Influenza-associated pulmonary aspergillosis (IAPA) is a severe complication of influenza infection associated with substantial mortality. Influenza disrupts pulmonary host defenses and alters innate immune responses, predisposing patients to invasive fungal infection. Interleukin-27 (IL-27) is an immunoregulatory cytokine with context-dependent antiviral and antifungal effects; however, its role during IAPA remains undefined. A mouse model of IAPA was established by infecting wild-type and IL-27 receptor α-deficient ( Il27ra - / - ) mice with influenza A, followed by Aspergillus fumigatus challenge. IL-27 and IL-27Rα expression were increased during IAPA. Single-cell RNA sequencing identified monocytes as the primary source of IL-27 and T cells as major IL-27rα-expressing cells. Il27ra - / - mice exhibited significantly increased pulmonary fungal and influenza viral burden, enhanced type 2 immune responses characterized by elevated IL-4, IL-5, IL-9, IL-13, eosinophils, Th2 cells, pathogenic Th2 cells, and ILC2s. Despite increased eosinophil abundance, eosinophil-mediated conidial killing was impaired in Il27ra - / - mice. IL-27Rα deficiency also reduced macrophage abundance and impaired macrophage conidial uptake. Conversely, timed administration of rIL-27 enhanced fungal clearance, improved survival, and increased macrophage conidial uptake and augmented eosinophil killing capacity during IAPA. IL-27 signaling is a protective immunoregulatory cytokine during IAPA that limits pathological type 2 inflammation and enhances antifungal effector function of both eosinophils and macrophages. These findings identify IL-27 as a potential therapeutic in IAPA.

Severe antibody-mediated rejection (AMR) is associated with chronic lung allograft dysfunction (CLAD) and death in lung transplant recipients. However, AMR diagnostics are imprecise, and new biomarkers are needed. We assessed whether airway inflammation gene signatures could distinguish AMR cases from controls. We analyzed small airway brush RNA sequencing from 16 AMR cases and 39 controls across 2 centers. We compared gene signatures for complement activation, molecular target of rapamycin (mTOR) signaling, and natural killer cell-mediated injury. Differential gene expression and pathway analyses sought AMR molecular features. Additionally, we evaluated the airway inflammation 2 (AI2) score, previously associated with CLAD and graft failure, in relation to AMR clinical features and survival outcomes. AMR airway brushes demonstrated transcriptional evidence of airway inflammation and upregulation of complement and natural killer cell pathways. The AI2 score was significantly elevated in AMR cases (P < .001) and was associated with AMR-compatible histology, donor-specific antibodies, complement binding, and acute graft dysfunction. Increasing AI2 scores were associated with worse retransplant-free survival, independent of other AMR features. In small airways, lung transplant AMR predominantly exhibits molecular features of cellular rejection rather than a distinct humoral rejection profile. Airway brush transcriptomics may provide a valuable tool for characterizing and prognosticating suspected AMR.

NK and T cells are key effectors that eliminate cancer cells, but upregulation of the inhibitory receptor NKG2A on these cells attenuates antitumor immune responses. To counteract NKG2A inhibitory signaling, we identified two specific fully human monoclonal anti-NKG2A antibodies that block HLA-E ligand binding. These antibodies activated NK cells and enhanced antibody-dependent cellular cytotoxicity of tumor-targeting IgG1s both in vitro and in vivo. Bispecific engagers (BiNKs), generated by fusing NKG2A antibodies with tumor targeting binders, promoted immune synapse formation and directed cytotoxicity of NK and CD8+ T cells toward cancer cells. In a human PBMC-engrafted NSG mouse xenograft lung cancer model, an anti-HER2 × anti-NKG2A BiNK markedly inhibited tumor growth as a monotherapy or in combination with pertuzumab. Cell depletion studies revealed that the BiNK enhanced antitumor activity of both NK and T cells. NKG2A blockade with potent and specific, fully human antibodies and BiNKs show promise for further development as cancer immunotherapeutics.

Background:Influenza-associated pulmonary aspergillosis (IAPA) is a severe complication of influenza infection associated with prolonged intensive care unit stay and increased mortality. Although impaired antifungal immunity has been implicated in IAPA pathogenesis, the cell type-specific immune mechanisms driving susceptibility remain incompletely understood. We aimed to characterize the pulmonary immune landscape during IAPA using single-cell transcriptomics and functional neutrophil assays. Methods:Male C57BL/6 mice were assigned to naïve control, influenza A/PR/8/34 (H1N1) infection, A. fumigatus (ATCC 42202) infection, or IAPA groups. Lung CD45 + immune cells underwent single-cell RNA sequencing with downstream clustering and CellChat ligand-receptor interaction analysis. Differential gene expression analyses were performed across myeloid, lymphoid, and neutrophil populations. Functional neutrophil responses were evaluated using flow cytometry, myeloperoxidase activity assays, and FLARE (fluorescent Aspergillus reporter) conidia to assess fungal conidia uptake and killing. Cross-species validation was performed using gene set enrichment analysis compared with published human IAPA transcriptomic datasets. Results:IAPA broadly remodeled the pulmonary immune landscape across myeloid, lymphoid, and neutrophil compartments. Myeloid cells showed coordinated suppression of fungal pattern recognition receptors, lysosomal biogenesis programs, and inflammatory signaling. Lymphoid populations exhibited transcriptional signatures of T cell exhaustion and Th17 suppression. Within the neutrophil compartment, we identified two transcriptionally and functionally distinct populations, conventional and inflammatory neutrophils, with divergent antifungal effector capacities. Inflammatory neutrophils showed selective killing defects, while both subsets exhibited impaired phagocytic uptake during IAPA. Murine transcriptomic findings demonstrated strong concordance with immune dysfunction signatures identified in human IAPA. Conclusion:IAPA susceptibility arises from coordinated transcriptional dysfunction spanning innate and adaptive immune compartments. Distinct neutrophil subset dysfunction, impaired fungal recognition pathways, and T-cell exhaustion signatures collectively contribute to defective fungal clearance, providing mechanistic insight into IAPA susceptibility and potential therapeutic targets.

Virus-like nanoparticles (VLPs) are naturally occurring polymeric nanomaterials formed by the self-assembly of one or more viral capsid proteins (CPs). VLPs have garnered significant attention in a wide range of nanotechnology-based diagnostics and therapies, including immunotherapy and drug delivery. VLPs are Exhibiting high biocompatibility and biodegradability, alone with uniform structure and controlled assembly, VLPs represent a complex and versatile therapeutic platform. This review provides an overview of the fundamental aspects of VLPs, including their types, structures, immune mechanisms, as well as expression and purification method. Recent advances in the development and application of engineered VLPs for drug delivery, gene therapy, immunology studies and multifunctional therapeutics are discussed. This paper also summarised research advances in the use of virus-like particles as chimaeric vectors and highlights their potential as efficient delivery vehicles. Finally, we present several successful examples of VLPs-based drug delivery system and provide a comprehensive analysis of VLPs that takes advantage of both viral and non-viral delivery methods for efficient theranostic application.

Viral lower respiratory tract infections are common early in life and are associated with long-term development of asthma, a chronic condition defined by reversible airflow obstruction secondary to inflammation. Understanding the immunological mechanism connecting these two pathologies observed early in life becomes imperative to guide therapeutic measures. To investigate this connection, neonatal (days 4-6) or adult mice were infected with human metapneumovirus (HMPV) followed by a secondary HMPV infection 6 weeks later. Mice initially infected as neonates demonstrated increased mucus production, eosinophil recruitment, airway hyperresponsiveness, and Th2 T cell differentiation after rechallenge compared with adult mice rechallenged with HMPV. Neonatal HMPV infection led to formation of Th2 clonally expanded tissue-resident memory (TRM) T cells that were absent after adult HMPV. FTY720-mediated disruption of lymphocyte circulation demonstrated that TRMs contributed to pathology. Local depletion of lung CD4+ T cells and JAK2 inhibition mitigated pathology. These findings suggest TRMs uniquely generated after early-life viral infection can contribute to Th2-driven asthma pathology.

Chronic lung allograft dysfunction (CLAD) is the major barrier for long-term survival in lung transplant recipients (LTRs). CLAD remains a diagnosis of exclusion with poor responses to therapies. A molecular diagnostic for CLAD is needed to risk-stratify LTRs for prognosis and identify new targets to mitigate CLAD progression. We used weighted gene correlation network analysis on the airway brush-derived airway transcriptome to identify immune pathways and markers relevant to CLAD. Weighted gene correlation network analysis was performed on RNA sequencing from airway brushings of 37 LTRs with CLAD compared with 37 stable LTRs. We analyzed gene coexpression networks (modules) for their biological significance and association with CLAD. Three gene modules were positively correlated with CLAD, its severity, allograft dysfunction, and survival. These enriched components of the acute phase response, type 1 adaptive immunity, and innate immunity, respectively. A fourth module correlated with protection and was inversely correlated with the other modules. We validated our findings by identification of downstream protein and eicosanoid levels in the bronchoalveolar lavage, and an external validation cohort where module expression differentiated LTRs with CLAD and correlated with worse survival. The CLAD airway transcriptome enriches for coexpression networks associated with network modules that correlate with allograft dysfunction and survival.

Staphylococcus aureus is a leading cause of lethal bacteremia and pneumonia, which are driven by potent virulence factors such as T-cell superantigens and alpha hemolysin. S. aureus has among the highest rates of antibiotic resistance, yet no vaccines or alternative therapies are available. Here, we developed a repertoire of potent, high-affinity nanobodies (Nbs) targeting key toxins in S. aureus infection, including Hla and superantigens SEB, SEC, and TSST-1. Comprehensive cryo-EM and AlphaFold3 analyses of these Nbs, which were elicited with clinical cocktail vaccines, revealed diverse neutralizing epitopes and mechanisms that provide insights for immunotherapy and vaccine strategies. Guided by these findings, we engineered stable, multivalent, and multifunctional Nb constructs. These constructs included an aerosolizable trimeric Nb with enhanced neutralization activity against Hla and SEC, and a decameric Nb-IgG-Fc fusion construct with pM or better potencies against a wide range of major toxins in S. aureus sepsis (SEB, SEC, TSST-1, and Hla). These multifunctional Nbs demonstrated protective activity in murine models of pneumonia and sepsis, underscoring their potential as versatile immunotherapies that address the complex virulence of S. aureus. Our work lays a foundation for precision immunotherapies beyond current treatment options to combat complex bacterial infections with multiple virulence mechanisms.

Abstract Introduction Identifying tissue resident memory T cells (TRM) in human lungs has traditionally relied on imprecise surrogate markers of tissue retention, markers that can change based on activation state. Using ex vivo lung perfusion (EVLP), we can identify circulating and tissue resident T cells in a functional manner. Better knowledge of differential gene regulation in TRM will allow for improved analysis of their role in various disease states, as well as identify potential therapeutic targets. Methods Human lungs declined for organ donation were ventilated and attached to a normothermic EVLP system for 2-4 hours (2 experiments with 2 human lungs each). A biotinylated CD45 antibody was instilled into the perfusate to label circulating cells for the final 20 minutes; tissue-resident cells were identified as those cells “protected” from the circulating antibody. Immune cells were isolated and then stained with oligo-tagged streptavidin as well as oligo-tagged antibodies against several traditional TRM markers in order to perform simultaneous single cell RNA sequencing and CITE-seq. Results UMAP clustering of scRNA-seq data identified 13 discrete T cell clusters with differential proportions of labeled vs. protected cells. Clusters corresponding to canonical CD4+ and CD8+ TRM populations contained almost no labeled cells. Differential gene analysis of protected vs labeled CD8+ T cells show that functionally defined CD8+ TRM have a gene expression signature similar, but not identical, to canonical marker-defined TRM. Of particular interest is upregulation of galectin-3 (LGALS3), a member of the lectin family previously implicated in inflammatory and fibrotic disease. Conclusion EVLP allows for effective functional identification of tissue resident T cells in the human lung, which overlap with but are not identical to TRM defined by canonical surface markers. Future directions include kinetics of antibody labeling, validation of differentially regulated gene targets, and analysis of TCR sequencing data. Funding Source T32HL007563 Topic Categories Immune Mechanisms of Human Disease (HUM)

Elucidating the mechanisms underlying vascular injury and repair may guide development of therapies against vascular cognitive impairment and dementia (VD). We employed single-cell RNA sequencing to map cell populations and explore vascular responses in mouse brains collected 42 days after asymmetric common carotid artery stenosis (ACAS)-induced chronic cerebral hypoperfusion. A unique tip cell type was identified among endothelial cell (EC) clusters and validated by immunostaining. Gene ontology analyses suggested tip cell enrichment in angiogenesis and the involvement of Apln/Aplnr signaling. Immunoblotting confirmed an increase in apelin (Apln) protein after ACAS. In EC cultures, [Pyr1]-Apelin-13 (Apln13), a selective endogenous apelin receptor agonist, enhanced EC proliferation, migration, and tube formation. Treatment with Apln13 also improved angiogenesis, white matter integrity, and cognitive functions in ACAS mice. Cell-cell interaction analyses highlighted astrocyte-tip cell crosstalk via Vegfa-Vegfr interactions.

The activation of IL-17 signaling has been linked to the pathogenesis of many chronic, inflammatory lung diseases including Cystic Fibrosis (CF). Through unbiased single-cell RNAseq screening, we found that IL-17+ T cells highly express Srm and Smox, which encode two key enzymes for spermidine synthesis. Spermidine has been shown to reduce inflammation by regulating macrophage activation and balancing Th17/Treg differentiation, but its direct effects on Th17 cytokine production has not been carefully investigated. Here, using already differentiated Th17 cells from cultured mouse splenocytes, we found that exogenous spermidine directly inhibits IL-1β/IL-23 induced IL-17 production. Blockade of endogenous spermidine synthesis enhanced IL-17 production above native levels, further supporting that spermidine is a direct regulator of cytokine secretion independent of differentiation. In vivo, spermidine alleviates lung inflammation in both PA infection and LPS induced acute lung injury models. Further RNA-seq analysis suggests spermidine suppression of Th17 cytokine production is mediated through its PRDX1 dependent antioxidant activity. Our data establishes that spermidine is a direct regulator of Type-17 T cell cytokine production and has potent anti-inflammatory effects against lung inflammation.

Chronic lung allograft dysfunction (CLAD) substantially limits long-term survival following lung transplantation. To identify potential targets for CLAD prevention, T cells from explanted CLAD lungs and lung-draining lymph nodes, as well as diseased and nondiseased controls were isolated and single-cell RNA sequencing and TCR sequencing were performed. TCR sequencing revealed a clonally expanded population of CD8+ tissue-resident memory T cells (TRMs) with high cytotoxic potential, including upregulation of KLRK1, encoding the co-receptor NKG2D. These cytotoxic CD8+ TRMs accumulated around the CLAD airways and had a 100-fold increase in clonal overlap with lung-draining lymph nodes when compared with non-CLAD lungs. Using a murine model of orthotopic lung transplantation, we confirmed that cytotoxic CD8+ TRM accumulation was due to chronic rejection and not transplantation alone. Furthermore, blocking NKG2D in vivo attenuated the airway remodeling following transplantation and diminished airway accumulation of CD8+ T cells. Our findings support NKG2D as a potential therapeutic target for CLAD, affecting cytotoxic CD8+ TRM accumulation.

ABSTRACT Human metapneumovirus (HMPV) is a primary cause of acute respiratory infection, yet there are no approved vaccines or antiviral therapies for HMPV. Early host responses to HMPV are poorly characterized, and further understanding could identify important antiviral pathways. Type III interferon (IFN-λ) displays potent antiviral activity against respiratory viruses and is being investigated for therapeutic use. However, its role in HMPV infection remains largely unknown. Here, we show that IFN-λ is highly upregulated during HMPV infection in vitro in human and mouse airway epithelial cells and in vivo in mice. We found through several immunological and molecular assays that type II alveolar cells are the primary producers of IFN-λ. Using mouse models, we show that IFN-λ limits lung HMPV replication and restricts virus spread from upper to lower airways but does not contribute to clinical disease. Moreover, we show that IFN-λ signaling is predominantly mediated by CD45 - non-immune cells. Mice lacking IFN-λ signaling showed diminished loss of ciliated epithelial cells and decreased recruitment of lung macrophages in early HMPV infection along with higher inflammatory cytokine and interferon-stimulated gene expression, suggesting that IFN-λ may maintain immunomodulatory responses. Administration of IFN-λ for prophylaxis or post-infection treatment in mice reduced viral load without inflammation-driven weight loss or clinical disease. These data offer clinical promise for IFN-λ in HMPV treatment. IMPORTANCE Human metapneumovirus (HMPV) is a common respiratory pathogen and often contributes to severe disease, particularly in children, immunocompromised people, and the elderly. There are currently no licensed HMPV antiviral treatments or vaccines. Here, we report novel roles of host factor IFN-λ in HMPV disease that highlight therapeutic potential. We show that IFN-λ promotes lung antiviral responses by restricting lung HMPV replication and spread from upper to lower airways but does so without inducing lung immunopathology. Our data uncover recruitment of lung macrophages, regulation of ciliated epithelial cells, and modulation of inflammatory cytokines and interferon-stimulated genes as likely contributors. Moreover, we found these roles to be distinct and non-redundant, as they are not observed with knockout of, or treatment with, type I IFN. These data elucidate unique antiviral functions of IFN-λ and suggest IFN-λ augmentation as a promising therapeutic for treating HMPV disease and promoting effective vaccine responses.

Spatial transcriptomics technologies have shed light on the complexities of tissue structures by accurately mapping spatial microenvironments. Nonetheless, a myriad of methods, especially those utilized in platforms like Visium, often relinquish spatial details owing to intrinsic resolution limitations. In response, we introduce TransformerST, an innovative, unsupervised model anchored in the Transformer architecture, which operates independently of references, thereby ensuring cost-efficiency by circumventing the need for single-cell RNA sequencing. TransformerST not only elevates Visium data from a multicellular level to a single-cell granularity but also showcases adaptability across diverse spatial transcriptomics platforms. By employing a vision transformer-based encoder, it discerns latent image-gene expression co-representations and is further enhanced by spatial correlations, derived from an adaptive graph Transformer module. The sophisticated cross-scale graph network, utilized in super-resolution, significantly boosts the model's accuracy, unveiling complex structure-functional relationships within histology images. Empirical evaluations validate its adeptness in revealing tissue subtleties at the single-cell scale. Crucially, TransformerST adeptly navigates through image-gene co-representation, maximizing the synergistic utility of gene expression and histology images, thereby emerging as a pioneering tool in spatial transcriptomics. It not only enhances resolution to a single-cell level but also introduces a novel approach that optimally utilizes histology images alongside gene expression, providing a refined lens for investigating spatial transcriptomics.

Klebsiella pneumoniae (KP) is an extracellular Gram-negative bacterium that causes infections in the lower respiratory and urinary tracts and the bloodstream. STAT1 is a master transcription factor that acts to maintain T cell quiescence under homeostatic conditions. Although STAT1 helps defend against systemic spread of acute KP intrapulmonary infection, whether STAT1 regulation of T cell homeostasis impacts pulmonary host defense during acute bacterial infection and injury is less clear. Using a clinical KP respiratory isolate and a pneumonia mouse model, we found that STAT1 deficiency led to an early neutrophil-dominant transcriptional profile and neutrophil recruitment in the lung preceding widespread bacterial dissemination and lung injury development. Yet, myeloid cell STAT1 was dispensable for control of KP proliferation and dissemination, because myeloid cell-specific STAT1-deficient (LysMCre/WT;Stat1fl/fl) mice showed bacterial burden in the lung, liver, and kidney similar to that of their wild-type littermates. Surprisingly, IL-17-producing CD4+ T cells infiltrated Stat1-/- murine lungs early during KP infection. The increase in Th17 cells in the lung was not due to preexisting immunity against KP and was consistent with circulating rather than tissue-resident CD4+ T cells. However, blocking global IL-17 signaling with anti-IL-17RC administration led to increased proliferation and dissemination of KP, suggesting that IL-17 provided by other innate immune cells is essential in defense against KP. Contrastingly, depletion of CD4+ T cells reduced Stat1-/- murine lung bacterial burden, indicating that early CD4+ T cell activation in the setting of global STAT1 deficiency is pathogenic. Altogether, our findings suggest that STAT1 employs myeloid cell-extrinsic mechanisms to regulate neutrophil responses and provides protection against invasive KP by restricting nonspecific CD4+ T cell activation and immunopathology in the lung.

Single-cell RNA sequencing (scRNA-seq) is a high-throughput transcriptomic approach with the power to identify rare cells, discover new cellular subclusters, and describe novel genes. scRNA-seq can simultaneously reveal dynamic shifts in cellular phenotypes and heterogeneities in cellular subtypes. Since the publication of the first protocol on scRNA-seq in 2009, this evolving technology has continued to improve, through the use of cell-specific barcodes, adoption of droplet-based systems, and development of advanced computational methods. Despite induction of the cellular stress response during the tissue dissociation process, scRNA-seq remains a popular technology, and commercially available scRNA-seq methods have been applied to the brain. Recent advances in spatial transcriptomics now allow the researcher to capture the positional context of transcriptional activity, strengthening our knowledge of cellular organization and cell-cell interactions in spatially intact tissues. A combination of spatial transcriptomic data with proteomic, metabolomic, or chromatin accessibility data is a promising direction for future research. Herein, we provide an overview of the workflow, data analyses methods, and pros and cons of scRNA-seq technology. We also summarize the latest achievements of scRNA-seq in stroke and acute traumatic brain injury, and describe future applications of scRNA-seq and spatial transcriptomics.

Respiratory infection by Pseudomonas aeruginosa, common in hospitalized immunocompromised and immunocompetent ventilated patients, can be life-threatening because of antibiotic resistance. This raises the question of whether the host's immune system can be educated to combat this bacterium. Here we show that prior exposure to a single low dose of lipopolysaccharide (LPS) protects mice from a lethal infection by P. aeruginosa. LPS exposure trained the innate immune system by promoting expansion of neutrophil and interstitial macrophage populations distinguishable from other immune cells with enrichment of gene sets for phagocytosis- and cell-killing-associated genes. The cell-killing gene set in the neutrophil population uniquely expressed Lgals3, which encodes the multifunctional antibacterial protein, galectin-3. Intravital imaging for bacterial phagocytosis, assessment of bacterial killing and neutrophil-associated galectin-3 protein levels together with use of galectin-3-deficient mice collectively highlight neutrophils and galectin-3 as central players in LPS-mediated protection. Patients with acute respiratory failure revealed significantly higher galectin-3 levels in endotracheal aspirates (ETAs) of survivors compared to non-survivors, galectin-3 levels strongly correlating with a neutrophil signature in the ETAs and a prognostically favorable hypoinflammatory plasma biomarker subphenotype. Taken together, our study provides impetus for harnessing the potential of galectin-3-expressing neutrophils to protect from lethal infections and respiratory failure. This study reports training by lipopolysaccharide to expand neutrophils expressing the anti-bacterial galectin-3 protein defending mice from a lethal bacterial infection, a similar signature associated with survivors of respiratory failure in humans.