Objectives/Goals: The Human Immune Dysregulation Evaluation Framework (Hi-DEF) quantifies myeloid dysregulation in infectious and non-infectious illness using peripheral blood gene expression (Moore, Nat Med). We evaluated immune dysregulation in obesity and quantified the changes in dysregulation due to lifestyle and medical interventions. Methods/Study Population: We assessed the association of obesity with myeloid dysregulation in the Framingham Cohort using linear regression. In two additional datasets (GSE66175, GSE19790), we assessed the effect of dietary interventions and bariatric surgery on myeloid dysregulation using paired Wilcoxon rank sum test pre- and post- intervention and Pearson’s correlation. Results/Anticipated Results: In the Framingham cohort (n=5321), myeloid dysregulation score was associated with BMI (p=2.2e-6) after correcting for age and sex. Patients assigned to diet/exercise experienced significant reductions in myeloid score after 3 months (n=63, p=1.2e-7), which was not seen in controls (n=63, p=0.29). Change in myeloid score was significantly correlated with change in weight loss (r=0.32, p=0.006). This reduction in myeloid dysregulation was also seen post-bariatric surgery (n=11, p=0.0001). Discussion/Significance of Impact: Patients with obesity have significantly higher myeloid dysregulation compared to healthy subjects, which is modifiable with lifestyle and medical interventions. These results suggest that we may be able to quantify and monitor immune dysregulation from obesity which may place these patients at risk for worse outcomes.

The hyperinflammatory syndromes in critically ill patients, including trauma, sepsis, and acute lung injury, are characterized by dysregulated neutrophil responses that contribute to tissue damage and poor outcomes. Using murine models of cytokine storm induced by trauma and lung injury, we identified transforming growth factor β (TGFβ) as a central regulator of immune checkpoint in neutrophils. TGFβ signaling modulates neutrophil activation and upregulates the expression of programmed death-ligand 1 (PDL1). Disruption of TGFβ signaling during hyperinflammation restores the migratory capacity of neutrophils but leads to excessive activation, severe pulmonary tissue damage, and increased susceptibility to spontaneous bacterial infection in the lung. Mechanistically, PDL1 expression alters neutrophil behavior within lung capillaries, promoting intravascular clustering and restricting tissue infiltration. Targeted deletion of PDL1 in neutrophils reverses hyperinflammation-induced clustering, restores effective trafficking to infectious foci, and enhances host-protective immune function while limiting pathological neutrophil hyperactivation. These findings define a TGFβ-PDL1 regulatory axis that restrains the pathogenicity of neutrophils during hyperinflammation, revealing a checkpoint mechanism that balances host defense and tissue integrity.

Human vaccine responses vary widely, but the determinants remain incompletely defined. Here we analyzed 66 cytokines across four inactivated influenza vaccine (IIV) cohorts over five seasons (n = 581) and identified baseline serum interleukin (IL)-18 and interferon (IFN)-β as correlates of day 28 antibody responses. To test causality, we evaluated 19 cytokines in human tonsil and spleen organoids and found that type I IFNs, IL-21 and IL-12, but not IL-18 or IFNγ, enhanced antibody production. The addition of IFNβ to IIV recapitulated key features of the live-vaccine cytokine program. IL-12 and IL-21 defined a parallel pathway independent of type I IFNs, with IL-12 inducing IL-21 in humans, unlike in mice. Delivery of IL-21 or IFNβ via mRNA lipid nanoparticles in vivo promoted long-lived plasma cell formation. Together, these findings define parallel pathways that regulate vaccine immunity. Our approach unites high-throughput organoid testing and human cohort studies, establishing a human-centric platform to identify adjuvant candidates.

Abstract Introduction We recently described a framework to quantify immune dysregulation in peripheral blood using cell-specific gene expression scores across the severity spectrum in sepsis (Moore et al, Nature Medicine). Given the associations between elevated neutrophil proportion in blood and bronchoalveolar lavage fluid and poor outcomes in idiopathic pulmonary fibrosis (IPF), we investigated whether the myeloid dysregulation score (MDS), which includes neutrophil-specific genes, generalizes to IPF and whether this differs depending on the collection technique. Methods We calculated the MDS at baseline in bulk RNA sequencing data from whole blood (containing all peripheral blood cells, including neutrophils) or peripheral blood mononuclear cell (PBMC) samples for 409 subjects (295 IPF patients, 114 healthy) in four publicly available IPF datasets (GSE93606, GSE33566, GSE38958, and GSE28042) and assigned high versus low myeloid dysregulation using median a threshold. We determined the difference in MDS between IPF patients and controls at baseline using Wilcoxon rank sum-test. When available, we assessed the correlation between forced vital capacity (FVC) and MDS using Spearman correlation. Using Kaplan-Meier estimator, we evaluated survival in cohorts with complete outcome information stratified by high versus low MDS. Results Of the 409 subjects, PBMCs or whole blood samples were available for 209 and 200 patients, respectively. 171 of 295 IPF patients had outcomes available. Of the combined 409 individuals in the four cohorts, MDS was significantly higher in IPF patients than healthy controls (p = 6.1e-06). This difference was significant in the whole blood cohort (p = 1.5 x 10-7) but not the PBMC cohort (p = 0.2). MDS was inversely correlated with baseline FVC (r = -0.1883, p = 0.026). In the whole blood cohort, transplant-free-survival within the first year was significantly higher for patients with low MDS than those with high MDS at baseline (HR = 4.4, 95% CI 1.4-14.0, p = 0.005), but not in the PBMC cohort (p > 0.9). Conclusion MDS in peripheral blood, which was derived predominantly in sepsis, is associated with severity and outcomes in IPF. The prognostic association is seen only in whole blood and not PBMCs, suggesting neutrophils may be associated with disease severity and outcomes in IPF. These results support shared immune dysregulation in sepsis and IPF that may contribute to disease trajectory in both conditions. This abstract is funded by: none

Abstract Introduction Inferring molecular signatures of disease progression usually requires costly and time-consuming longitudinal samples. Single-cohort transcriptomic studies also suffer from the curse of dimensionality, as the number of genes profiled is much larger than the number of samples. The availability of numerous independent, heterogeneous datasets in public databases presents a unique opportunity to model disease progression trajectories by leveraging cross-sectional data that collectively capture all stages of a disease. Methods We introduce TIDEPOOL, a method that uses a disease-defining gene signature to define consensus disease trajectories within independent, heterogeneous datasets. Results To demonstrate the utility of TIDEPOOL, we applied it to 24 bulk transcriptomic datasets comprising 2,222 blood samples from patients with viral or bacterial infections of varying severities. We identified two diverging trajectories separating patients with severe outcomes from those with non-severe outcomes. We found 54 genes, clustered into four gene modules, that differentiate these two trajectories. We validated that these genes also differentiate patients with severe and non-severe outcomes in 1,913 samples of patients with infection from 10 external datasets (AUROC = 0.80). Through single-cell analysis of an integrated whole blood dataset, we identified that 3 of these gene modules were highly expressed in neutrophils and the other in B and dendritic cells. In-depth analysis of the 3 neutrophil-associated modules in the Single-Cell Atlas of Human Neutrophils (SCAHN) showed that the modules originate from different neutrophil subtypes including both protective and detrimental neutrophils. Conclusion Using the novel TIDEPOOL framework, we have identified a 54-gene signature that distinguishes infectious disease patients with severe and non-severe outcomes. Many of these genes originate from neutrophils, including multiple neutrophil subsets which are associated with both favorable and adverse outcomes. Funding Source n/a Topic Categories Computational and Systems Immunology (COMP)

Abstract Introduction We recently described the Human Immune Dysregulation Evaluation Framework (Hi-DEF) to quantify myeloid and lymphoid dysregulation across infectious and non-infectious illness using peripheral blood gene expression (Moore et al, Nature Medicine). Using the Hi-DEF framework, we evaluated the baseline immune dysregulation in patients with obesity and quantified the changes in immune dysregulation due to lifestyle changes and medical interventions. Methods We assessed the association of obesity with myeloid dysregulation in the Framingham Cohort using linear regression. In two additional datasets (GSE66175, GSE19790), we assessed the effect of dietary interventions and bariatric surgery on myeloid dysregulation using paired Wilcoxon rank sum test pre- and post- intervention and Pearson correlation. Results In the Framingham cohort (n = 5321), myeloid dysregulation score was associated with BMI (p = 2.2e-6) after correcting for age and sex. Patients assigned to diet/exercise experienced significant reductions in myeloid score after 3 months (n = 63, p = 1.2e-7), which was not seen in controls (n = 63, p = 0.29). Change in myeloid score was significantly correlated with change in weight loss (r = 0.32, p = 0.006). This reduction in myeloid dysregulation was also seen post-bariatric surgery (n = 11, p = 0.0001). Conclusion Patients with obesity have significantly higher myeloid dysregulation compared to healthy subjects, which is modifiable with lifestyle and medical interventions. Funding Source 5K12TR004930-02 Topic Categories Computational and Systems Immunology (COMP)

Abstract Introduction Post Acute Sequelae of COVID-19 (PASC), also called Long COVID, is an infection-associated chronic syndrome. Despite proposed viral persistence mechanisms, no therapeutic benefit was observed in randomized placebo-controlled trials of nirmatrelvir/ritonavir (NMV/r) in adults with Long COVID, including the Selective Trial of Paxlovid for PASC (STOP-PASC). This systems immunology analysis aimed to characterize immune profiles of participants during clinical trial intervention, identify biomarkers associated with patient-reported outcomes, and investigate potential mechanisms underlying Long COVID. Methods We performed comprehensive immunological profiling of 152 STOP-PASC trial participants using plasma proteomics (Olink® Explore HT 5400 panel), autoantigen arrays, viral serology, and microclot assays at baseline, day 15, and week 10. We assessed associations between immune features and patient-reported outcomes. We also conducted meta-analysis of nine independent Long COVID proteomics cohorts (n = 590 total samples) to identify conserved inflammatory signatures. Results NMV/r treatment at day 15 compared with baseline induced transient changes in plasma proteins that normalized by week 10, primarily impacting myeloid cell/monocyte, lysosome, and complement activation pathways. Cardiovascular symptoms were negatively associated with SARS-CoV-2 antibody levels at baseline. No widespread differences in autoantibody profiles, Epstein-Barr virus (EBV) reactivation, or microclotting were observed between STOP-PASC Long COVID participants, pre-pandemic controls, and individuals without Long COVID. Meta-analysis of publicly available Olink® data from Long COVID cohorts identified a conserved 60-protein Long COVID Signature (LCS) score revealing multi-compartment immune activation involving monocyte, neutrophil, and T/NK cell modules. Conclusion These findings advance our understanding of Long COVID immunology and may help direct future proteomic biomarker endpoints for Long COVID clinical trials. Funding Source Pfizer Topic Categories Computational and Systems Immunology (COMP)

The aging brain exhibits a decline in the regenerative populations of neural stem cells (NSCs). While mechanisms that restore old NSC function have started to be identified, the role of lipids-especially complex lipids-in NSC aging remains largely unclear. Using lipidomic profiling by mass spectrometry, we identify age-related changes in complex lipids in quiescent NSCs in vitro and in vivo. Moreover, several polyunsaturated fatty acids increase across lipid classes in quiescent NSCs during aging. Using spatial lipidomics, we find that some of the changes in complex lipids are also observed in situ. Several age-related changes in complex lipids and side chain composition are occurring at the plasma membrane, as revealed by lipidomic profiling of isolated plasma membrane vesicles. Experimentally, we show that aging is accompanied by a decrease in plasma membrane order, a key membrane biophysical property, in old quiescent NSCs in vitro and in vivo. To determine the functional role of plasma membrane lipids in aging NSCs, we performed genetic and supplementation studies. Knocking out the phospholipid acyltransferase MBOAT2 exacerbates age-related lipidomic changes in old quiescent NSCs and impedes their ability to activate. Mboat2 overexpression reverses age-related lipidomic changes in old quiescent NSCs and boosts their ability to activate in vitro and in vivo. Moreover, supplementation of plasma membrane lipids from young NSCs improves the ability of old quiescent NSCs to activate. Our work could lead to lipid-based strategies for restoring the regenerative potential of NSCs, which has important implications for countering brain decline during aging.

Alveolar growth and repair are central processes in development and chronic lung disease, such as bronchopulmonary dysplasia (BPD), a neonatal lung disease without curative therapy. Alveolar epithelial type 2 (AT2) cells are the endogenous progenitor pool giving rise to alveolar epithelial type 1 cells and promoting alveolar repair. Since netrin-1, a regulator of cell homeostasis and stemness, has been linked to lung diseases, we now investigated its signaling and function in AT2 cells in a hyperoxia-based model of BPD and in lungs of infants with BPD. First, we demonstrated that prolonged hyperoxia reduced both netrin-1 and its receptor Unc5b in neonatal mouse lungs and in primary AT2 cells. Second, ex vivo studies using precision-cut lung slices (PCLS) and primary murine AT2 cell culture showed that netrin-1 regulates AT2 cell survival and the expression of Krüppel-like factor 4 (Klf4) through Unc5b, a transcription factor regulating cell survival. Third, single-cell and bulk transcriptomic analysis, as well as proximity-dependent biotin identification assay, showed Klf4 to be upregulated in AT2 cells during alveolarization, downstream of netrin-1, and to regulate AT2 cell survival. In vivo, Klf4 gene expression and protein abundance was significantly reduced in total lung homogenates and in AT2 cells of neonatal mice exposed to hyperoxia. Finally, KLF4+ cells, KLF4+ epithelial, and specifically KLF4+ AT2 cells were reduced in clinical BPD. In summary, our data identify a novel netrin-1-Unc5b-Klf4 axis in AT2 cells that is disrupted in BPD and could offer a novel target for endogenous alveolar repair.

Abstract Introduction Malaria manifests in a spectrum of clinical outcomes shaped by the complex heterogeneity of both the human host and the Plasmodium parasite, its causative agent. This dynamic and multifactorial host—parasite interaction poses major challenges for effective diagnostics, leading to fragmented observations that hinder the generalizability of findings, and consequently, clinical translation. We hypothesized that a conserved immune response underlies malaria disease trajectories and diverges across patient outcomes. Methods We conducted the largest multicohort analysis of the host transcriptional response to malaria, integrating >3,600 blood transcriptomes from 42 cohorts across 20 countries on six continents spanning children and adults with diverse clinical presentations. Using TIDEPOOL (Trajectory Inference for Disease Endotyping from Pooled data), we applied trajectory modeling to resolve gene modules associated with protective versus detrimental responses, delineating the transcriptional continuum from asymptomatic to severe malaria. Results We identified a robust 208-gene blood signature that distinguishes malaria-infected individuals from uninfected controls (AUROC = 0.875, validation cohort), providing a generalizable framework for molecular diagnosis. TIDEPOOL found that severe malaria was defined by a blood transcriptional program marked by expansion of granulocytes, neutrophils, and monocytes, coupled with depletion of eosinophils, NK cells, B cells, and T cells–reflecting broad immune dysregulation. Within severe cases, we resolved a novel conserved gene signature diagnostic of cerebral malaria. These signatures were consistent across age groups, geographic regions, and parasite strains, underscoring universal host responses to Plasmodium infection. Conclusion Together, our findings reveal a unified framework linking immune dynamics to clinical severity and offer actionable biomarkers to guide next-generation diagnostic and host-targeted therapeutic strategies for malaria elimination. Funding Source Gate Foundation, NIAID Topic Categories Computational and Systems Immunology (COMP)

OBJECTIVES:Immunoglobulin (Ig) G4-related disease (IgG4-RD) is commonly treated with glucocorticoids and B-cell depletion, but cumulative toxicity and relapse underscore the need for alternative approaches. We evaluated the efficacy, safety, and immunological effects of Bruton's tyrosine kinase (BTK) inhibition with zanubrutinib in active IgG4-RD. METHODS:In this phase 2, open-label, proof-of-concept trial, 10 participants with lacrimal and submandibular gland IgG4-RD received zanubrutinib 80 mg twice daily for up to 24 weeks without glucocorticoid induction or background immunosuppression. The primary endpoint was change in lacrimal and submandibular gland volume at week 24 by blinded fluorodeoxyglucose positron emission tomography/magnetic resonance imaging (evaluable n = 8). Secondary endpoints included metabolic imaging parameters, clinical disease activity, serologic biomarkers, and safety. Single-cell RNA sequencing with immune repertoire profiling was performed to define cellular mechanisms. RESULTS:At week 24, mean gland volume decreased by 46.7% (lacrimal) and 29.9% (submandibular) (both P = .008), with concordant reductions in total lesion glycolysis (-91.6 g; P = .05) and total metabolic lesion volume (-20.7 cm³; P = .05). Clinical disease activity improved (IgG4-RD Responder Index -6.0 points; P = .01), alongside reductions in serum IgG4 (-417 mg/dL; P = .008) and circulating plasmablasts. Imaging and serologic changes were strongly correlated. Single-cell analyses demonstrated treatment-associated modulation of B-cell transcriptional programmes, reductions in IgG4-skewed plasmablasts, and attenuation of cytotoxic CD4⁺ T cells. Adverse events were predominantly mild; 1 serious event (COVID-19) occurred off treatment. CONCLUSIONS:Zanubrutinib monotherapy produced substantial imaging-defined and clinical improvements in active glandular IgG4-RD without glucocorticoid induction. BTK inhibition was associated with modulation of B-cell differentiation states and downstream immune programmes, supporting its development as a steroid-sparing, non-B-cell-depleting therapeutic strategy.

Early sepsis recognition is challenging due to its heterogeneous presentations and limited rapid diagnostic and prognostic tools. The TriVerityTM Test, a high-multiplex host-response mRNA point-of-care test, supports clinical decision-making by estimating infection likelihood and illness severity. This study assesses TriVerity’s accuracy in Dutch emergency department (ED) patients. Adult ED patients with suspected infection and Systemic Inflammatory Response Syndrome score (SIRS) ≥2 or quick Sequential Organ Failure Assessment (qSOFA) ≥2 were prospectively included by the Acutelines data/biobank. The TriVerity test provides three scores: likelihood of bacterial infection, viral infection, and illness severity. Infection status was determined post hoc by independent physicians through clinical consensus and forced adjudication. Illness severity was defined as ICU admission or death within 7 days after ED admission. Among the 467 patients included, 234 (50

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers and shows resistance to any therapeutic strategy used. Here we tested small-molecule inhibitors targeting chromatin regulators as possible therapeutic agents in PDAC. We show that JQ1, an inhibitor of the bromodomain and extraterminal (BET) family of proteins, suppresses PDAC development in mice by inhibiting both MYC activity and inflammatory signals. The histone deacetylase (HDAC) inhibitor SAHA synergizes with JQ1 to augment cell death and more potently suppress advanced PDAC. Finally, using a CRISPR-Cas9-based method for gene editing directly in the mouse adult pancreas, we show that de-repression of p57 (also known as KIP2 or CDKN1C) upon combined BET and HDAC inhibition is required for the induction of combination therapy-induced cell death in PDAC. SAHA is approved for human use, and molecules similar to JQ1 are being tested in clinical trials. Thus, these studies identify a promising epigenetic-based therapeutic strategy that may be rapidly implemented in fatal human tumors.

Suppl. Figure 6. A. Left. Protein expression levels of phospho-ERK and ERK in HPAFII and PATU8902 cells treated with MEKi (50 nM) for 24 h. Right. Protein expression levels of phospho-AXL and AXL in HPAFII and PATU8902 cells treated with AXLi (0.2 μM) for 24 h. Quantification: pAXL levels normalized to total AXL levels and housekeeping protein levels. B. Heatmap of the Fa fraction of CAPAN2 PDAC human cell line treated with single MEKi, AXLi, or both at indicated concentrations for 72 h. C. 2D surface plot of Bliss synergy scores from B. Red regions indicate synergistic interactions.

FOXP3+ Treg cells are critical for immune tolerance. Genetic deletion of the Forkhead domain-containing proteins of the FOXP-subfamily member FOXP1 from Tregs results in impaired function associated with reduced CD25 expression and IL-2 signaling, but to date the only other FOXP family member expressed in Tregs, FOXP4, has been minimally studied. To investigate the potential functional interactions among FOXP family members in Tregs, we specifically deleted Foxp1, Foxp4, or both in FOXP3+ committed Tregs in mice. Our findings show that mice with combined, but not individual, deficiency in FOXP1 and FOXP4 exhibit lymphoproliferation, inflammation, autoimmunity, and early lethality. The combined absence of FOXP1 and FOXP4 in Tregs results in an activated/effector-like phenotype with compromised suppressive function in peripheral lymphoid organs, an enhanced germinal center response, and proinflammatory cytokine production. We further show that FOXP1 and FOXP4 bind to Il2ra promoter regions to regulate CD25 expression in Tregs. Through pairwise comparison among mouse strains with Treg-specific deletion of Foxp1, Foxp4, or both, our findings indicate a nonredundant but insufficient role of FOXP4 in Treg function.

Biomarkers based on host response signatures are currently under development for the critically ill. We applied a 29-mRNA classifier for the diagnosis and prognosis of suspected acute infection and sepsis (TriVerity™, Inflammatix Inc.) in patients hospitalized with COVID-19. We applied three scores from locked classifiers (IMX-BVN-4 and IMX-SEV-4) from the 29-mRNA TriVerity™ blood test in participants of the SAVE-MORE randomized clinical trial (ClinicalTrials.gov NCT04680949) at baseline and days 4 and 7 of treatment, to classify bacterial infection, viral infection and decompensation. Participants were adults hospitalized with confirmed COVID-19 pneumonia and plasma soluble urokinase plasminogen activator receptor (suPAR) levels of ≥ 6 ng/ml, randomized to placebo or anakinra treatment. A total of 471 patients were studied. At baseline nearly 90