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Dendritic cells (DCs) mature in an immunogenic or tolerogenic manner depending on the context in which an antigen is perceived, preserving the balance between immunity and tolerance. Whereas the pathways driving immunogenic maturation in response to infectious insults are well-characterized, the signals that drive tolerogenic maturation during homeostasis are still poorly understood. We found that the engulfment of apoptotic cells triggered homeostatic maturation of type 1 conventional DCs (cDC1s) within the spleen. This maturation process could be mimicked by engulfment of empty, nonadjuvanted lipid nanoparticles (LNPs), was marked by intracellular accumulation of cholesterol, and was highly specific to cDC1s. Engulfment of either apoptotic cells or cholesterol-rich LNPs led to the activation of the liver X receptor (LXR) pathway, which promotes the efflux of cellular cholesterol, and repressed genes associated with immunogenic maturation. In contrast, simultaneous engagement of TLR3 to mimic viral infection via administration of poly(I:C)-adjuvanted LNPs repressed the LXR pathway, thus delaying cellular cholesterol efflux and inducing genes that promote T cell–mediated immunity. These data demonstrate that conserved cellular cholesterol efflux pathways are differentially regulated in tolerogenic versus immunogenic cDC1s and suggest that administration of nonadjuvanted cholesterol-rich LNPs may be an approach for inducing tolerogenic DC maturation.

The development of controlled biodegradable materials is of fundamental importance in immunodrug delivery to spatiotemporally controlled immune stimulation but avoid systemic inflammatory side effects. Based on this, polycarbonate nanogels are developed as degradable micellar carriers for transient immunoactivation of lymph nodes. An imidazoquinoline-type TLR7/8 agonist is covalently conjugated via reactive ester chemistry to these nanocarriers. The nanogels not only provide access to complete disintegration by the hydrolysable polymer backbone, but also demonstrate a gradual disintegration within several days at physiological conditions (PBS, pH 6.4–7.4, 37 °C). These intrinsic properties limit the lifetime of the carriers but their payload can still be successfully leveraged for immunological studies in vitro on primary immune cells as well as in vivo. For the latter, a spatiotemporal control of immune cell activation in the draining lymph node is found after subcutaneous injection. Overall, these features render polycarbonate nanogels a promising delivery system for transient activation of the immune system in lymph nodes and may consequently become very attractive for further development toward vaccination or cancer immunotherapy. Due to the intrinsic biodegradability combined with the high chemical control during the manufacturing process, these polycarbonate-based nanogels may also be of great importance for clinical translation.

The search for vaccines that protect from severe morbidity and mortality because of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Here we describe an amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile. It is water-soluble and exhibits massive translocation to lymph nodes upon local administration through binding to albumin, affording localized innate immune activation and reduction in systemic inflammation. The adjuvanticity of IMDQ-PEG-CHOL was validated in a licensed vaccine setting (quadrivalent influenza vaccine) and an experimental trimeric recombinant SARS-CoV-2 spike protein vaccine, showing robust IgG2a and IgG1 antibody titers in mice that could neutralize viral infection in vitro and in vivo in a mouse model.

Antigen-presenting conventional dendritic cells (cDCs) are broadly divided into type 1 and type 2 subsets that further adapt their phenotype and function to perform specialized tasks in the immune system. The precise signals controlling tissue-specific adaptation and differentiation of cDCs are currently poorly understood. We found that mice deficient in the Ste20 kinase Thousand and One Kinase 3 (TAOK3) lacked terminally differentiated ESAM(+) CD4(+) cDC2s in the spleen and failed to prime CD4(+) T cells in response to allogeneic red-blood-cell transfusion. These NOTCH2- and ADAM10-dependent cDC2s were absent selectively in the spleen, but not in the intestine of Taok3(−/−) and CD11c-cre Taok3(fl/fl) mice. The loss of splenic ESAM(+) cDC2s was cell-intrinsic and could be rescued by conditional overexpression of the constitutively active NOTCH intracellular domain in CD11c-expressing cells. Therefore, TAOK3 controls the terminal differentiation of NOTCH2-dependent splenic cDC2s.

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.

Small-molecular Toll-like receptor 7/8 (TLR7/8) agonists hold promise as immune modulators for a variety of immune therapeutic purposes including cancer therapy or vaccination. However, due to their rapid systemic distribution causing difficult-to-control inflammatory off-target effects, their application is still problematic, in particular systemically. To address this problem, we designed and robustly fabricated pH-responsive nanogels serving as versatile immunodrug nanocarriers for safe delivery of TLR7/8-stimulating imidazoquinolines after intravenous administration. To this aim, a primary amine-reactive methacrylamide monomer bearing a pendant squaric ester amide is introduced, which is polymerized under controlled RAFT polymerization conditions. Corresponding PEG-derived squaric ester amide block copolymers self-assemble into precursor micelles in polar protic solvents. Their cores are amine-reactive and can sequentially be transformed by acid-sensitive cross-linkers, dyes, and imidazoquinolines. Remaining squaric ester amides are hydrophilized affording fully hydrophilic nanogels with profound stability in human plasma but stimuli-responsive degradation upon exposure to endolysosomal pH conditions. The immunomodulatory behavior of the imidazoquinolines alone or conjugated to the nanogels was demonstrated by macrophages in vitro. In vivo, however, we observed a remarkable impact of the nanogel: After intravenous injection, a spatially controlled immunostimulatory activity was evident in the spleen, whereas systemic off-target inflammatory responses triggered by the small-molecular imidazoquinoline analogue were absent. These findings underline the potential of squaric ester-based, pH-degradable nanogels as a promising platform to permit intravenous administration routes of small-molecular TLR7/8 agonists and, thus, the opportunity to explore their adjuvant potency for systemic vaccination or cancer immunotherapy purposes.

Triggering antibody-mediated innate immune mechanisms to kill cancer cells is an attractive therapeutic avenue. In this context, recruitment of endogenous antibodies to the cancer cell surface could be a viable alternative to the use of monoclonal antibodies. We report on antibody-recruiting polymers containing multiple antibody-binding hapten motifs and cyclooctynes that can covalently conjugate to azides introduced onto the glycocalyx of cancer cells by metabolic labeling with azido sugars.

Background The most common endotype of asthma is type 2–high asthma, which is sometimes driven by adaptive allergen-specific TH2 lymphocytes that react to allergens presented by dendritic cells (DCs), or sometimes by an innate immune response dominated by type 2 innate lymphocytes (ILC2s). Understanding the underlying pathophysiology of asthma is essential to improve patient-tailored therapy. The STE20 kinase thousand-and-one kinase 3 (TAOK3) controls key features in the biology of DCs and lymphocytes, but to our knowledge, its potential usefulness as a target for asthma therapy has not yet been addressed. Objective We examined if and how loss of Taok3 affects the development of house dust mite (HDM)-driven allergic asthma in an in vivo mouse model. Methods Wild-type Taok3+/+ and gene-deficient Taok3−/− mice were sensitized and challenged with HDM, and bronchoalveolar lavage fluid composition, mediastinal lymph node cytokine production, lung histology, and bronchial hyperreactivity measured. Conditional Taok3fl/fl mice were crossed to tissue- and cell-specific specific deletor Cre mice to understand how Taok3 acted on asthma susceptibility. Kinase-dead (KD) Taok3KD mice were generated to probe for the druggability of this pathway. Activation of HDM-specific T cells was measured in adoptively transferred HDM-specific T-cell receptor–transgenic CD4+ T cells. ILC2 biology was assessed by in vivo and in vitro IL-33 stimulation assays in Taok3−/− and Taok3+/+, Taok3KD, and Red5-Cre Taok3fl/fl mice. Results Taok3−/− mice failed to mount salient features of asthma, including airway eosinophilia, TH2 cytokine production, IgE secretion, airway goblet cell metaplasia, and bronchial hyperreactivity compared to controls. This was due to intrinsic loss of Taok3 in hematopoietic and not epithelial cells. Loss of Taok3 resulted in hampered HDM-induced lung DC migration to the draining lymph nodes and defective priming of HDM-specific TH2 cells. Strikingly, HDM and IL-33–induced ILC2 proliferation and function were also severely affected in Taok3-deficient and Taok3KD mice. Conclusions Absence of Taok3 or loss of its kinase activity protects from HDM-driven allergic asthma as a result of defects in both adaptive DC-mediated TH2 activation and innate ILC2 function. This identifies Taok3 as an interesting drug target, justifying further testing as a new treatment for type 2–high asthma.

Peptide-based subunit vaccines are attractive in view of personalized cancer vaccination with neo-antigens, as well as for the design of the newest generation of vaccines against infectious diseases. Key to mounting robust antigen-specific immunity is delivery of antigen to antigen-presenting (innate immune) cells in lymphoid tissue with concomitant innate immune activation to promote antigen presentation to T cells and to shape the amplitude and nature of the immune response. Nanoparticles that co-deliver both peptide antigen and molecular adjuvants are well suited for this task. However, in the context of peptide-based antigen, an unmet need exists for a generic strategy that allows for co-encapsulation of peptide and molecular adjuvants due to the stark variation in physicochemical properties based on the amino acid sequence of the peptide. These properties also strongly differ from those of many molecular adjuvants. Here, we devise a lipid nanoparticle (LNP) platform that addresses these issues. Key in our concept is poly(L-glutamic acid) (PGA), which serves as a hydrophilic backbone for conjugation of, respectively, peptide antigen (Ag) and an imidazoquinoline (IMDQ) TLR7/8 agonist as a molecular adjuvant. Making use of the PGA's polyanionic nature, we condensate PGA-Ag and PGA-IMDQ into LNP by electrostatic interaction with an ionizable lipid. We show in vitro and in vivo in mouse models that LNP encapsulation favors uptake by innate immune cells in lymphoid tissue and promotes the induction of Ag-specific T cells responses both after subcutaneous and intravenous administration.

The search for vaccines that protect from severe morbidity and mortality because of infection with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the virus that causes coronavirus disease 2019 (COVID‐19) is a race against the clock and the virus. Here we describe an amphiphilic imidazoquinoline (IMDQ‐PEG‐CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol‐poly(ethylene glycol) macromolecular amphiphile. It is water‐soluble and exhibits massive translocation to lymph nodes upon local administration through binding to albumin, affording localized innate immune activation and reduction in systemic inflammation. The adjuvanticity of IMDQ‐PEG‐CHOL was validated in a licensed vaccine setting (quadrivalent influenza vaccine) and an experimental trimeric recombinant SARS‐CoV‐2 spike protein vaccine, showing robust IgG2a and IgG1 antibody titers in mice that could neutralize viral infection in vitro and in vivo in a mouse model., Amphiphilic TRL7/8 agonist adjuvanted recombinant Spike protein vaccine protects against SARS‐CoV‐2 infection in mouse models.

Small molecule immuno-modulators such as agonists of Toll-like receptors (TLRs) are attractive compounds to stimulate innate immune cells toward potent antiviral and antitumor responses. However, small molecules rapidly enter the systemic circulation and cause "wasted inflammation". Hence, synthetic strategies to confine their radius of action to lymphoid tissue are of great relevance, to both enhance their efficacy and concomitantly limit toxicity. Here, we demonstrate that covalent conjugation of a small molecule TLR7/8 agonist immunomodulatory to a micelle-forming amphiphilic block copolymer greatly alters the pharmacokinetic profile, resulting in highly efficient lymphatic delivery. Moreover, we designed amphiphilic block copolymers in such a way to form thermodynamically stable micelles through π-π stacking between aromatic moieties, and we engineered the block copolymers to undergo an irreversible amphiphilic to hydrophilic transition in response to the acidic endosomal pH.

Improving the immunogenicity of subunit vaccines, in particular skewing of the immune response towards Th1 type immunity, is crucial for the development of effective vaccines against intracellular infections and for the development of anti-cancer vaccines. Small molecule TLR7/8 agonist hold high potential for this purpose, but suffer from an undesirable pharmacokinetic profile, resulting in systemic inflammatory responses . An effective solution to this problem is covalent ligation to a larger carrier. Here, a degradable nanogel carrier containing a covalently linked imidazoquinoline (IMDQ) TLR7/8 agonist is explored as adjuvant for vaccination against the respiratory syncytial virus (RSV). In vitro and in vivo experiments in mice provide a solid rational base for preferring nanogels over soluble polymers as IMDQ carrier in terms of cellular uptake and lymph node accumulation.

Notch2 and B cell antigen receptor (BCR) signaling determine whether transitional B cells become marginal zone B (MZB) or follicular B (FoB) cells in the spleen, but it is unknown how these pathways are related. We generated Taok3 -/- mice, lacking the serine/threonine kinase Taok3, and found cell-intrinsic defects in the development of MZB but not FoB cells. Type 1 transitional (T1) B cells required Taok3 to rapidly respond to ligation by the Notch ligand Delta-like 1. BCR ligation by endogenous or exogenous ligands induced the surface expression of the metalloproteinase ADAM10 on T1 B cells in a Taok3-dependent manner. T1 B cells expressing surface ADAM10 were committed to becoming MZB cells in vivo, whereas T1 B cells lacking expression of ADAM10 were not. Thus, during positive selection in the spleen, BCR signaling causes immature T1 B cells to become receptive to Notch ligands via Taok3-mediated surface expression of ADAM10.

A crystal-clear ingredient for allergy? Charcot-Leyden crystals (CLCs) are formed from the eosinophil granule protein galectin-10 (Gal10) and found in severe eosinophil-associated diseases like asthma and chronic rhinosinusitis. Whether CLCs actively contribute to disease pathogenesis is unknown. Persson et al. found that lab-grown Gal10 crystals are biosimilar to CLCs (see the Perspective by Allen and Sutherland). When given to mice, the crystals acted as a type 2 adjuvant, mimicking many of the features of human asthma. In contrast, a Gal10 mutein unable to crystallize had no effect. Antibodies against epitopes crucial for Gal10 autocrystallization could dissolve both in vitro–generated Gal10 crystals and patient-derived CLCs. Furthermore, these anti-Gal10 antibodies reversed the effects of Gal10 crystals in a humanized mouse model of asthma, suggesting a potential therapeutic approach for crystallopathies more broadly. Science , this issue p. eaaw4295 ; see also p. 738

Uncontrolled systemic inflammatory immune triggering has hampered the clinical translation of several classes of small-molecule immunomodulators, such as imidazoquinoline TLR7/8 agonists for vaccine design and cancer immunotherapy. By taking advantage of the inherent serum-protein-binding property of lipid motifs and their tendency to accumulate in lymphoid tissue, we designed amphiphilic lipid–polymer conjugates that suppress systemic inflammation but provoke potent lymph-node immune activation. This work provides a rational basis for the design of lipid–polymer amphiphiles for optimized lymphoid targeting.

Interactive materials that can respond to a trigger by changing their morphology, but that can also gradually degrade into a fully soluble state, are attractive building blocks for the next generation of biomaterials. Herein, we design such transiently responsive polymers that exhibit UCST behaviour while gradually losing this property in response to a hydrolysis reaction in the polymer side chains. The polymers operate within a physiologically relevant window in terms of temperature, pH, and ionic strength. Whereas such behaviour has been reported earlier for LCST systems, it is at present unexplored for UCST polymers. Furthermore, we demonstrate that, in contrast to LCST polymers, in aqueous medium the UCST polymer forms a coacervate phase below the UCST, which can entrap a hydrophilic model protein, as well as a hydrophobic dye. Because of their non-toxicity, we also provide in vivo proof of concept of the use of this coacervate as a protein depot, in view of sustained-release applications.

The study of Vetters et al. identifies the ubiquitin-modifying enzyme A20 as a critical regulator of mTOR. Loss of A20 unleashes mTOR activity and induces NK cell death, underscoring the need for a tightly controlled mTOR pathway for proper NK cell homeostasis., The ubiquitin-editing enzyme A20 is a well-known regulator of immune cell function and homeostasis. In addition, A20 protects cells from death in an ill-defined manner. While most studies focus on its role in the TNF-receptor complex, we here identify a novel component in the A20-mediated decision between life and death. Loss of A20 in NK cells led to spontaneous NK cell death and severe NK cell lymphopenia. The few remaining NK cells showed an immature, hyperactivated phenotype, hallmarked by the basal release of cytokines and cytotoxic molecules. NK-A20−/− cells were hypersensitive to TNF-induced cell death and could be rescued, at least partially, by a combined deficiency with TNF. Unexpectedly, rapamycin, a well-established inhibitor of mTOR, also strongly protected NK-A20−/− cells from death, and further studies revealed that A20 restricts mTOR activation in NK cells. This study therefore maps A20 as a crucial regulator of mTOR signaling and underscores the need for a tightly balanced mTOR pathway in NK cell homeostasis.

This mouse study demonstrates that repetitive inhalation of a single major house dust mite (HDM) allergen prevents HDM-induced allergic asthma development through suppressing the function of lung dendritic cells, thus providing an alternative to classical allergen-specific immunotherapy.

BACKGROUND: Genome-wide association studies in asthma have repeatedly identified single nucleotide polymorphisms in the ORM (yeast)-like protein isoform 3 (ORMDL3) gene across different populations. Although the ORM-homologues in yeast are well-known inhibitors of sphingolipid (SL) synthesis, it is still unclear whether and how mammalian ORMDL3 regulates SL metabolism and whether altered SL synthesis would be causally related to asthma risk. OBJECTIVE: To examine the in vivo role of ORMDL3 in SL metabolism and allergic asthma METHODS: Ormdl3-LacZ reporter mice, gene deficient Ormdl3(-/-) and overexpressing Ormdl3 (Tg/wt) mice were exposed to physiologically relevant aeroallergens such as house dust mite or Alternaria alternata to induce experimental asthma. Mass spectrometry based sphingolipidomics was performed, and airway eosinophilia, Th2 cytokine production, Ig synthesis, airway remodeling and bronchial hyperreactivity measured. RESULTS: HDM challenge significantly increased total sphingolipids in the lung of HDM-sensitized mice compared to controls. In Ormdl3(Tg/wt) mice, the allergen-induced increase in lung ceramides was significantly reduced, whereas total sphingolipid levels were not affected. Conversely, in liver and serum, total sphingolipids including ceramides were increased in Ormdl3(-/-) mice, while they were lowered in Ormdl3(Tg/wt) mice. This difference was independent of allergen exposure. Despite these changes, all features of asthma were identical between wildtype, Ormdl3(Tg/wt) and Ormdl3(-/-) mice, across several models of experimental asthma. CONCLUSION: ORMDL3 regulates systemic ceramide levels, but genetically interfering with Ormdl3. expression does not result in altered experimental asthma. CAPSULE SUMMARY: ORMDL3 regulates systemic ceramide levels, but genetically interfering with Ormdl3 expression does not result in altered experimental asthma.

Immunogens carried in lymphatic fluid drain via afferent vessels into regional lymph nodes and facilitate the efficient induction of appropriate immune responses. The lymphatic system possesses receptors recognizing hyaluronic acid (HA). Covalent conjugates of small-molecule TLR7/8 agonists with HA are entirely devoid of immunostimulatory activity in vitro. In murine models of immunization, however, such conjugates traffic to lymph nodes, where they are “unmasked”, releasing the small molecule TLR7/8 agonist from the carrier polysaccharide. The resulting focal immunostimulation is manifested in potent adjuvantic effects with negligible systemic exposure. The efficient delivery of immunogens has been a major challenge in the development of subunit vaccines, and enhancing targeted delivery of immunogens to secondary lymphoid organs might be a promising approach for improving vaccine efficacy, as well as safety.

Degradable synthetic crosslinking is a versatile strategy to harness nanomaterials against disassembly in a complex physiological medium prompted by dilution effects or competitive interaction. In particular, chemical bonds such as ketals that are stable at physiological conditions but are cleaved in response to disease-mediated or intracellular conditions (e.g., a mildly acidic pH) are of great relevance for biomedical applications. Despite the range of spectroscopic or chromatographic analyses methods that allow chemical degradation in solution to be assessed, it is much less straightforward to interrogate synthetic nanomaterials for their degradation state when located inside a living organism. We demonstrate a method based on FRET analysis to monitor intracellular disassembly of block-copolymer-derived nanoparticles engineered with a FRET couple on separate polymer chains, which after self-assembly are covalently crosslinked with a pH-sensitive ketal-containing crosslinker.

Localized therapeutic modalities that subvert the tumor microenvironment from immune-suppressive to pro-immunogenic can elicit systemic antitumor immune responses that induce regression of directly treated as well as nontreated distal tumors. A key toward generating robust antitumor T cell responses is the activation of dendritic cells (DCs) in the tumor microenvironment. Treatment with agonists triggering various pattern recognition receptors is very efficient to activate DCs, yet suffers from the induction of serious immune-related adverse effects, which is closely linked to their unfavorable PK/PD profile causing systemic immune activation and cytokine release. Here, it is reported that nanoparticle conjugation of a highly potent TLR7/8 agonist restricts immune activation to the tumor bed and its sentinel lymph nodes without hampering therapeutic antitumor efficacy. On a mechanistic level, it is confirmed that localized treatment with a nanoparticle-conjugated TLR7/8 agonist leads to potent activation of DCs in the sentinel lymph nodes and promotes proliferation of tumor antigen-specific CD8 T cells. Furthermore, therapeutic improvement upon combination with anti-PDL1 checkpoint inhibition and Flt3L, a growth factor that expands and mobilizes DCs from the bone marrow, is demonstrated. The findings provide a rational base for localized tumor engineering by nanomedicine strategies that provide spatial control over immune-activation.

How farming protects against allergies People who grow up on dairy farms only rarely develop asthma or allergies. This is probably because as children, they breathe air containing bacterial components, which reduce the overall reactivity of the immune system. Schuijs et al. chronically exposed mice to bacterial endotoxin before they received an allergic stimulus. The protocol indeed protected them from developing an allergic response. Protection relied on a particular enzyme: A20. In humans, a variant of A20 correlates with increased susceptibility to asthma and allergy in children growing up on farms. Science , this issue p. 1106

Due to their capacity to skew T cell responses towards Th1 oriented immunity, oligonucleotides containing unmethylated CpG motifs (CpG) have emerged as interesting adjuvants for vaccination. Whereas the signalling pathways in response to CpG mediated TLR9 activation have been extensively documented at the level of the individual cell, little is however known on the precise identity of the innate immune cells that govern T cell priming and polarisation to CpG adjuvanted protein antigens in vivo. In this study, we demonstrate that optimal induction of Th1 oriented immunity to CpG adjuvanted protein vaccines requires the coordinated actions of conventional DCs and of monocytes. Whilst conventional DCs were required for antigen presentation and initial T cell priming, monocytes constitute the main source of the Th1 polarising cytokine IL-12.

Targeting immunomodulatory pathways has ushered a new era in lung cancer therapy. Further progress requires deeper insights into the biology of immune cells in the lung cancer micro-environment. Dendritic cells (DCs) represent a heterogeneous and highly plastic immune cell system with a central role in controlling immune responses. The intratumoral infiltration and activation status of DCs are emerging as clinically relevant parameters in lung cancer. In this study, we used an orthotopic preclinical model of lung cancer to dissect how the lung tumor micro-environment affects tissue-resident DCs and extract novel biologically and clinically relevant information. Lung tumor-infiltrating leukocytes expressing generic DC markers were found to predominantly consist of CD11b(+) cells that, compare with peritumoral lung DC counterparts, strongly overexpress the T-cell inhibitory molecule PD-L1 and acquire classical surface markers of tumor-associated macrophages (TAMs). Transcriptome analysis of these CD11b(+) tumor-infiltrating DCs (TIDCs) indicates impaired antitumoral immunogenicity, confirms the skewing toward TAM-related features, and indicates exposure to a hypoxic environment. In parallel, TIDCs display a specific microRNA (miRNA) signature dominated by the prototypical lung cancer oncomir miR-31. In vitro, hypoxia drives intrinsic miR-31 expression in CD11b(+) DCs. Conditioned medium of miR-31 overexpressing CD11b(+) DCs induces pro-invasive lung cancer cell shape changes and is enriched with pro-metastatic soluble factors. Finally, analysis of TCGA datasets reveals that the TIDC-associated miRNA signature has a negative prognostic impact in non-small cell lung cancer. Together, these data suggest a novel mechanism through which the lung cancer micro-environment exploits the plasticity of the DC system to support tumoral progression.

Summary Naive CD4+ T cells differentiate into functionally diverse T helper (Th) cell subsets. Th2 cells play a pathogenic role in asthma, yet a clear picture of their transcriptional profile is lacking. We performed single-cell RNA sequencing (scRNA-seq) of T helper cells from lymph node, lung, and airways in the house dust mite (HDM) model of allergic airway disease. scRNA-seq resolved transcriptional profiles of naive CD4+ T, Th1, Th2, regulatory T (Treg) cells, and a CD4+ T cell population responsive to type I interferons. Th2 cells in the airways were enriched for transcription of many genes, including Cd200r1, Il6, Plac8, and Igfbp7, and their mRNA profile was supported by analysis of chromatin accessibility and flow cytometry. Pathways associated with lipid metabolism were enriched in Th2 cells, and experiments with inhibitors of key metabolic pathways supported roles for glucose and lipid metabolism. These findings provide insight into the differentiation of pathogenic Th2 cells in the context of allergy.

Alveolar macrophages differentiate from fetal monocytes in a GM-CSF–dependent fashion and colonize the alveolar space within a few days after birth., Tissue-resident macrophages can develop from circulating adult monocytes or from primitive yolk sac–derived macrophages. The precise ontogeny of alveolar macrophages (AMFs) is unknown. By performing BrdU labeling and parabiosis experiments in adult mice, we found that circulating monocytes contributed minimally to the steady-state AMF pool. Mature AMFs were undetectable before birth and only fully colonized the alveolar space by 3 d after birth. Before birth, F4/80hiCD11blo primitive macrophages and Ly6ChiCD11bhi fetal monocytes sequentially colonized the developing lung around E12.5 and E16.5, respectively. The first signs of AMF differentiation appeared around the saccular stage of lung development (E18.5). Adoptive transfer identified fetal monocytes, and not primitive macrophages, as the main precursors of AMFs. Fetal monocytes transferred to the lung of neonatal mice acquired an AMF phenotype via defined developmental stages over the course of one week, and persisted for at least three months. Early AMF commitment from fetal monocytes was absent in GM-CSF–deficient mice, whereas short-term perinatal intrapulmonary GM-CSF therapy rescued AMF development for weeks, although the resulting AMFs displayed an immature phenotype. This demonstrates that tissue-resident macrophages can also develop from fetal monocytes that adopt a stable phenotype shortly after birth in response to instructive cytokines, and then self-maintain throughout life.

Inducible bronchus-associated lymphoid tissue (iBALT) is a long lasting tertiary lymphoid tissue that can be induced following influenza A virus (IAV) infection. Previous studies have shown that iBALT structures containing germinal center (GC) B cells protect against repeated infection by contributing locally to the cellular and humoral immune response. If we are to exploit this in vaccination strategies, we need a better understanding on how iBALT structures are induced. One hypothesis is that the strength of the initial innate response dictates induction of iBALT. In the present study, we investigated the role of interleukin (IL)-1 and IL-1R signaling on iBALT formation. Mice lacking the IL-1R had a delayed viral clearance and, thus, a prolonged exposure to viral replication, leading to increased disease severity, compared to wild-type mice. Contradictorily, iBALT formation following clearance of the virus was heavily compromised in Il1r1(-/-) mice. Quantification of gene induction after IAV infection demonstrated induction of IL-1 alpha and to a much lesser extent of IL-1 beta. Administration of recombinant IL-1 alpha to the lungs of wild-type mice, early but not late, after IAV infection led to more pronounced iBALT formation and an increased amount of GC B cells in the lungs. Bone marrow chimeric mice identified the stromal compartment as the crucial IL -1 responsive cell for iBALT induction. Mechanistically, Q-PCR analysis of lung homogenates revealed a strongly diminished production of CXCL13, a B cell-attracting chemokine, in Il1r(-/-) mice during the early innate phase of IAV infection. These experiments demonstrate that appropriate innate IL-1 alpha-IL-1R signaling is necessary for IAV clearance and at the same time instructs the formation of organized tertiary lymphoid tissues through induction of CXCL13 early after infection. These findings are discussed in the light of recent insights on the pathogenesis of tertiary lymphoid organ formation in the lung in various diseases where the IL-1 axis is hyperactive, such as rheumatoid arthritis and COPD.

Agonists of Toll-like receptors (TLRs) are potent activators of the innate immune system and hold promise as vaccine adjuvant and for anticancer immunotherapy. Unfortunately, in soluble form they readily enter systemic circulation and cause systemic inflammatory toxicity. Here we demonstrate that by covalent ligation of a small-molecule imidazoquinoline-based TLR7/8 agonist to 50-nm-sized degradable polymeric nanogels the potency of the agonist to activate TLR7/8 in in vitro cultured dendritic cells is largely retained. Importantly, imidazoquinoline-ligated nanogels focused the in vivo immune activation on the draining lymph nodes while dramatically reducing systemic inflammation. Mechanistic studies revealed a prevalent passive diffusion of the nanogels to the draining lymph node. Moreover, immunization studies in mice have shown that relative to soluble TLR7/8 agonist, imidazoquinoline-ligated nanogels induce superior antibody and T-cell responses against a tuberculosis antigen. This approach opens possibilities to enhance the therapeutic benefit of small-molecule TLR agonist for a variety of applications.

IL-1α promotes a cascade of cytokine production from epithelial cells culminating in Th2 immunity to house dust mite allergens., House dust mite (HDM) is one of the most common allergens worldwide. In this study, we have addressed the involvement of IL-1 in the interaction between HDM and the innate immune response driven by lung epithelial cells (ECs) and dendritic cells (DCs) that leads to asthma. Mice lacking IL-1R on radioresistant cells, but not hematopoietic cells, failed to mount a Th2 immune response and did not develop asthma to HDM. Experiments performed in vivo and in isolated air–liquid interface cultures of bronchial ECs showed that TLR4 signals induced the release of IL-1α, which then acted in an autocrine manner to trigger the release of DC-attracting chemokines, GM-CSF, and IL-33. Consequently, allergic sensitization to HDM was abolished in vivo when IL-1α, GM-CSF, or IL-33 was neutralized. Thymic stromal lymphopoietin (TSLP) became important only when high doses of allergen were administered. These findings put IL-1α upstream in the cytokine cascade leading to epithelial and DC activation in response to inhaled HDM allergen.

To cite this article: Krysko O, Holtappels G, Zhang N, Kubica M, Deswarte K, Derycke L, Claeys S, Hammad H, Brusselle GG, Vandenabeele P, Krysko DV, Bachert C. Alternatively activated macrophages and impaired phagocytosis of S. aureus in chronic rhinosinusitis. Allergy 2011; 66: 396–403. Abstract Background: Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by biased Th2 inflammation and CRS without nasal polyps (CRSsNP) by a Th1 immune response. Colonization by Staphylococcus aureus is increased in CRSwNP. We aimed to determine macrophage phenotypes in nasal mucosa of CRSwNP and CRSsNP and to examine phagocytosis of S. aureus in these pathologies. Methods: Macrophage phenotyping was performed by immunohistochemical staining on nasal mucosa sections from 28 patients; in addition flow cytometry analysis was performed. Tissue homogenate protein levels of IFN-γ, IL-5, IL-6, IL-1β, TGF-β, eosinophil cationic protein (ECP) and total IgE were analyzed and correlated with macrophage subtypes. Phagocytosis of S. aureus was analyzed by flow cytometry. Survival of S. aureus in Thp1 cells in the presence of polarizing cytokines was studied in vitro. Results: By immunohistochemical analysis more M2 macrophages were present in CRSwNP than in CRSsNP. This also was positively correlated with increased levels of IL-5, ECP and locally produced IgE and decreased levels of IL-6, IL-1β and IFN-γ. FACS analysis of dissociated nasal tissue confirmed the presence of increased numbers of M2 macrophages (CD206+HLADR+CD14+CD11c+CD20−) in CRSwNP as compared to controls, while the number of M1 macrophages (CD206−HLADR+CD14+CD11cintCD16−CD20−) was not different. Phagocytosis of S. aureus by human tissue derived macrophages was reduced in CRSwNP as compared to macrophages from the control inferior turbinates. Conclusions: Decreased phagocytosis of S. aureus and an M2 activation phenotype in CRSwNP could potentially contribute to persistence of chronic inflammation in CRSwNP.

It is unclear how Th2 immunity is induced in response to allergens like house dust mite (HDM). Here, we show that HDM inhalation leads to the TLR4/MyD88-dependent recruitment of IL-4 competent basophils and eosinophils, and of inflammatory DCs to the draining mediastinal nodes. Depletion of basophils only partially reduced Th2 immunity, and depletion of eosinophils had no effect on the Th2 response. Basophils did not take up inhaled antigen, present it to T cells, or express antigen presentation machinery, whereas a population of FceRI+ DCs readily did. Inflammatory DCs were necessary and sufficient for induction of Th2 immunity and features of asthma, whereas basophils were not required. We favor a model whereby DCs initiate and basophils amplify Th2 immunity to HDM allergen.

© 2015 Macmillan Publishers Limited. All rights reserved.Foxp3+ regulatory T cells (Tregs) maintain immune homoeostasis through mechanisms that remain incompletely defined. Here by two-photon (2P) imaging, we examine the cellular dynamics of endogenous Tregs. Tregs are identified as two non-overlapping populations in the T-zone and follicular regions of the lymph node (LN). In the T-zone, Tregs migrate more rapidly than conventional T cells (Tconv), extend longer processes and interact with resident dendritic cells (DC) and Tconv. Tregs intercept immigrant DCs and interact with antigen-induced DC:Tconv clusters, while continuing to form contacts with activated Tconv. During antigen-specific responses, blocking CTLA4-B7 interactions reduces Treg-Tconv interaction times, increases the volume of DC:Tconv clusters and enhances subsequent Tconv proliferation in vivo. Our results demonstrate a role for altered cellular choreography of Tregs through CTLA4-based interactions to limit T-cell priming.

Asthma is a T helper 2 (Th2)-cell-mediated disease; however, recent findings implicate Th17 and innate lymphoid cells also in regulating airway inflammation. Herein, we have demonstrated profound interleukin-21 (IL-21) production after house dust mite (HDM)-driven asthma by using T cell receptor (TCR) transgenic mice reactive to Dermatophagoides pteronyssinus 1 and an IL-21GFP reporter mouse. IL-21-producing cells in the mediastinal lymph node (mLN) bore characteristics of T follicular helper (Tfh) cells, whereas IL-21(+) cells in the lung did not express CXCR5 (a chemokine receptor expressed by Tfh cells) and were distinct from effector Th2 or Th17 cells. Il21r(-/-) mice developed reduced type 2 responses and the IL-21 receptor (IL-21R) enhanced Th2 cell function in a cell-intrinsic manner. Finally, administration of recombinant IL-21 and IL-25 synergistically promoted airway eosinophilia primarily via effects on CD4(+) lymphocytes. This highlights an important Th2-cell-amplifying function of IL-21-producing CD4(+) T cells in allergic airway inflammation.

To date, the mRNA delivery field has been heavily dominated by lipid-based systems. Reports on the use of nonlipid carriers for mRNA delivery in contrast are rare in the context of mRNA vaccination. This paper describes the potential of a cell-penetrating peptide containing the amphipathic RALA motif to deliver antigen-encoding mRNA to the immune system. RALA condenses mRNA into nanocomplexes that display acidic pH-dependent membrane disruptive properties. RALA mRNA nanocomplexes enable mRNA escape from endosomes and thereby allow expression of mRNA inside the dendritic cell cytosol. Strikingly, RALA mRNA nanocomplexes containing pseudouridine and 5-methylcytidine modified mRNA elicit potent cytolytic T cell responses against the antigenic mRNA cargo and show superior efficacy in doing so when compared to RALA mRNA nanocomplexes containing unmodified mRNA. RALA's unique sequence and structural organization are vital to act as mRNA vaccine vehicle, as arginine-rich peptide variants that lack the RALA motif show reduced mRNA complexation, impaired cellular uptake and lose the ability to transfect dendritic cells in vitro and to evoke T cell immunity in vivo.

Tumor-derived micro-RNA expression patterns are known to convey prognostic information in lung cancer. However, the cellular source of these signatures is unclear. In parallel, immunological parameters such as the presence and activation status of dendritic cells (DCs) emerge as important prognostic factors in this disease as well, presumably by affecting the quality of immune surveillance.We hypothesized that tumor-associated DCs are involved in the way tumor-derived miRNA expression patterns impact on disease outcome.Using a preclinical model of lung cancer featuring orthotopic tumor growth in syngeneic, immunocompetent hosts, we found that lung tumors were preferentially infiltrated by CD11c+/MHCII+/CD11b+/CD103- DCs (TIDCs). Compared to their peritumoral counterparts, displayed a dramatic overexpression of PD-L1 along with markers associated with tumor-associated macrophages (TAM). Transcriptome analysis of these CD11b+ TIDCs vs peritumoral CD11b+ DCs using GSEA and ISMARA indicated loss of immunogenicity, upregulation of features related to immunosuppressive TAMs, and exposure to hypoxia. Global miRNA profiling of CD11b+TIDCs revealed a specific signature dominated by a strong overexpression of mir-31, a prototypical lung cancer oncomir. Exposure to hypoxia appeared as a powerful driver of endogenous miR-31 expression in DCs. Confocal microscopy of intrapulmonary tumors confirmed that CD11c+ dendritic-shaped leukocytes colocalize within hypoxic regions. We found that miR-31 overexpression and hypoxia alike cause DCs to secrete the tumor-supporting factors VEGF, S100A8 and S100A9. Conditioned medium of miR-31-overexpression DCs induced pro-invasive cell morphology changes in lung carcinoma cells cultured on a collagen matrix, a phenomenon that could be reproduced by exposure to recombinant S100A8 protein. By re-processing TCGA expression data for non-coding RNA sequences, we discovered that the lung TIDC miRNA signature imparts a strong negative impact on overall survival in non-squamous lung cancer. Moreover, the TIDC-derived miRNA signature induced a profound difference in outcome among lung cancer patients with regional lymph node invasion.In summary, lung tumors predominantly recruit inflammatory-type DCs that are reprogrammed at the level of immuno-phenotype and micro-RNA repertoire. The miRNA signature of lung tumor-infiltrating DCs is dominated by the miR-31, a hypoxia-induced oncomir stimulating the release of pro-metastatic proteins by DCs. Finally, we show for the first time that a miRNA signature derived from a stromal immune cell has a dramatic impact on long term outcome in lung cancer. Citation Format: Elisabeth Brabants, Lotte Pyfferoen, Celine Everaert, Simon Tavernier, Kelly Heyns, Nancy De Cabooter, Glenn Wagemans, Kim Deswarte, Hamida Hammad, Olivier De Wever, Jo Vandesompele, Bart Lambrecht, Pieter Mestdagh, Karim Vermaelen. Specific myelomonocytic cells heavily infiltrate orthotopic lung tumors and display a hypoxia-driven miRNA expression signature that directs tumor-supporting functions and negatively impacts on clinical outcome [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A091.

To determine the role of vascular endothelial growth factor (Vegf) in embryonic erythroid development we have deleted or overexpressed Vegf specifically in the erythroid lineage using the EpoR-iCre transgenic line in combination with Cre/loxP conditional gain and loss of function Vegf alleles. ROSA26 promoter-based expression of the Vegf164 isoform in the early erythroid lineage resulted in a differentiation block of primitive erythroid progenitor (EryP) development and a partial block in definitive erythropoiesis between the erythroid burst-forming unit and erythroid colony-forming unit stages. Decreased mRNA expression levels of the key erythroid transcription factor Gata1 were causally linked to this phenotype. Conditional deletion of Vegf within the erythroid lineage was associated with increased Gata1 levels and increased erythroid differentiation. Expression of a ROSA26-based GATA2 transgene rescued Gata1 mRNA levels and target genes and restored erythroid differentiation in our Vegf gain of function model. These results demonstrate that Vegf modulates Gata1 expression levels in vivo and provides new molecular insight into Vegf's ability to modulate erythropoiesis.

The bloodstream is an important route of dissemination of invading pathogens. Most of the small bloodborne pathogens, like bacteria or viruses, are filtered by the spleen or liver sinusoids and presented to the immune system by dendritic cells (DCs) that probe these filters for the presence of foreign antigen (Ag). However, larger pathogens, like helminths or infectious emboli, that exceed 20 µm are mostly trapped in the vasculature of the lung. To determine if Ag trapped here can be presented to cells of the immune system, we used a model of venous embolism of large particulate Ag (in the form of ovalbumin [OVA]-coated Sepharose beads) in the lung vascular bed. We found that large Ags were presented and cross-presented to CD4 and CD8 T cells in the mediastinal lymph nodes (LNs) but not in the spleen or liver-draining LNs. Dividing T cells returned to the lungs, and a short-lived infiltrate consisting of T cells and DCs formed around trapped Ag. This infiltrate was increased when the Toll-like receptor 4 was stimulated and full DC maturation was induced by CD40 triggering. Under these conditions, OVA-specific cytotoxic T lymphocyte responses, as well as humoral immunity, were induced. The T cell response to embolic Ag was severely reduced in mice depleted of CD11chi cells or Ly6C/G+ cells but restored upon adoptive transfer of Ly6Chi monocytes. We conclude that the lung vascular filter represents a largely unexplored site of immune induction that traps large bloodborne Ags for presentation by monocyte-derived DCs.

Despite the major role played by regulatory T cells (Tregs) in immune homeostasis, our knowledge on mechanisms involved is still incomplete. Using two-photon microscopy we characterize the behavior of endogenous Tregs under steady-state conditions, during inflammation, and during an antigen-specific T cell response in lymph nodes. Under steady-state conditions, endogenous Tregs maintain the highest average velocities of any cells in the lymph node and exist as two non-overlapping populations that explore the T-zone and the B cell follicle. In the T-zone, Tregs migrate more rapidly than conventional T cells (Tconv), extend longer processes, and interact with resident dendritic cells (DC) and Tconv. Under inflammatory conditions, Tregs engage migratory DCs near the lymph node capsule, occasionally form long-lived Treg-DC interactions. During antigen specific immune response, Tregs interact with antigen-induced DC:Tconv clusters, while continuing to form contacts with activated Tconv. We investigated the role of CD80/86-CTLA4 interactions during Tconv-Treg cell interactions. Blocking CTLA-4 reduces Treg-Tconv interaction times, increases the volume of DC:Tconv clusters, and enhances proliferation of antigen-specific Tconv in vivo. Furthermore, genetic deletion of CD80/CD86 on Tconv makes them less susceptible to inhibition by Tregs. Our results demonstrate a role for altered cellular choreography of Tregs through CTLA4-based interactions to limit helper T cell priming.