Immunology & Cell Biology

Acquired immunity in mammals depends upon capture and presentation of antigens by specialised macrophage populations in splenic marginal zone and lymph node sinuses and follicular dendritic cells (FDC) within germinal centres. Cells referred to as FDC in chickens express CSF1R, the receptor for macrophage colony-stimulating factor (CSF1) and IL34. We utilised single cell RNA-seq on CSF1R+ cells from chicken spleen to identify monocytes and two distinct populations of macrophages. TIMD4/C1Q/MAFB+ macrophages were enriched for expression of genes involved in iron metabolism. A MARCO/VSIG4+ population expressed SPIC, a transcription factor associated with red pulp macrophages in mammals but also expressed receptors (CR2) and trophic factors (TNFSF13, CXCL13) associated with mammalian FDC. SPIC+ cells were located within follicles in spleen, caecal tonsil and bursa. We generated a CSF1R knockout in the chicken germ line. Mutant birds lack macrophages in the embryo. They were indistinguishable from wild type at hatch and behaved and fed normally but from day 5-6 post hatch they failed to thrive. Loss of CSF1R function in hatchlings led to monocytopenia and granulocytosis and the loss of macrophage subpopulations in lymphoid organs. Consistent with their expression of B cell trophic factors, the loss of follicular macrophages in the bursa was associated with involution and severe B cell deficiency in the circulation and spleen. In summary, lymphoid tissues of chickens contain specialised macrophage populations with distinct expression profiles. The details of regulation by CSF1R, specialised functions and underlying transcriptional regulation are quite different between birds and mammals.

Sex-based differences in respiratory disease outcomes are well recognized. However, the underlying immunological mechanisms driving this dimorphism remain incompletely understood. While sex hormones influence immune cell development and function, the role of commensal microbes in shaping sex-specific lung immunity has not been explored. Here, we used single-cell RNA sequencing (scRNAseq) and flow cytometry to profile lung immune cells in male and female mice housed under specific pathogen-free (SPF) or germ-free (GF) conditions. Under SPF conditions, males exhibited a striking myeloid bias, with increased monocytes and macrophages, along with broad upregulation of inflammatory mediators, including S100a8, S100a9, and Il1b, across multiple cell types, and enrichment of TNF and interferon (IFN) signaling pathways. In contrast, females displayed lymphocyte-skewed profiles, with higher frequencies of T cells and natural killer (NK) cells. Interestingly, these sex-based differences in immune composition and inflammatory programs were largely absent in GF mice, indicating that microbial exposure amplifies baseline immunological dimorphism between males and females. Notably, select sex-associated features, including female-biased NK cell enrichment, persisted irrespective of microbial status, suggesting intrinsic, microbiota-independent programming. Together, these findings indicate that commensal microbes modulate sex-specific lung immunity by amplifying pre-existing intrinsic differences, highlighting the intersection of extrinsic (microbial) and intrinsic (sex-linked) factors in shaping baseline mucosal immunity.

Here we describe a 16-parameter, 14-color surface staining panel optimized for murine bronchoalveolar lavage cells that enables reproducible identification of major innate and adaptive immune populations relevant to pulmonary infections or other inflammatory conditions of the airways. The panel enables confident identification of neutrophils, eosinophils, B cells, T cells and subtypes, NK cells, and distinguishes between tissue resident and monocyte-derived macrophages populations. The panel was carefully designed for BAL samples that vary in cell number, are rich in debris, and often autofluorescent. Antibody concentrations are optimized to provide reproducible results regardless of sample-variable cell numbers allowing for the preparation of a single antibody cocktail master mix and rapid sample staining time, thereby cutting down on sample preparation and optimizing cell viability of analyzed samples. The panel facilitates robust cross-sectional and longitudinal comparison of airway inflammation across different airway inflammatory conditions, infections by different respiratory pathogens, impact of vaccination or therapeutics on the inflammatory landscape, and more. It facilitates hypothesis generation by revealing recruitment kinetics and remodeling of myeloid compartments, supports downstream sorting for transcriptomic or functional assays, and provides a standardized baseline for labs to adopt or extend for activation or intracellular cytokine analyses. We have successfully utilized this panel to identify differential host responses to different respiratory Mycoplasma pathogens as well as to longitudinally track the progression of inflammatory response to Mycoplasma pneumoniae over a 21-day time course study. This panel provides an economic immunophenotyping option by utilizing only 14 markers and a two laser (Blue and Violet) full spectrum cytometer to provide comprehensive immunophenotyping power of both myeloid and lymphoid cells. Furthered by lacking the requirement for advanced unmixing for sample analysis, the panel can be easily adopted by the community, enabling comparative meta-analyses of host responses across murine respiratory infection models.

BackgroundNeutrophils play an important role in the immune system and sense environmental perturbations including pathogens. Upon pathogen detection, neutrophils extrude their chromatin, forming neutrophil extracellular traps (NETs) trapping and removing pathogens. Previous studies have shown that controlled chromatin decondensation occurs during NET formation, reflecting NET inducing pathways, and the cellular environment. While NET inducing stimuli like phorbol 12-myristate 13-acetate (PMA) is commonly used to study NET formation, it bypassing regulatory mechanisms, limiting insights. MethodsWe used the Assay for Transposase-Accessible Chromatin with sequencing (ATAC-Seq) to profile chromatin accessibility in neutrophils stimulated in whole blood with PMA and physiologically relevant inflammatory factors (NFs), including TNF-, GM-CSF, fMLP, C5a, and IL-1{beta}, alone and in combination. Chromatin responses were compared across conditions and integrated with publicly available transcriptomic sepsis cohorts. ResultsWe found that NF stimulation induced stimulus specific chromatin accessibility programs distinct from PMA. Individual NFs increased specific transcription factor (TF) motif enrichments in a stimulus dependent manner, with GM-CSF increasing STATs, TNF- increasing NF-{kappa}B, C5a/fMLP increasing AP-1, and Combined with a cooperative response including CEBP. Integration with sepsis transcriptomic datasets revealed that promoter accessibility changes within NF stimulations correspond to transcriptional states associated with sepsis disease severity, highlighting the upstream regulatory programs linked to clinical outcomes. ConclusionsThese findings demonstrate that NF stimulation in whole blood reveals chromatin accessibility programs in neutrophils that correlate with disease severity in sepsis. This approach provides a framework for linking cytokine driven neutrophil regulation to heterogenous inflammatory states in sepsis and other NET-associated diseases.

Breastfeeding provides health benefits in childhood, reducing the frequency of gastrointestinal and respiratory infections. Breastmilk (BM) is a rich source of bioactive molecules including extracellular vesicles (EVs), which exert immunomodulatory signalling in recipient cells, with cargo that is affected by maternal characteristics. Here we investigated the biophysical characteristics of BM-EVs from mothers with (asthmatic BM-EVs) or without asthma (control BM-EVs) and their effect on the release of cytokines from primary human hTERT-immortalized airway smooth muscle cells (hASMs) from asthmatic or non-asthmatic (control) donors. BM-EVs were isolated using size exclusion chromatography (N=5/group), characterized biophysically and by EV-specific protein markers. In addition, BM-EV were co-cultured (48h) with primary hASM cells from both non-asthmatic (control) and asthmatic donors to determine the effect on cytokine release. All participants were Caucasian and the BM was collected 12-15 weeks postpartum. BM-EVs showed the presence of intact and small-EVs ([~]100 nm). Asthmatic BM-EVs appeared to have a smaller average EV size (135.6 nm) vs. controls (148.3 nm, p=0.0613), but [~]5-fold higher concentration of both total (p=0.0014) and small EVs (p=0.0016). The expression of EV subtype protein expression was reduced in asthmatic BM-EVs vs. control BM-EVs: CD63 by 86% (p=0.0224), flotillin-1 by 40% (p=0.0196), CD9 by 24% (p=0.0646) and HSP70 by 69% (p=0.0873). Asthmatic BM-EVs co-cultured with hASMs from control donors decreased pro-inflammatory cytokine release: MCP-1 by 55% (p=0.0286), IL-6 by 45% (p=0.0801) and IL-2 by 32% (p=0.0970) vs. control-BM-EVs. Conversely, asthmatic BM-EVs co-cultured with hASMs from asthmatic donors increased secretion of anti-inflammatory cytokine IL-10 by 32% (p=0.0660), and IL-1Ra by 75% (p=0.0875), and pro-inflammatory IL-2 by 57% (p=0.0688) vs. control-BM-EVs. Internalization of control and asthmatic BM-EVs was confirmed by labelled EV uptake experiments. No detrimental effects on cell viability with BM-EV treatment were observed. In summary, asthmatic BM-EVs are smaller and enriched in BM, and exert differential effects on cytokine release in a BM-donor and recipient-cell specific manner. Given that BM can enter infant airways, the immunomodulatory effects of BM-EVs on hASMs warrants further investigation to delineate the under underlying mechanisms.

Background and Aims: Alterations in immunoglobulin G (IgG) N-glycosylation are implicated in inflammatory bowel disease (IBD); however, the robustness of IgG glycan signatures across IBD cohorts with diverse demographics and geographic origins remains underexplored. We aimed to determine whether compositional data analysis (CoDA) and machine learning (ML) can identify IBD-related IgG N-glycan signatures and whether these signatures capture disease-associated acceleration of biological aging. Methods: We analyzed the IgG glycome profiles of 1,367 plasma samples collected from healthy controls (HC), symptomatic controls (SC), and people with newly diagnosed Crohn's (CD), and ulcerative colitis (UC) across four cohorts (UK, Italy, United States, and Netherlands). IgG glycosylation was analyzed by ultra-high-performance liquid chromatography, yielding 24 total-area-normalized glycan peaks (GPs). Analyses were performed using cross-sectional data obtained at baseline. CoDA-powered association analyses were used to identify disease-related effects on GPs while controlling for demographic covariates. ML models were trained and evaluated to assess generalizability to unseen cohorts and demographic subgroups, with a focus on discrimination and reliability. Results: Across all cohorts, people with IBD demonstrated accelerated biological aging as quantified by the GlycanAge index. This was accompanied by consistent reductions in IgG galactosylation, with effects partially modulated by age. Classification models trained on glycomics and demographics achieved robust discrimination (AUROC~0.80) between non-IBD (HC+SC) and IBD across cohorts. Conclusion: These findings reveal accelerated biological aging in people with IBD and support the translational potential of IgG glycans as biomarkers and a novel route toward clinically interpretable personalized risk estimates.

Unbiased classification of equine dendritic cells (DC) is necessary to address various research questions such as the role of DC subsets in immune-mediated diseases of horses. We applied single-cell RNA sequencing (scRNA-seq) on DC enriched from the blood of two horses. All main DC subsets were detected by key gene expression, including conventional DC type 1 (cDC1; XCR1) and type 2 (cDC2; FCER1A, CD1E) as well as plasmacytoid DC (pDC; TCF4). In addition, we detected a small cluster of hematopoietic progenitors, as well as transitional DC (tDC; FCER1A, TCF4) and putative DC type 3 (DC3; FLT3, CD163). Our data confirms the previously reported phenotype of equine pDC (Flt3+MHC-IIlowCADM1lowCD172aint), cDC1 (Flt3+MHC-IIhighCADM1highCD172alow-int) and cDC2 (Flt3+MHC-IIhighCADM1intCD172ahigh), while also highlighting considerable CD14 expression for cDC2. Two subclusters of equine cDC2 were found to be enriched in FCER1A or CX3CR1 transcripts (cDC2.1 and cDC2.2, respectively), with suggested enhanced extravasation and T-cell stimulatory capacities of the latter. Conservation of DC subsets across species (horse, pig, human, mouse) was illustrated by enrichment analyses with subset-specific gene signatures and by cross-species data integration with publicly available scRNA-seq datasets. Our atlas of equine blood DC is a valuable resource for comparative analyses, and it forms the foundation for understanding the involvement of distinct DC subsets in infections and immune-mediated pathologies.

Autophagy has been implicated in several lung diseases, either protecting tissues or driving pathology. Hypersensitivity pneumonitis (HP) is a complex inflammatory lung disease, and autophagy is heavily involved in regulating inflammation. The role of autophagy in HP remains unclear. The aim of our study was to understand the role of autophagy in HP pathogenesis. GFP-LC3 transgenic mice were exposed intranasally to Saccharopolyspora rectivirgula (SR) to induce HP and follow autophagy activation in the lung. Then, we take advantage of our Atg4b-deficient mouse model to assess how autophagy disruption impacts lung inflammation in response to SR antigen challenge. Increased autophagy activation was observed in epithelial and inflammatory cells after SR antigen exposure in GFP-LC3 transgenic lungs. GFP-LC3 puncta colocalized with ATG4B and ATG5 in epithelial and inflammatory cells after antigen exposure. Autophagy impairment limits the inflammatory response after SR antigen exposure in the lungs from the Atg4b-deficient mice when compared to WT mice. To evaluate whether lipopolysaccharide (LPS) exacerbates the inflammatory response in the Atg4b-deficient, a SR+LPS combined treatment was developed and we discovered that LPS aggravates the SR-induced HP in WT but not in Atg4b-deficient mice. Reduced HP severity in Atg4b-deficient mice was associated with decreased expression of NFkB, CCL1, CCL25, CXCL1, TNFR1, IL-13, and IL-17A, diminished CD4+ T cell recruitment and expansion, reduced M2-like macrophages, and decreased granuloma and iBALT development. Our findings highlight autophagy as a critical driver in HP pathogenesis and as a potetial target for novel theraphy development.

BackgroundGroup 2 innate lymphoid cells (ILC2s) are key effector cells of type-2 immunity. A subset of ILC2s expresses KIT (CD117), which display increased phenotypic plasticity and were previously linked to severe asthma and psoriasis. However, the molecular mechanisms promoting a KIT+ ILC2 state remain poorly understood. ObjectiveDefine the molecular basis for the enhanced plasticity of KIT+ ILC2s and identify signals that induce this phenotype, including links with immune disease susceptibility. MethodsWe combine bulk as well as single-cell transcriptome (RNA-seq) and epigenome (ATAC-seq) with in vitro culture assays using primary human KIT+ or KITneg ILC2s and multipotent ILC precursors (ILCPs). Epigenomic data were integrated with genetic risk variants for major human immune diseases. ResultsMulti-omics analyses revealed that KIT+ ILC2s maintain a unique hybrid character marked by expression and open chromatin of genes linked to both ILCP and ILC2 biology. KIT+ ILC2s showed extensive epigenomic priming at gene loci related to naive lymphocyte biology, tissue homing, and ILC3 effector functions, including IL17 and IL23R - explaining why KIT+ ILC2s are poised to adopt an ILC3-like phenotype. Genetic risk variants for asthma and autoimmunity are enriched in the poised epigenome of KIT+ ILC2s. Common {gamma}-chain cytokines IL-2 and IL-7 induced a KIT+ phenotype in KITneg ILC2s through STAT5 activation. ConclusionsOur study defines KIT+ ILC2s as a developmentally immature state carrying a precursor-like epigenome that promotes phenotypic plasticity and is linked to immune disease susceptibility. Importantly, we identify STAT5-mediated cytokine signals as candidates for therapeutic targeting of KIT+ ILC2s.

BackgroundGestational exposures may contribute to the newborns lifetime risk of inflammatory bowel disease (IBD). While gestational influences are associated with IBD onset, the causality and confounding of such exposures are difficult to ascertain. The neonatal metabolome provides a metabolic snapshot of gestational influences. ObjectiveWe tested the neonatal metabolomes ability to predict future IBD, to assess whether gestational exposures are reflected in early molecular precursors of the disease. MethodsWe profiled dried blood spots from 520 newborns who later developed IBD and matched controls using high-resolution untargeted mass spectrometry metabolomics (1,350 QC-passing metabolites). Genotyping was available for 1,009 of these individuals. PERMANOVA confirmed assay sensitivity to gestational exposures, gradient boosting was used for prediction. ResultsThe neonatal metabolome significantly captured maternal smoking, birth weight, and gestational age (p < 0.001), but explained minimal variance in IBD status (R2 = 0.09%, p = 0.390) and showed no predictive power for IBD (AUC = 0.51, 95% CI 0.50-0.52, p = 0.585). Stratifying by disease subtype and age of onset did not improve performance. In contrast, genetic risk scores were modestly predictive (CD: AUC = 0.64, p < 5.11x10-14; UC: AUC = 0.63, p < 7.65x10-{superscript 1}{superscript 2}), but uncorrelated with neonatal metabolomic profiles (CD: p = 0.650; UC: p = 0.970), suggesting a later-age effect. ConclusionsUsing a large, comprehensively profiled cohort, we demonstrate that neonatal metabolomic profiles sensitively capture gestational signatures, but not the overall future IBD risk. Our findings suggest that most IBD risk accumulates later in life, beyond gestational molecular imprints.

Inflammation is a key driver of hematopoietic dysfunction in myeloid malignancies, but its role in the context of hypomethylating therapy remains incompletely understood. Although 5-Azacytidine is used posttransplant in high-risk myelodysplastic syndrome (MDS), only 50% of patients show a clinical response. We provide evidence that inherent inflammatory properties of healthy donor CD34+ stem cells exist that are likely to contribute to the "response" seen in MDS patients. These are linked to epigenetic priming of the myeloid niche, resulting in S100A8/A9-driven inflammatory program that promotes functionality of immature NK cells. Using in vitro differentiation systems, multi-omic profiling, and a S100A9-/- mouse model, we find that 5-AzaC modulates inflammatory transcriptional programs through epigenetic rewiring of upstream regulatory elements. Loss of S100A9 disrupts myeloid differentiation, impairs NK cell maturation, and alters key developmental regulators including CEBPB, JUN, and NFIL3. In vivo, 5-AzaC restores these defects and primes NK cells in a time- and context-dependent manner. Re-analysis of the published Australian MDS/CMML cohort shows that "responders" display increased S100A8/A9 expression together with enhanced IFN-{gamma}, IL6-JAK-STAT3, and TNF signaling. These findings suggest that inflammatory myeloid programs may serve as predictive biomarkers and therapeutic targets to enhance NK cell-mediated graft-versus-leukemia activity posttransplant. SummaryO_LIWe provide compelling evidence that inherent properties of healthy donor CD34+ hematopoietic stem cells (SCs) exist that are likely to contribute to the "response" seen upon pre-emptive posttransplant 5-AzaC therapy of patients with high-risk myelodysplastic syndrome (MDS). C_LIO_LIThese properties are linked to a distinct form of epigenetic plasticity at upstream-located transcription factor (TF) binding sites. This may indirectly contribute to acute S100A8/A9-driven inflammation, which is demonstrable in distinct monocyte subsets and, importantly, also in NK cells thereby determining the characteristics of inflammatory monocyte-NK cell crosstalk. C_LIO_LIMice with a targeted deletion of S100A9 fail to upregulate CEBPB / JUN and NFIL3 which results in impaired myeloid priming and dysfunctional NK cell maturation, respectively. C_LIO_LIRe-analysis of the Australian MDS/CMML cohort confirms that MDS patients that "respond" to 5-AzaC exhibit activated IFN-{gamma}, IL6-JAK-STAT3, and TNF-signaling pathways in the context of upregulated S100A8/A9 after six months of treatment. C_LIO_LIOur study indicates that screening of healthy donors SCs for specific inflammatory markers in early developing monocytes could be used as a marker to predict which donor will have the potential of generating a S100A8/A9-driven inflammatory response. This may help identify patients with MDS as well as AML who are likely to benefit from low-dose, short-term 5-AzaC therapy as early as day 7 after transplantation, potentially resulting in increased graft-versus-leukemia (GvL) activity. C_LI

Premature birth and cesarean section are major perinatal factors influencing immune development and are associated with increased morbidity and inflammatory diseases. However, their impact on neonatal immunity remains incompletely defined. To determine how gestational age and mode of delivery shape early immune programming, we analyzed CD4 T cells, central regulators of adaptive responses, from preterm neonates and full-term neonates born by cesarean section or natural birth. We performed transcriptomic profiling (mRNA-seq) and functional assessment of T cell activation, proliferation, and cytokine production following stimulation. The mode of delivery exerted a dominant effect on the CD4 T cell transcriptome and function. CD4 T cells from full-term neonates delivered by natural birth exhibited an immune activation signature, produced higher levels of multiple cytokines, but showed reduced proliferative capacity. In contrast, prematurity induced modest changes in basal gene expression relative to full- term cesarean section neonates. CD4+ T cells from preterm neonates displayed enhanced proliferation and increased secretion of inflammatory cytokines IL-13, TNF, IL-6, and IL- 17F upon stimulation, indicating heightened responsiveness. Collectively, our findings show that CD4 T cells from preterm neonates exhibit augmented inflammatory capacity, which becomes more regulated at term. The mode of delivery further refines this developmental trajectory: cesarean section is associated with a restrained functional profile, whereas natural birth is associated with an immune activation signature and increased responsiveness. These results provide evidence that neonatal CD4 T cell trajectories are established during fetal life and subsequently modulated at birth, underscoring the layered influence of perinatal factors on immune development.

Macrophages are now recognised as key players in a range of tissues and biological processes, responding to injury and infection, and facilitating development and regeneration. As the importance of macrophage crosstalk within these processes has been revealed, so too has the significance of studying the spatial positioning of macrophages within the tissue of interest. As such, immunofluorescent microscopy-based analysis is becoming an increasingly attractive technique for immunology research. While tissue fixation preserves the tissue architecture and immobilises target antigens, prolonged fixation can negatively impact protein recognition. We report that prolonged exposure to a paraformaldehyde-based fixative profoundly impacts detection of cell surface markers that define macrophage subsets in the mouse submandibular gland, in contrast to epithelial cell markers which appear more robust. We find that this that this is not exclusive to the salivary gland, and similar effects are seen in the pancreas and kidney. Importantly, a short duration of fixation allowed the detection of macrophage subsets in both mouse and human tissue without compromising the detection of other markers. Adoption of a short fixation approach enables accurate detection of a wide range of cell types in tissues, and facilitates exploration of spatial positioning and cell proximity by immunofluorescent microscopy analysis.

BackgroundMaternal obesity is a global health challenge with profound consequences for offspring health. While its impact on metabolic programming has been widely studied, far less is known about how maternal obesity shapes the fetal immune system. The fetal bone marrow (FBM) is the central site of hematopoietic stem and progenitor cell (HSPC) development, and disruptions in this niche can have lifelong effects on immunity, infection susceptibility, and inflammatory disease risk. In this study, we examined FBM hematopoiesis in a nonhuman primate model of spontaneous maternal obesity. MethodsUsing spectral flow cytometry, single-cell RNA sequencing, and functional differentiation assays, we mapped progenitor composition, lineage trajectories, and immune function in offspring exposed to maternal obesity compared with lean controls. These complementary approaches allowed us to capture cellular frequencies and transcriptional programs, while trajectory and signaling analyses provided insight into how progenitor maturation and intercellular communication are disrupted by maternal obesity. ResultsOur findings reveal that maternal obesity decreases CD34+ HSPCs and common lymphoid progenitor populations, while expanding megakaryocyte-erythroid and granulocyte-monocyte progenitors. Pseudotime analysis demonstrated altered maturation, with cells accumulating at early differentiation states. Transcriptional profiling uncovered a strong inflammatory bias, with myeloid progenitors upregulating alarmins, interferon-stimulated genes, and proinflammatory mediators. Functionally, monocytes derived from obese FBM showed impaired migratory and colony-stimulating capacity, coupled with exaggerated TNF responses to LPS stimulation. ConclusionTogether, these results demonstrate that maternal obesity, even in the absence of obesogenic diet, disrupts fetal bone marrow hematopoiesis by altered HSPC maturation, reprogramming lineage trajectories, and inducing inflammatory bias.

Spatial imaging technologies provide an expansive view of tissue microenvironments through high-plex profiling of protein and molecular targets in situ. Imaging mass cytometry (IMC; Standard BioTools) is a trusted method for defining immune phenotypes based on up to 40 protein targets, whilst Xenium in situ spatial transcriptomics (Xenium; 10x Genomics) is an emerging platform that can measure up to 5000 mRNA markers simultaneously. Although these platforms can reveal valuable insights on their own, there is an increasing need to analyse samples using a multi-omics approach to further our understanding of complex biological processes. To address this, we have assessed a novel dual-platform workflow that combines Xenium and IMC on a single formalin-fixed paraffin-embedded tissue section to enable the spatial profiling of both mRNA and protein targets at single-cell resolution. The feasibility of the workflow was determined by comparing the staining quality of IMC performed after Xenium to that of IMC performed alone on an adjacent tissue section, confirming that Xenium has little to no negative impact on subsequent IMC protein staining. Although the location of transcripts picked up by Xenium correlated with the corresponding proteins picked up by IMC at a global scale, discrepancies between the two technologies were apparent at the single-cell level. This is to be expected, as biologically transcript expression does not always correlate with protein, and both platforms have their own technical limitations. However, when we analyse T cells identified by both technologies, as opposed to T cells identified by Xenium or IMC alone, it produces the most biologically meaningful results at both the transcript and protein level for specific T cell markers. These results highlight how integration of the two platforms, identifying the presence of both RNA and protein, can foster a more comprehensive view of cellular landscapes and provide a greater depth of functional capabilities and cellular interactions.

BackgroundHyperinflammatory syndromes such as viral acute respiratory distress syndrome (ARDS) demand immunotherapies that are safe and effective at suppressing cytokine storm. As a novel treatment for ARDS, PRS CK STORM is proposed as a next-generation cell-free secretome derived from co-cultures of M2 macrophages and mesenchymal stromal cells. MethodsWe performed a double-blind, randomized controlled trial to test the effects of PRS CK STORM in K18-hACE2 transgenic mice infected intranasally with 105 PFU of SARS-CoV-2 BE.1.1. This was complemented by a mechanistic analysis of this secretome using transcriptomic and COX2 enzymatic activity studies, as well as a compositional analysis of its proteomic and miRNA profile. ResultsIn a lethal SARS-CoV-2 ARDS mouse model, PRS CK STORM significantly improved lung histopathology to a degree on par with corticosteroids, while stimulating angiogenesis in damaged lung tissue. Early and late cytokine profiling showed marked reductions in IFN-{gamma}. In response to PRS CK STORM treatment, transcriptomic analyses in inflamed macrophages revealed robust downregulation of key proinflammatory drivers (MyD88, TRAF6, IKK2, NF-{kappa}B, COX2). In vitro enzymatic assays confirmed potent, dose-dependent inhibition of COX2, with high inter-batch reproducibility. A compositional analysis revealed this secretome to be rich in anti-inflammatory miRNAs, immune-modulating proteins, and regenerative factors. ConclusionsPRS CK STORM operates as a multi-target immune recalibrator, enabling broad downregulation of pathological inflammation while promoting tissue repair. Its off-the-shelf, GMP-manufactured format ensures reproducibility and scalability, offering a novel resolution pharmacology approach for cytokine storm syndromes originated from ARDS and beyond.

Tracking mature neutrophils remains challenging due to the lack of reliable cell surface markers. Although CD101 is a promising candidate for mature neutrophils, its stability under pathological conditions is unclear. Using a CD101-tdTomato reporter mouse model, we confirmed that the reporting system does not alter CD101 expression, and tdTomato fluorescence is predominantly expressed in mature neutrophils across peripheral tissues. Further analysis revealed that CD101+ and tdTomato+ neutrophils display identical characteristics of mature neutrophil, including poly-segmented nuclei, cell size, and key functions under homeostasis. By comparing tdTomato fluorescence with CD101 protein levels, we demonstrate that reduced CD101 expression under pathological states was not attributed to shedding or degradation. Our finding enhances CD101 as a robust and reliable marker of neutrophil maturity, providing a foundation for future applications in spatial transcriptomics and lineage tracing studies to dissect neutrophil heterogeneity and function. Highlights of the studyO_LIIn CD101-tdTomato homozygous mice, tdTomato is predominantly expressed in neutrophils and labels nearly 100% of mature neutrophils, aligning with the phenotype of CD101+ mature neutrophils; C_LIO_LIThe CD101-tdTomato reporting system does not interrupt CD101 expression or neutrophil functions; C_LIO_LICD101 remains a stable and reliable cell surface marker for labeling mature neutrophils, even under pathological conditions. C_LI

Th17 cells (CD4+ T cells producing IL-17) are important for clearance of fungal infections and play a critical role in the development and exacerbation of numerous autoimmune diseases. Their differentiation, signalling pathways, cytokine production and metabolism are now well-characterised. As well as CD4+ T cells, CD8+ cells also produce IL-17 family members, and these have been named Tc17 cells. However, much less is known about their development, signalling or metabolism compared to their CD4+ counterparts. Here, we performed a series of in vitro and in vivo analyses of Tc17 cells as well as computational analysis of the published Tabula muris dataset, comparing Tc17 to IL-17- CD8+ T cells and to Th17 cells. We show that murine Tc17 cells are generated in the presence of TGF-{beta} and IL-6, and that cells produced by these culture conditions substantially reflect Tc17 cells seen in vivo; that is, with high expression of PD1, CD6, ICOS and CD161. Tc17 cells show phenotypic and functional differences to their Th17 counterparts, with increased production of IL-2 and IL-22 as well as an increased tendency to produce IL-17F as well as IL-17A. They show a more glycolytic profile than Th17 cells, with lowered mitochondrial membrane potential. This divergent phenotype and cytokine production suggests differential roles in vivo for these two cells.

The human neonatal immune system is developmentally specialized to balance the unique requirements of perinatal transition. Disruption of this finely tuned balance, as in preterm birth, may have profound consequences for immunity and overall health. However, the impact of prematurity on immune composition and functional responsiveness across gestational ages (GA) remains incompletely understood. Single-cell profiling has advanced our understanding of neonatal immunity, yet most studies were limited to unimodal readouts, narrow GA windows, or baseline function. Here, we present a comprehensive human neonatal CITE-seq atlas (82 samples from 25 neonates and 10 adults as controls) at the first days of life covering a wide GA range and integrating baseline and stimulated conditions. Most notably, we identify a GA-dependent immune transition point centered around 32 weeks of GA, which discriminates extremely and very preterm neonates (GA <32wks) from those of higher GA ([&ge;]32wks). In particular, early-life immunity in extremely and very preterm infants showed CD15+ granulocytic myeloid derived suppressor cell-like predominance, whereas more mature neonates exhibited interferon-primed transcriptional profiles. This resulted in divergent myeloid-to-lymphocyte signaling networks and qualitatively distinct NK- and T-cell bystander responses upon activation. Together, these findings show that intrauterine development imprints GA-specific immune programs. By defining a developmental transition around a GA of 32 weeks that regulates baseline and induced responses of neonatal immune cells, our atlas provides a framework for understanding the vulnerability of preterm infants and thus may pave the way for developing GA-adapted immunomodulatory strategies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/715643v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1db4534org.highwire.dtl.DTLVardef@9c9665org.highwire.dtl.DTLVardef@55f063org.highwire.dtl.DTLVardef@190a52_HPS_FORMAT_FIGEXP M_FIG C_FIG

DNA methylation is a stable epigenetic mark that critically influences the phenotype of immune cells. Identifying differentially methylated regions within immune cell lineages supports their phenotypic and functional characterization, leading to a better understanding of lineage-specific transcriptional regulation. Here, we performed a genome-wide methylation analysis of human innate lymphoid cells (ILCs), which allowed us to define specific epigenetic marker regions for ILC1, ILC2, and ILC3. These regions were associated with genes that have well-described functions in ILCs, such as TBX21 in ILC1, GATA3 and MAF in ILC2, RORC and IL23R in ILC3, but were also found in genetic loci that have not been previously associated with ILCs. In-depth analysis of ILC2-related marker regions within the HPGDS and NRROS gene loci confirmed their critical role in transcriptional regulation and suggested a novel role for NRROS in ILC2. Genome-wide methylation analysis of ILC2, derived from the blood of juvenile donors with atopy or asthma led to the identification of several disease-specific epigenetic regions associated with genes such as GIMAP4 and PTGS2. Together, our study not only provides novel epigenetic marker regions in human ILCs and confirms the functional role of ILC2-related markers, but also identifies promising markers for studying allergies in humans.