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BACKGROUND: Asthma is often accompanied by type 2 immunity rich in IL-4, IL-5 and IL-13 cytokines produced by TH2 lymphocytes or type 2 innate lymphoid cells (ILC2s). Interleukin-2 family cytokines play a key role in the differentiation, homeostasis and effector function of innate and adaptive lymphocytes. OBJECTIVE: IL-9 and IL-21 boost the activation and proliferation of TH2 and ILC2s, but the relative importance and potential synergism between these γc cytokines is currently unknown. METHODS: Using newly generated antibodies, we inhibited IL-9 and IL-21 alone or in combination, in various murine models of asthma. In a translational approach using segmental allergen challenge, we recently described elevated IL-9 levels in human allergic asthmatics in comparison to non-asthmatic controls. Here, we also measured IL-21 in both groups. RESULTS: IL-9 played a central role in controlling innate IL-33 induced lung inflammation by promoting proliferation and activation of ILC2s, in an IL-21 independent manner. Conversely, chronic house dust mite induced airway inflammation, mainly driven by adaptive immunity, was solely dependent on IL-21, that controlled TH2 activation, eosinophilia, total serum IgE and formation of tertiary lymphoid structures. In a model of innate on adaptive immunity driven by papain allergen, a clear synergy was found between both pathways, since combined anti-IL-9 or anti-IL-21 blockade was superior in reducing key asthma features. In human bronchoalveolar lavage (BAL) samples we measured elevated IL-21 protein within the allergic asthmatic group, compared with the allergic control group. We also found increased IL21R transcripts and predicted IL-21 ligand activity in various disease-associated cell subsets. CONCLUSION: IL-9 and IL-21 play important and non-redundant roles in allergic asthma by boosting ILC2s and TH2 cells, revealing a dual IL-9 and IL-21 targeting strategy as a new and testable approach.
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Cancer vaccines aim at generating cytotoxic CD8+ T cells that kill cancer cells and confer durable tumor regression. Hereto, CD8+ peptide epitopes should be presented by antigen presenting cells to CD8+ T cells in lymphoid tissue. Unfortunately, in unformulated soluble form, peptide antigens are poorly taken up by antigen presenting cells and do not efficiently reach lymph nodes. Hence, the lack of efficient delivery remains a major limitation for successful clinical translation of cancer vaccination using peptide antigens. Here we propose a generic peptide nanoformulation strategy by extending the amino acid sequence of the peptide antigen epitope with 10 glutamic acid residues. The resulting overall anionic charge of the peptide allows encapsulation into lipid nanoparticles (peptide-LNP) by electrostatic interaction with an ionizable cationic lipid. We demonstrate that intravenous injection of peptide-LNP efficiently delivers the peptide to immune cells in the spleen. Peptide-LNP that co-encapsulate an imidazoquinoline TLR7/8 agonist (IMDQ) induce robust innate immune activation in a broad range of immune cell subsets in the spleen. Peptide-LNP containing the minimal CD8+ T cell epitope of the HPV type 16 E7 oncoprotein and IMDQ induces high levels of antigen-specific CD8+ T cells in the blood, and can confer protective immunity against E7-expressing tumors in both prophylactic and therapeutic settings.
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Ratones Endogámicos C57BL , Nanopartículas , Receptor Toll-Like 7 , Receptor Toll-Like 8 , Animales , Nanopartículas/química , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/administración & dosificación , Ratones , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Lípidos/química , Péptidos/química , Femenino , Proteínas E7 de Papillomavirus/inmunología , Quinolinas/farmacología , Quinolinas/química , Imidazoles/química , Imidazoles/farmacologíaRESUMEN
Acute systemic inflammation critically alters the function of the immune system, often promoting myelopoiesis at the expense of lymphopoiesis. In the thymus, systemic inflammation results in acute thymic atrophy and, consequently, impaired T-lymphopoiesis. The mechanism by which systemic inflammation impacts the thymus beyond suppressing T-cell development is still unclear. Here, we describe how the synergism between TL1A and IL-18 suppresses T-lymphopoiesis to promote thymic myelopoiesis. The protein levels of these two cytokines were elevated in the thymus during viral-induced thymus atrophy infection with murine cytomegalovirus (MCMV) or pneumonia virus of mice (PVM). In vivo administration of TL1A and IL-18 induced acute thymic atrophy, while thymic neutrophils expanded. Fate mapping with Ms4a3-Cre mice demonstrated that thymic neutrophils emerge from thymic granulocyte-monocyte progenitors (GMPs), while Rag1-Cre fate mapping revealed a common developmental path with lymphocytes. These effects could be modeled ex vivo using neonatal thymic organ cultures (NTOCs), where TL1A and IL-18 synergistically enhanced neutrophil production and egress. NOTCH blockade by the LY411575 inhibitor increased the number of neutrophils in the culture, indicating that NOTCH restricted steady-state thymic granulopoiesis. To promote myelopoiesis, TL1A, and IL-18 synergistically increased GM-CSF levels in the NTOC, which was mainly produced by thymic ILC1s. In support, TL1A- and IL-18-induced granulopoiesis was completely prevented in NTOCs derived from Csf2rb-/- mice and by GM-CSFR antibody blockade, revealing that GM-CSF is the essential factor driving thymic granulopoiesis. Taken together, our findings reveal that TL1A and IL-18 synergism induce acute thymus atrophy while promoting extramedullary thymic granulopoiesis in a NOTCH and GM-CSF-controlled manner.
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Factor Estimulante de Colonias de Granulocitos y Macrófagos , Interleucina-18 , Timo , Miembro 15 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Animales , Interleucina-18/metabolismo , Timo/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Ratones , Miembro 15 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Ratones Endogámicos C57BL , Granulocitos/metabolismo , Mielopoyesis , Neutrófilos/inmunología , Neutrófilos/metabolismo , Receptores Notch/metabolismo , Linfopoyesis , AtrofiaAsunto(s)
Asma , Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos , Asma/inmunología , Humanos , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/genética , Inmunoterapia Adoptiva/métodos , Animales , Linfocitos T/inmunología , Ratones , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
The T cell population size is stringently controlled before, during, and after immune responses, as improper cell death regulation can result in autoimmunity and immunodeficiency. RIPK1 is an important regulator of peripheral T cell survival and homeostasis. However, whether different peripheral T cell subsets show a differential requirement for RIPK1 and which programmed cell death pathway they engage in vivo remains unclear. In this study, we demonstrate that conditional ablation of Ripk1 in conventional T cells (Ripk1ΔCD4) causes peripheral T cell lymphopenia, as witnessed by a profound loss of naive CD4+, naive CD8+, and FoxP3+ regulatory T cells. Interestingly, peripheral naive CD8+ T cells in Ripk1ΔCD4 mice appear to undergo a selective pressure to retain RIPK1 expression following activation. Mixed bone marrow chimeras revealed a competitive survival disadvantage for naive, effector, and memory T cells lacking RIPK1. Additionally, tamoxifen-induced deletion of RIPK1 in CD4-expressing cells in adult life confirmed the importance of RIPK1 in post-thymic survival of CD4+ T cells. Ripk1K45A mice showed no change in peripheral T cell subsets, demonstrating that the T cell lymphopenia was due to the scaffold function of RIPK1 rather than to its kinase activity. Enhanced numbers of Ripk1ΔCD4 naive T cells expressed the proliferation marker Ki-67+ despite the peripheral lymphopenia and single-cell RNA sequencing revealed T cell-specific transcriptomic alterations that were reverted by additional caspase-8 deficiency. Furthermore, Ripk1ΔCD4Casp8 ΔCD4 and Ripk1ΔCD4Tnfr1-/- double-knockout mice rescued the peripheral T cell lymphopenia, revealing that RIPK1-deficient naive CD4+ and CD8+ cells and FoxP3+ regulatory T cells specifically die from TNF- and caspase-8-mediated apoptosis in vivo. Altogether, our findings emphasize the essential role of RIPK1 as a scaffold in maintaining the peripheral T cell compartment and preventing TNFR1-induced apoptosis.
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Apoptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Receptores Tipo I de Factores de Necrosis Tumoral , Linfocitos T Reguladores , Animales , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Ratones , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Ratones Endogámicos C57BL , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Ratones Noqueados , Caspasa 8/metabolismo , Linfopenia/patología , Linfopenia/inmunologíaRESUMEN
The activities, ontogeny, and mechanisms of lineage expansion of eosinophils are less well resolved than those of other immune cells, despite the use of biological therapies targeting the eosinophilia-promoting cytokine interleukin (IL)-5 or its receptor, IL-5Rα. We combined single-cell proteomics and transcriptomics and generated transgenic IL-5Rα reporter mice to revisit eosinophilopoiesis. We reconciled human and murine eosinophilopoiesis and provided extensive cell-surface immunophenotyping and transcriptomes at different stages along the continuum of eosinophil maturation. We used these resources to show that IL-5 promoted eosinophil-lineage expansion via transit amplification, while its deletion or neutralization did not compromise eosinophil maturation. Informed from our resources, we also showed that interferon response factor-8, considered an essential promoter of myelopoiesis, was not intrinsically required for eosinophilopoiesis. This work hence provides resources, methods, and insights for understanding eosinophil ontogeny, the effects of current precision therapeutics, and the regulation of eosinophil development and numbers in health and disease.
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Linaje de la Célula , Eosinófilos , Interleucina-5 , Ratones Transgénicos , Proteómica , Análisis de la Célula Individual , Transcriptoma , Eosinófilos/inmunología , Eosinófilos/metabolismo , Animales , Interleucina-5/metabolismo , Interleucina-5/genética , Humanos , Ratones , Proteómica/métodos , Análisis de la Célula Individual/métodos , Diferenciación Celular/inmunología , Ratones Endogámicos C57BL , Perfilación de la Expresión Génica/métodos , Subunidad alfa del Receptor de Interleucina-5/metabolismo , Subunidad alfa del Receptor de Interleucina-5/genética , Mielopoyesis/genética , Factores Reguladores del Interferón/metabolismo , Factores Reguladores del Interferón/genética , Ratones NoqueadosRESUMEN
Asthma is a chronic, heterogeneous disease of the airways, often characterised by structural changes known collectively as airway remodelling. In response to environmental insults, including pathogens, allergens and pollutants, the epithelium can initiate remodelling via an inflammatory cascade involving a variety of mediators that have downstream effects on both structural and immune cells. These mediators include the epithelial cytokines thymic stromal lymphopoietin, interleukin (IL)-33 and IL-25, which facilitate airway remodelling through cross-talk between epithelial cells and fibroblasts, and between mast cells and airway smooth muscle cells, as well as through signalling with immune cells such as macrophages. The epithelium can also initiate airway remodelling independently of inflammation in response to the mechanical stress present during bronchoconstriction. Furthermore, genetic and epigenetic alterations to epithelial components are believed to influence remodelling. Here, we review recent advances in our understanding of the roles of the epithelium and epithelial cytokines in driving airway remodelling, facilitated by developments in genetic sequencing and imaging techniques. We also explore how new and existing therapeutics that target the epithelium and epithelial cytokines could modify airway remodelling.
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Remodelación de las Vías Aéreas (Respiratorias) , Asma , Humanos , Citocinas , Linfopoyetina del Estroma Tímico , EpitelioRESUMEN
Sublingual allergen immunotherapy (SLIT) is an emerging treatment option for allergic asthma and a potential disease-modifying strategy for asthma prevention. The key cellular events leading to such long-term tolerance remain to be fully elucidated. We administered prophylactic SLIT in a mouse model of house dust mite (HDM)-driven allergic asthma. HDM extract was sublingually administered over 3 weeks followed by intratracheal sensitization and intranasal challenges with HDM. Prophylactic SLIT prevented allergic airway inflammation and hyperreactivity with a low lab-to-lab variation. The HDM-specific T helper (Th)2 (cluster of differentiation 4 Th) response was shifted by SLIT toward a regulatory and Th17 response in the lung and mediastinal lymph node. By using Derp1-specific cluster of differentiation 4+ T cells (1-DER), we found that SLIT blocked 1-DER T cell recruitment to the mediastinal lymph node and dampened IL-4 secretion following intratracheal HDM sensitization. Sublingually administered Derp1 protein activated 1-DER T cells in the cervical lymph node via chemokine receptor7+ migratory dendritic cells (DC). DCs migrating from the oral submucosa to the cervical lymph node after SLIT-induced Foxp3+ regulatory T cells. When mice were sensitized with HDM, prior prophylactic SLIT increased Derp1 specific regulatory T cells (Tregs) and lowered Th2 recruitment in the lung. By using Foxp3-diphtheria toxin receptor mice, Tregs were found to contribute to the immunoregulatory prophylactic effect of SLIT on type 2 immunity. These findings in a mouse model suggest that DC-mediated functional Treg induction in oral mucosa draining lymph nodes is one of the driving mechanisms behind the disease-modifying effect of prophylactic SLIT.
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Antígenos Dermatofagoides , Asma , Células Dendríticas , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead , Pyroglyphidae , Inmunoterapia Sublingual , Linfocitos T Reguladores , Células Th2 , Animales , Linfocitos T Reguladores/inmunología , Células Dendríticas/inmunología , Ratones , Pyroglyphidae/inmunología , Factores de Transcripción Forkhead/metabolismo , Antígenos Dermatofagoides/inmunología , Asma/inmunología , Asma/terapia , Asma/prevención & control , Inmunoterapia Sublingual/métodos , Células Th2/inmunología , Proteínas de Artrópodos/inmunología , Femenino , Alérgenos/inmunología , Alérgenos/administración & dosificación , Ratones Endogámicos BALB C , Humanos , Desensibilización Inmunológica/métodos , Cisteína Endopeptidasas/inmunología , Cisteína Endopeptidasas/metabolismoRESUMEN
Spontaneous protein crystallization is a rare event, yet protein crystals are frequently found in eosinophil-rich inflammation. In humans, Charcot-Leyden crystals (CLCs) are made from galectin-10 (Gal10) protein, an abundant protein in eosinophils. Although mice do not encode Gal10 in their genome, they do form pseudo-CLCs, made from the chitinase-like proteins Ym1 and/or Ym2, encoded by Chil3 and Chil4 and made by myeloid and epithelial cells respectively. Here, we investigated the biological effects of pseudo-CLCs since their function is currently unknown. We produced recombinant Ym1 crystals which were shown to have identical crystal packing and structure by X-ray crystallography as in vivo native crystals derived from murine lung. When administered to the airways of mice, crystalline but not soluble Ym1 stimulated innate and adaptive immunity and acted as a type 2 immune adjuvant for eosinophilic inflammation via triggering of dendritic cells (DCs). Murine Ym1 protein crystals found at sites of eosinophilic inflammation reinforce type 2 immunity and could serve as a surrogate model for studying the biology of human CLCs.
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Inmunidad Adaptativa , Quitinasas , Animales , Humanos , Ratones , Adyuvantes Inmunológicos , Cristalización , InflamaciónRESUMEN
The filamentous fungus Aspergillus causes a wide spectrum of diseases in the human lung, with Aspergillus fumigatus being the most pathogenic and allergenic subspecies. The broad range of clinical syndromes that can develop from the presence of Aspergillus in the respiratory tract is determined by the interaction between host and pathogen. In this review, an oversight of the different clinical entities of pulmonary aspergillosis is given, categorized by their main pathophysiological mechanisms. The underlying immune processes are discussed, and the main clinical, radiological, biochemical, microbiological, and histopathological findings are summarized.
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Pulmón , Aspergilosis Pulmonar , Humanos , Aspergillus , Aspergilosis Pulmonar/diagnóstico , Aspergilosis Pulmonar/tratamiento farmacológico , Aspergilosis Pulmonar/etiología , Aspergillus fumigatusRESUMEN
INTRODUCTION: Intravenous vedolizumab is a widely used monoclonal antibody for outpatients with inflammatory bowel disease. Drug preparation is performed on the day of administration, but is time consuming, causing unnecessary in-hospital patient delay and inefficient logistics for preparation and distribution. Storage of vedolizumab ready-to-administer infusions and distribution via pneumatic air tubes could streamline logistics in the outpatient setting. The aim of this study was to test the shelf life and stability of ready-to-administer intravenous infusion bags containing vedolizumab. METHODS: For assessing in-use shelf life, the reconstituted product (300 mg fixed dose) was diluted to a concentration of 1.2 mg/mL in 0.9% NaCl under aseptic conditions, and stored in polyolefin infusion bags at 2-8°C prior to analysis. On replicate samples, we measured concentration, physical and chemical stability using sodium dodecyl sulphate polyacrylamide gel electrophoresis, size exclusion chromatography, and multi-angle laser light scattering, as well as biological activity using a biolayer interferometry assay to study target engagement, and endotoxin content to assess microbiological stability. Stability of ready-to-use vedolizumab was assessed also after transportation via pneumatic tube system. Samples were taken at different time points over an observation period of 30 days on four replicate samples. RESULTS: For all parameters assessed, the ready-to-use solution of vedolizumab remained stable over a period of at least 30 days. There were no signs of protein aggregation, chemical instability, or loss of binding of the antibody to the α4ß7 integrin target. There was no increase in endotoxin concentration over time. No significant difference was seen in antibody structural stability and protein aggregation between samples before and after transportation via pneumatic tube system. CONCLUSION: When prepared under aseptic conditions, dissolved ready-to-administer vedolizumab infusion bags can be stored long term at 2-8°C and transported via pneumatic air tube, without observable loss of antibody stability or binding activity.
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The unfolded protein response (UPR) aims to restore ER homeostasis under conditions of high protein folding load, a function primarily serving secretory cells. Additional, non-canonical UPR functions have recently been unraveled in immune cells. We addressed the function of the inositol-requiring enzyme 1 (IRE1) signaling branch of the UPR in NK cells in homeostasis and microbial challenge. Cell-intrinsic compound deficiency of IRE1 and its downstream transcription factor XBP1 in NKp46+ NK cells, did not affect basal NK cell homeostasis, or overall outcome of viral MCMV infection. However, mixed bone marrow chimeras revealed a competitive advantage in the proliferation of IRE1-sufficient Ly49H+ NK cells after viral infection. CITE-Seq analysis confirmed strong induction of IRE1 early upon infection, concomitant with the activation of a canonical UPR signature. Therefore, we conclude that IRE1/XBP1 activation is required during vigorous NK cell proliferation early upon viral infection, as part of a canonical UPR response.