of memory B cell; also, the high response was seen in TH(Helper) and TC(cytotoxic) cell population from the vaccine construct proposed in the current study. Hence, these constructs are suitable vaccine candidates that might be useful in developing a novel vaccine.

Large, Anionic Liposomes Enable Targeted Intraperitoneal Delivery of a TLR 7/8 Agonist To Repolarize Ovarian Tumors' Microenvironment.

Ovarian cancer is the most lethal gynecological malignancy in the United States. Current standard of treatment includes surgical debulking and chemotherapy, such as cisplatin and paclitaxel. However, the patients' response rate for chemotherapy in ovarian cancer is not optimal, and they often develop chemoresistance and suffer from side effects. Current clinical trials make extensive use of immune checkpoint blockade (ICB) as a novel cancer immunotherapeutic strategy against ovarian tumors. However, the response rates for ICB antibodies remain limited to 10-20% of treated ovarian cancer patients despite the success of this approach in melanoma, renal, head and neck, and nonsmall cell lung cancers. This lack of efficacy is often attributed to the "cold" immune status of ovarian tumors, as these tumors often have a low number of tumor-infiltrating lymphocytes (TILs) but a high number of suppressive immune cells, including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), or regulatory T cells (Tregs). Repolarizing TAMs could be a promising strategy to reshape the tumor immune microenvironment and promote antitumor activity when combined with ICBs. Toll-like receptor (TLR) 7 and 8 agonists, such as imiquimod and resiquimod, are potent immunostimulatory molecules with potential to repolarize macrophages. However, these small molecules have poor pharmacokinetic profiles and can induce severe side effects when administered systemically. Previously, our group demonstrated that various large, anionic nanomaterials (silica, PLGA, and polystyrene) specifically target TAMs when administered intraperitoneally (IP) to ovarian tumor-bearing mice. In the present study, we demonstrate that large, anionic liposomes administered IP also efficiently localize to TAMs and can be used to target the delivery of resiquimod. Resiquimod delivered in this targeted fashion promoted activation of M1 macrophages and T cell infiltration, while reducing the percentage of Tregs in the tumor microenvironment. Finally, liposome-formulated resiquimod significantly enhanced the efficacy of PD1 blockade against syngeneic ovarian tumors. We anticipate that further optimization of our liposomal delivery strategy can generate a clinically relevant strategy for more effective and safer immunotherapy for ovarian cancer patients.

TLR7 Ligation Inhibits TLR8 Responsiveness in IL-27-Primed Human THP-1 Monocytes and Macrophages.

Regulation of proinflammatory cytokine expression is critical in the face of single-stranded RNA (ssRNA) virus infections. Many viruses, including coronavirus and influenza virus, wreak havoc on the control of cytokine expression, leading to the formation of detrimental cytokine storms. Understanding the regulation and interplay between inflammatory cytokines is critical to the identification of targets involved in controlling the induction of cytokine expression. In this study, we focused on how the antiviral cytokine interleukin-27 (IL-27) regulates signal transduction downstream of Toll-like receptor 7 (TLR7) and TLR8 ligation, which recognize endosomal single-stranded RNA. Given that IL-27 alters bacterial-sensing TLR expression on myeloid cells and can inhibit replication of single-stranded RNA viruses, we investigated whether IL-27 affects expression and function of TLR7 and TLR8. Analysis of IL-27-treated THP-1 monocytic cells and THP-1-derived macrophages revealed changes in mRNA and protein expression of TLR7 and TLR8. Although treatment with IL-27 enhanced TLR7 expression, only TLR8-mediated cytokine secretion was amplified. Furthermore, we demonstrated that imiquimod, a TLR7 agonist, inhibited cytokine and chemokine production induced by a TLR8 agonist, TL8-506. Delineating the immunomodulatory role of IL-27 on TLR7 and TLR8 responses provides insight into how myeloid cell TLR-mediated responses are regulated during virus infection.

Landscape of toll-like receptors expression in tumor microenvironment of triple negative breast cancer (TNBC): Distinct roles of TLR4 and TLR8.

TNBC is the most aggressive and hormone receptor-negative subtype of breast cancer with molecular heterogeneity in bulk tumors hindering effective treatment. Toll-like receptors (TLRs) have the potential to ignite diverse immune responses in the tumor microenvironment (TME). This encouraged us to screen their transcript expression in the publically available TCGA datasets. Reported molecular subtypes of TNBC may represent different TMEs and we observed differentially expressed TLRs (DETs) i.e. TLR3/4/6/8/9 have unique expression pattern in the TNBC subtypes, particularly in Immunomodulatory (IM) TNBC subtype. We then dissected expression of the DETs in immune and other components of the TME. TLR4 and TLR8 showed significant (p-value ≤ 0.05) negative partial correlation with tumor purity compared to other DETs. Interestingly, TLR4 and TLR8 expression showed a significant (adjusted p-value ≤ 0.05) correlation with different subsets of immune infiltrating cells having the highest correlation with monocytes/macrophage/dendritic cell populations mediating both innate and adaptive response in TNBC. The co-expression network identified genes correlated with these immune cells. Further, GSEA analysis of co-expressed genes showed a significant association of TLR8 partners with 'Peptide ligand binding', 'Gά-signaling', and 'Cytokine-cytokine interaction' while TLR4 associated genes correlated with 'Adaptive immune system' and 'Systemic lupus erythematosus' interactome. Finally, the expression of TLR4 protein was validated in a panel of TNBC cell lines. TLR4 expression in chemoresponsive TNBC was also validated in TNBC cell lines upon Paclitaxel (PTX) treatment. Collectively, the present study identified specific DETs in TNBC and discovered a prospective role of TLR4 and TLR8 in the maintenance of tumor-immune-microenvironment.

A tractable covalent linker strategy for the production of immunogenic antigen-TLR7/8L bioconjugates.

Despite the ease of production and improved safety profiles of recombinant vaccines, the inherently low immunogenicity of unadjuvanted proteins remains an impediment to their widespread adoption. The covalent tethering of TLR agonists to antigenic proteins offers a unique approach to co-deliver both constituents to the same cell-enhancing vaccine efficacy while minimizing reactogenicity. However, the paucity of simple and effective linker chemistries continues to hamper progress. Here, we present a modular, PEG-based linker system compatible with even extremely lipophilic and challenging TLR7/8 agonists. To advance the field and address previous obstacles, we offer the most straightforward and antigen-preserving linker system to date. These antigen-adjuvant conjugates enhance antigen-specific immune responses in mice, demonstrating the power of our approach within the context of modern vaccinology.

Successful Direct Acting Antiviral Therapy in Chronic Hepatitis C Normalizes IFNγ and IL2 Production in T Cells Together with TLR8 Expression and Functionality in Peripheral Blood Mononuclear Cells.

Chronic hepatitis C infection (HCV) activates a systemic cell-mediated immune response characterized by the production of IFNγ and an innate immune response addressed by the activation of TLR signaling. We aimed to investigate whether HCV eradication by direct acting antivirals (DAA) leads to a recovery in cell-mediated immune response and TLR expression and functionality. Blood samples were obtained in HCV infected patients before DAA treatment and at week +48 after the end of treatment. Results were compared to healthy controls. Cell surface expression of TLR8 was assessed on peripheral blood mononuclear cells (PBMCs) by flow cytometry. Freshly isolated PBMCs were cultured with specific TLR8 agonists and intracellular production of cytokines was determined by flow-cytometry after ex vivo TLR8 activation with ssRNA 40. Production of IFNγ, IL2 and IL17 was assessed by flow cytometry in T cells after polyclonal activation. Included were 50 HCV-infected patients and 15 controls. TLR8 expression in PBMCs was significantly increased before treatment and recovered normal levels at week +48. Production of IL1b, IL6 and TNFα dependent on the activation of TLR8 in PBMCs was also increased in patients before DAA treatment, with a significant reduction at week +48. Combined expression of IFNγ and IL2 in CD4+ T cells in HCV-infected patients was significantly increased compared to controls and recovered normal levels at week +48. DAA-mediated clearance of HCV is associated with a decreased expression and activation of TLR8 in PBMCs until healthy control levels which is accompanied by a reduction in the Th1 response.

Ex vivo peripheral blood mononuclear cell response to R848 in children after supplementation with the probiotic Lactobacillus acidophilus NCFM/Bifidobacterium lactis Bi-07.

Several studies have demonstrated a decrease in upper respiratory infection (URI) frequency and severity in subjects taking probiotic supplements. We hypothesised beneficial effects of probiotics on viral URI in children are due to modulation of inflammatory innate immune responses. We tested this hypothesis, providing children with a probiotic combination of Lactobacillus acidophilus/Bidfidobacterium animalis ssp. lactis Bi-07 (NCFM/Bi-07) and measuring levels of cytokines in response to stimulation of peripheral blood mononuclear cells (PBMCs) to toll-like receptor (TLR) 7/8 agonist resiquimod (R848). In this open label study, 21 (2 dropouts) children received probiotic containing 5×109 cfu each of NCFM/(Bi-07) daily for 30 days. Whole blood was taken from each subject at study entry and 30 days for culture of PBMCs. PBMCs stimulated with resiquimod (R848) or unstimulated were incubated and a panel of immune markers was measured. There was a significant decrease in the net (stimulated-null) level of myeloid progenitor inhibitory factor 1 (MPIF-1) (mean decrease 0.1 ng/ml, 95% confidence interval 0.01-0.24, P=0.032) following probiotic supplementation. The change in immune marker levels after supplementation, when analysed together with respect to expected inflammatory/anti-inflammatory effects, was increased for interleukin (IL)-10 and decreased for MPIF-1, IL-8, interferon gamma induced protein 10, macrophage inflammatory protein 3 alpha (MIP-3α) and E-selectin (P=0.01). Adverse events were mild. In conclusion, supplementation with this probiotic combination was safe and resulted in significant modulation of PBMC limited immune response to TLR7/8 agonist R848 and in levels of MPIF-1 and MIP-3α. The anti-inflammatory effect may be one mechanism by which probiotics modulate the immune system however further study is needed.

Novel TLR 7/8 agonists for improving NK cell mediated antibody-dependent cellular cytotoxicity (ADCC).

There is a significant interest in designing therapeutic agents that can enhance ADCC and thereby improve clinical responses with approved antibodies. We recently reported the combination of an imidazoquinoline-based TLR7/8 agonist (522) with a monoclonal antibody improved ADCC in vitro and in vivo. In the present study, we tested several new small molecule TLR7/8 agonists that induce significantly higher cytokines compared to both the FDA-approved TLR7 agonist, imiquimod, and 522. We evaluated these agonists in combination with monoclonal antibody therapy, with the main goal of enhancing ADCC. Our studies show these TLR7/8 agonists induce robust pro-inflammatory cytokine secretion and activate NK cells. Specifically, we found the agonists 574 and 558 significantly enhanced NK cell-mediated ADCC in vitro as well as enhanced the anti-cancer efficacy of monoclonal antibodies in two different in vivo mouse models. Additionally, we found the agonists were able to stimulate CD8 T cells, likely indicative of an early adaptive immune response.

Escape from X Chromosome Inactivation and the Female Predominance in Autoimmune Diseases.

Women represent 80% of people affected by autoimmune diseases. Although, many studies have demonstrated a role for sex hormone receptor signaling, particularly estrogens, in the direct regulation of innate and adaptive components of the immune system, recent data suggest that female sex hormones are not the only cause of the female predisposition to autoimmunity. Besides sex steroid hormones, growing evidence points towards the role of X-linked genetic factors. In female mammals, one of the two X chromosomes is randomly inactivated during embryonic development, resulting in a cellular mosaicism, where about one-half of the cells in a given tissue express either the maternal X chromosome or the paternal one. X chromosome inactivation (XCI) is however not complete and 15 to 23% of genes from the inactive X chromosome (Xi) escape XCI, thereby contributing to the emergence of a female-specific heterogeneous population of cells with bi-allelic expression of some X-linked genes. Although the direct contribution of this genetic mechanism in the female susceptibility to autoimmunity still remains to be established, the cellular mosaicism resulting from XCI escape is likely to create a unique functional plasticity within female immune cells. Here, we review recent findings identifying key immune related genes that escape XCI and the relationship between gene dosage imbalance and functional responsiveness in female cells.

RNA-Cholesterol Nanoparticles Function as Potent Immune Activators via TLR7 and TLR8.

The innate immune system senses viral and bacterial ribonucleic acid (RNA) via pattern recognition receptors (PRR) leading to subsequent activation of the immune system. One group of RNA sensors is formed by endosomal/lysosomal Toll-like receptors (TLR) such as TLR7 and TLR8. During viral or bacterial infection, immunostimulatory RNA is part of the pathogen reaching the endosomal/lysosomal compartment after cellular uptake. Synthetic single-stranded or double-stranded oligoribonucleotides (ORN) can mimic RNA from pathogens and are widely used as activating ligands for TLR7 and TLR8. However, one limitation in the use of synthetic ORN driven immune stimulation is the need for transfection reagents for RNA delivery into cells. Here we demonstrate that the conjugation of cholesterol to a double-stranded version of immunostimulatory RNA40 strongly enhanced RNA uptake into monocytes and plasmacytoid dendritic cells when compared to naked RNA. Cholesterol-conjugated RNA (RNA-chol) formed nanoparticles that were superior to RNA-liposomes complexes in regard to induction of type I interferon from human and murine plasmacytoid dendritic cells as well as proinflammatory cytokine production (e.g. TNF-α, IL12p70 or IL-6) in human monocytes. Furthermore, the RNA40-chol induced cytokines in human monocyte cultures supported TH1 and TFH cell differentiation underscoring a strong adjuvant function of RNA-chol nanoparticles for adaptive immune responses. In summary, cholesterol-conjugated immunostimulatory RNA forms nanoparticles and functions as a potent immune adjuvant in human and murine immune cells. It further simplifies the use of immunostimulatory RNA by avoiding the need for liposomal transfection reagents.

MAIT Cells Display a Specific Response to Type 1 IFN Underlying the Adjuvant Effect of TLR7/8 Ligands.

Mucosal-associated invariant T (MAIT) cells constitute a highly conserved subset of effector T cells with innate-like recognition of a wide array of bacteria and fungi in humans. Harnessing the potential of these cells could represent a major advance as a new immunotherapy approach to fight difficult-to-treat bacterial infections. However, despite recent advances in the design of potent agonistic ligands for MAIT cells, it has become increasingly evident that adjuvants are required to elicit potent antimicrobial effector functions by these cells, such as IFNγ production and cytotoxicity. Indeed, TCR triggering alone elicits mostly barrier repair functions in MAIT cells, whereas an inflammatory milieu is required to drive the antibacterial functions. Cytokines such as IL-7, IL-12 and IL-18, IL-15 or more recently type 1 IFN all display an apparently similar ability to synergize with TCR stimulation to induce IFNγ production and/or cytotoxic functions in vitro, but their mechanisms of action are not well established. Herein, we show that MAIT cells feature a build-in mechanism to respond to IFNα. We confirm that IFNα acts directly and specifically on MAIT cells and synergizes with TCR/CD3 triggering to induce maximum cytokine production and cytotoxic functions. We provide evidences suggesting that the preferential activation of the Stat4 pathway is involved in the high sensitivity of MAIT cells to IFNα stimulation. Finally, gene expression data confirm the specific responsiveness of MAIT cells to IFNα and pinpoints specific pathways that could be the target of this cytokine. Altogether, these data highlight the potential of IFNα-inducing adjuvants to maximize MAIT cells responsiveness to purified ligands in order to induce potent anti-infectious responses.

Potent and Prolonged Innate Immune Activation by Enzyme-Responsive Imidazoquinoline TLR7/8 Agonist Prodrug Vesicles.

Synthetic immune-stimulatory drugs such as agonists of the Toll-like receptors (TLR) 7/8 are potent activators of antigen-presenting cells (APCs), however, they also induce severe side effects due to leakage from the site of injection into systemic circulation. Here, we report on the design and synthesis of an amphiphilic polymer-prodrug conjugate of an imidazoquinoline TLR7/8 agonist that in aqueous medium forms vesicular structures of 200 nm. The conjugate contains an endosomal enzyme-responsive linker enabling degradation of the vesicles and release of the TLR7/8 agonist in native form after endocytosis, which results in high in vitro TLR agonist activity. In a mouse model, locally administered vesicles provoke significantly more potent and long-lasting immune stimulation in terms of interferon expression at the injection site and in draining lymphoid tissue compared to a nonamphiphilic control and the native TLR agonist. Moreover, the vesicles induce robust activation of dendritic cells in the draining lymph node in vivo.

Human neutrophils activated by TLR8 agonists, with or without IFNγ, synthesize and release EBI3, but not IL-12, IL-27, IL-35, or IL-39.

The IL-12 family of cytokines plays crucial functions in innate and adaptive immunity. These cytokines include heterodimers sharing distinct α (IL-12A, IL-23A, and IL-27A) with two ß (IL-12B and Epstein-Barr virus induced gene 3 [EBI3]) chains, respectively, IL-12 (IL-12B plus IL-12A) and IL-23 (IL-12B plus IL-23A) sharing IL-12B, IL-27 (EBI3 plus IL-27A), IL-35 (EBI3 plus IL-12A), and IL-39 (EBI3 plus IL-23A) sharing EBI3. In this context, we have recently reported that highly pure neutrophils incubated with TLR8 agonists produce functional IL-23. Previously, we showed that neutrophils incubated with LPS plus IFNγ for 20 h produce IL-12. Herein, we investigated whether highly pure, TLR8-activated, neutrophils produce EBI3, and in turn IL-27, IL-35, and IL-39, the IL-12 members containing it. We report that neutrophils incubated with TLR8 ligands, TNFα and, to a lesser extent, LPS, produce and release remarkable amounts of EBI3, but not IL-27A, consequently excluding the possibility for an IL-27 production. We also report a series of unsuccessful experiments performed to investigate whether neutrophil-derived EBI3 associates with IL-23A to form IL-39. Furthermore, we show that neutrophils incubated with IFNγ in combination with either TLR8 or TLR4 ligands express/produce neither IL-12, nor IL-35, due to the inability of IFNγ, contrary to previous findings, to activate IL12A transcription. Even IL-27 was undetectable in supernatants harvested from IFNγ plus R848-treated neutrophils, although they were found to accumulate IL27A transcripts. Finally, by immunohistochemistry experiments, EBI3-positive neutrophils were found in discrete pathologies only, including diverticulitis, cholecystitis, Gorham disease, and Bartonella Henselae infection, implying a specific role of neutrophil-derived EBI3 in vivo.