The appearance of diabetes-associated autoantibodies is the first detectable sign of the disease process leading to type 1 diabetes (T1D). Evidence suggests that T1D is a heterogenous disease, where the type of antibodies first formed implies subtypes. Here, we leverage longitudinal samples collected from 98 European TRIGR participants (49 children who subsequently presented with T1D, and 49 matched controls), and profile single-cell epigenomics at different time points of disease development. Quantitation of cell and nuclei populations, complemented by analysis of transcriptome and open-chromatin states, indicates robust, early, replicable monocyte lineage differences between cases and controls, suggesting the early emergence of heightened pro-inflammatory cytokine secretion among cases. The order of autoantibody emergence in cases shows variation across lymphoid and myeloid cells, potentially indicating divergence in the cellular immune response. The strong monocytic lineage representation in peripheral blood immune cells before seroconversion and the weaker differential coordination of these gene networks close to clinical diagnosis emphasize the importance of early life as a critical phase in T1D development.

Abstract Introduction Human metapneumovirus (hMPV) is a leading cause of severe respiratory illness in infants, and can lead to the rare development of acute neurological complications such as encephalitis and seizures. Severe early-life viral infections with hMPV, RSV and other respiratory viruses have been linked to long-term neurodevelopmental impairments, including cognitive, motor, and behavioral deficits. However, the viral-host mechanisms driving neurotropism and neuroinflammation associated with these clinical outcomes remain poorly understood. Methods Here, we have modeled HMPV-infection of the central nervous system (CNS) using human primary neuronal culture, human brain organoid models and in vivo mouse models of intranasal HMPV infection. In these models we used proteomics, single-cell genomics and functional studies to determine the mechanisms of neuroinflammation driven by HMPV infection. We then utilized a high-throughput small-molecule drug screen to identify novel inhibitors of HMPV infection. Results We demonstrated that hMPV can directly infect and replicate within neuronal and nonneuronal cells in the CNS leading to activation of proinflammatory innate immune responses and markers of neuronal damage using human cell and organoid models. Moreover, we identified different signatures of HMPV innate immune activation compared to a related respiratory virus, RSV. In a mouse model, we found that after Intranasal hMPV infection, virus was detected in both lung and brain tissues with inflammatory signatures. Finally, we performed a drug repurposing screen to identify novel antiviral drugs that could be used to block HMPV infection of both respiratory and CNS tissues. Conclusion These findings establish HMPV as a virus capable of CNS infection and inflammation, identify me mechanisms of host-virus immune interactions and identified novel antiviral drugs that could be used as therapies to mitigate both respiratory and neurological complications associated with HMPV infection. Funding Source Paul Henson Endowment, Children’s Mercy Kansas City Topic Categories Viral Immunology (VIR)

Natural killer (NK) cells are integral to the innate immune system, playing a crucial role in immune surveillance and the rapid response to virally infected and tumor cells. Epigenetic gene expression regulation significantly influences NK cell function and differentiation. Using a high-throughput small-molecule drug screening approach, we identified bromodomain and extra-terminal domain (BET) inhibitors (BETi) as potent modulators of NK cell function, reducing proinflammatory cytokine secretion while increasing markers of NK cell maturation and cytotoxicity. During NK lineage specification from hematopoietic stem cells, we demonstrated that BETi reduced NK cell fate and promoted increased myeloid cell differentiation. Moreover, differentiated NK cell types had more functionally differentiated gene expression programs. Thus, BET proteins are crucial for both mature NK cell functions and controlling NK cell lineage development from progenitors in the bone marrow. These findings suggest that BETi can fine-tune NK cell responses, offering promising therapeutic potential for cancer immunotherapy and the treatment of inflammatory and autoimmune diseases. Our study underscores the critical role of BET inhibitors in regulating NK cell function and opens new avenues for targeted immune modulation.

Using single-cell transcriptomics of bronchoalveolar lavage cells from Mtb/SIV co-infected rhesus macaques on cART, we reveal profound immune dysregulation during early SIV co-infection of latent tuberculosis. SIV induces a sharp decline in CD4+ T cells, NK, and NKT cells, with incomplete recovery of Mtb-specific TH1 effector responses despite viral suppression. Instead, a persistent TH17-skewed environment emerges, alongside sustained myeloid inflammation driven by Type I interferon signaling and pro-inflammatory regulators such as KLF6 and NFKB1. Ligand-receptor network analyses demonstrate expanded CD4+ T cell-macrophage crosstalk and loss of immune homeostasis that cART fails to fully restore. These findings expose how SIV remodels the pulmonary immune landscape to impair protective immunity against Mtb, providing a transcriptomic framework to explain TB reactivation in HIV infection. Our work highlights the urgent need for adjunctive immunotherapies to complement cART, aiming to rebalance immune responses and improve TB control in co-infected individuals.

The appearance of diabetes-associated autoantibodies is the first detectable sign of the disease process leading to type 1 diabetes (T1D). Evidence suggests that T1D is a heterogenous disease, where the type of antibodies first formed imply subtypes. Here, we followed 49 children, who subsequently presented with T1D and 49 matched controls, profiling single-cell epigenomics at different time points of disease development. Quantitation of cell and nuclei populations as well as transcriptome and open-chromatin states indicated robust, early, replicable monocyte lineage differences between cases and controls, suggesting heightened pro-inflammatory cytokine secretion early among cases. The order of autoantibody emergence in cases showed variation across lymphoid and myeloid cells, potentially indicating cellular immune response divergence. The strong monocytic lineage representation in peripheral blood immune cells before seroconversion and the weaker differential coordination of these gene networks close to clinical diagnosis emphasizes the importance of early life as a critical phase in T1D development.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has varied presentations from asymptomatic to death. Efforts to identify factors responsible for differential COVID-19 severity include but are not limited to genome wide association studies (GWAS) and transcriptomic analysis. More recently, variability in host epigenomic profiles have garnered attention, providing links to disease severity. However, whole epigenome analysis of the respiratory tract, the target tissue of SARS-CoV-2, remains ill-defined. We interrogated the nasal methylome to identify pathophysiologic drivers in COVID-19 severity through whole genome bisulfite sequencing (WGBS) of nasal samples from COVID-19 positive individuals with severe and mild presentation of disease. We noted differential DNA methylation in intergenic regions and low methylated regions (LMRs), demonstrating the importance of distal regulatory elements in gene regulation in COVID-19 illness. Additionally, we demonstrated differential methylation of pathways implicated in immune cell recruitment and function, and the inflammatory response. We found significant hypermethylation of the FUT4 promoter implicating impaired neutrophil adhesion in severe disease. We also identified hypermethylation of ELF5 binding sites suggesting downregulation of ELF5 targets in the nasal cavity as a factor in COVID-19 phenotypic variability. This study demonstrated DNA methylation as a marker of the immune response to SARS-CoV-2 infection, with enhancer-like elements playing significant roles. It is difficult to discern whether this differential methylation is a predisposing factor to severe COVID-19, or if methylation differences occur in response to disease severity. These differences in the nasal methylome may contribute to disease severity, or conversely, the nasal immune system may respond to severe infection through differential immune cell recruitment and immune function, and through differential regulation of the inflammatory response.

Background: Antibody-dependent cell-mediated cytotoxic (ADCC) response mediated by natural killer (NK) cells correlates with decreased infection risk in studies involving simian immunodeficiency virus (SIV)/simian-human immunodeficiency virus (SHIV), and human immunodeficiency virus (HIV) vaccine candidates. Currently, the heterogeneities of the functional subset of rhesus macaque natural killer (RMNK) cells are under-characterized. Method: We engaged the RMNK cells with ADCC-mediating anti-HIV-1 monoclonal antibodies (ADCCAbs) or anti-CD16 antibodies and used CD107a expression as the surrogate marker for RMNK cells actively involved in ADCC. CD107a+ and CD107a– populations were analyzed individually using single-cell RNA sequencing. Results: Subsets of CD107a+ RMNK cells produced more chemokines than the others, suggesting that these cells not only eliminate infected cells but also provide immunoregulatory signals and potentially curb HIV-1 replication. Crosslinking of Fc gamma receptor IIIa via anti-CD16 antibodies resulted in a significantly higher percentage of degranulating cells than via ADCCAbs. However, the magnitude of degranulation and chemokine production was reduced by 6- to 30-fold. Conclusion: The quality and quantity of receptor engagement are important determinants of achieving an optimal level of the RMNK response.

Autoantibodies are host-targeting antibodies typically removed through immune tolerance mechanisms. The presence of autoantibodies is commonly associated with autoimmune conditions such as systemic lupus erythematosus and rheumatoid arthritis, however recent large cohort studies have identified healthy individuals possess autoantibodies with ability to regulate immune response. The origin and functions of autoantibodies in individuals without autoimmune disease is not well characterized. Here, we identified autoantibodies directed against intracellular adhesion molecule 1 (ICAM-1), a cell surface glycoprotein important for inflammation and immune cell adhesion that is widely exploited by pathogens for cell entry. Serum from healthy individuals maintained heterogenous levels of anti-ICAM-1 antibodies and ICAM-1 autoantibody levels were associated with age. Individuals with inflammatory conditions, such as severe COVID-19 or autoimmune disease, had significantly less anti-ICAM-1 antibodies compared to healthy controls. Using peptide microarrays, we determined immunodominant epitopes targeted across individuals by ICAM-1 autoantibodies. Finally, we identified that anti-ICAM-1 antibodies could inhibit cellular adhesion of immune cells, disrupting immune system function. The findings of this study elucidate autoantibodies against ICAM-1 could play a role in immunoregulation during inflammation and may provide a future target for managing immune response. Supported by Children’s Mercy Research Partners, Children’s Mercy Research Institute, and Paul Henson Endowment Clinical Scholar Award Cellular Adhesion, Migration, and Inflammation (CAM)

Intracellular adhesion molecule 1 (ICAM-1) is a cell surface glycoprotein that regulates cell-cell interactions, signaling, and immune processes. ICAM-1 expression has been shown to be elevated in many types of infections and inflammatory diseases. Strategies to block ICAM-1 function, including monoclonal anti-ICAM-1 antibodies, have been successful in treating the effects of chronic respiratory, autoimmune, and cardiovascular diseases. Naturally occurring autoantibodies targeting cytokines, endothelial cells, and other host receptors have been identified, and have been found to play a role in immunoregulation in health and disease. In this study, we determined the presence and levels of ICAM-1 autoantibodies across different age groups and disease states. We found that ICAM-1 autoantibody levels increased with age and were lower in individuals with various inflammatory states, suggesting a dynamic role in immune regulation. Using peptide microarrays, we identified high-resolution epitopes targeted by ICAM-1 autoantibodies that overlap with critical functional ICAM-1 binding sites. Finally, we determined that ICAM-1 autoantibodies were enriched for the IgG2 subclass that is critical for the response to bacterial antigens and chronic inflammation. This could indicate that molecular mimicry of bacterial antigens or underlying immune dysregulation could trigger the generation of anti-ICAM-1 antibodies. Understanding the functional implications of ICAM-1 autoantibodies could provide new insights into their contribution to immune homeostasis and their potential impact on inflammatory and autoimmune conditions.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory illness. While most symptoms of RSV infection are linked to airway inflammation, emerging clinical data reveals that RSV can infiltrate the central nervous system (CNS), and lead to neurological manifestations such as seizures and central apnea, and in rare cases, encephalopathy, that can have long-term neurodevelopmental consequences. Despite detection of viral infection in the CNS, the pathophysiological mechanisms of RSV-mediated CNS disease are not well defined. Here, we have modeled RSV-infection of the CNS using human primary neuronal cultures and human brain organoid models of infection. Using confocal microscopy, biochemical and single-cell genomics approaches, we have identified that RSV could productively infect neurons, caused cell death and inflammatory responses, and altered the overall transcriptome of neurons and non-neuronal CNS cell populations after infection. Moreover, we have identified key cell-surface adhesion molecules that are critical for RSV infection in the CNS, and optimized soluble forms of these cell-surface glycoproteins to block viral infection. Our findings advance our understanding of RSV dissemination into the CNS and highlight a potential soluble receptor-based therapy to mitigate against lung and CNS inflammation. This work paves the way for new therapeutic strategies against RSV, and establishes an approach for characterizing viral infection in the human CNS. Supported by Children’s Mercy Research Partners, Children’s Mercy Research Institute and Paul Henson Endowment Clinical Scholar Award. Viral Immunology (VIR)

Abstract Natural killer (NK) cells are cytotoxic innate lymphocytes that provide defense against pathogens and malignancy. New evidence identified that NK cells are capable of memory-like immune responses in certain settings. This innate immune memory is thought to be imprinted through epigenetic modifications. However, precise epigenic pathways or strategies to modulate long-term NK cell activity are not well defined. In this study, we performed a screen of an epigenetic library containing 160 drug compounds with well-characterized epigenetic regulatory mechanisms, to identify drugs and pathways that could train or tolerize NK cell activation. One compound that we identified was the H3K27 methyltransferase Ezh2, which regulated NK cell lineage commitment from bone marrow hematopoietic stem cells and altered the phenotype of differentiated NK cells to increased cytotoxicity. This outcome provides an option to train or induce stronger cytotoxic effects on NK cells. In contrast, another compound we identified was the histone deacetylase inhibitor, Givinostat, which did not alter NK cell commitment from bone marrow stem cells, but significantly decreased NK cell function in the periphery resulting in a more tolerant innate immune state to secondary NK cell stimulation. Identification and exploration of these epigenome altering drugs may offer further insight into downstream pathways of NK cell function or provide novel therapies for tolerizing or priming innate immune responses.

The consequences of gestational coronavirus disease 2019(COVID-19) exposure on neonatal immunity are incompletely understood. While vertical transmission to the fetus is rare, maternal COVID-19 exposure can adversely impact the neonate by inducing placental pathology, or indirectly, via neonatal immune programming. To investigate the latter, Gilley et al. analyzed cytokine levels in cord blood mononuclear cells (CBMCs) obtained from neonates exposed to chorioamnionitis, gestational COVID-19, and unexposed controls. They observed that fetal exposure to maternal inflammation, by either SARS-CoV-2 infection or chorioamnionitis, exaggerated the pro-inflammatory cytokine response to neonatal bacterial pathogens in cultured CBMCs. While both chorioamnionitis and COVID-19 exposure exaggerated cytokine expression, there were differences in immune cell populations and cell-type specific responses. These interesting findings indicate that fetal exposure to inflammatory stimuli, even without fetal infection, results in changes in the neonatal immune cell phenotype and function leading to heightened immune activation. The results of this study differ from a trend towards a muted pro-inflammatory response seen in pediatric COVID-19, which is attributed to cause decreased disease severity. The mechanisms underlying neonatal immune programming, the timing and severity of maternal exposure in relationship to immune programming and long-term health consequences need to be explored in future studies.

Fcγ receptors (FcγRs) are membrane-bound glycoproteins that bind to the fragment crystallizable (Fc) constant regions of IgG antibodies. Interactions between IgG immune complexes and FcγRs can initiate signal transduction that mediates important components of the immune response including activation of immune cells for clearance of opsonized pathogens or infected host cells. In humans, many studies have identified associations between FcγR gene polymorphisms and risk of infection, or progression of disease, suggesting a gene-level impact on FcγR-dependent immune responses. Rhesus macaques are an important translational model for most human health interventions, yet little is known about the breadth of rhesus macaque FcγR genetic diversity. This lack of knowledge prevents evaluation of the impact of FcγR polymorphisms on outcomes of preclinical studies performed in rhesus macaques. In this study we used long-read RNA sequencing to define the genetic diversity of FcγRs in 206 Indian-origin Rhesus macaques, Macaca mulatta. We describe the frequency of single nucleotide polymorphisms, insertions, deletions, frame-shift mutations, and isoforms. We also index the identified diversity using predicted and known rhesus macaque FcγR and Fc-FcγR structures. Future studies that define the functional significance of this genetic diversity will facilitate a better understanding of the correlation between human and macaque FcγR biology that is needed for effective translation of studies with antibody-mediated outcomes performed in rhesus macaques.

Background Increased inflammation caused by SARS-CoV-2 infection can lead to severe coronavirus disease 2019 (COVID-19) and long-term disease manifestations. The mechanisms of this variable long-term immune activation are poorly defined. One feature of this increased inflammation is elevated levels of proinflammatory cytokines and chemokines. Autoantibodies targeting immune factors such as cytokines, as well as the viral host cell receptor, angiotensin-converting enzyme 2 (ACE2), have been observed after SARS-CoV-2 infection. Autoantibodies to immune factors and ACE2 could interfere with normal immune regulation and lead to increased inflammation, severe COVID-19, and long-term complications. Methods Here, we deeply profiled the features of ACE2, cytokine, and chemokine autoantibodies in samples from patients recovering from severe COVID-19. We measured the levels of immunoglobulin subclasses (IgG, IgA, IgM) in the peripheral blood against ACE2 and 23 cytokines and other immune molecules. We then utilized an ACE2 peptide microarray to map the linear epitopes targeted by ACE2 autoantibodies. Results We demonstrate that ACE2 autoantibody levels are increased in individuals with severe COVID-19 compared with those with mild infection or no prior infection. We identify epitopes near the catalytic domain of ACE2 targeted by these antibodies. Levels of autoantibodies targeting ACE2 and other immune factors could serve as determinants of COVID-19 disease severity, and represent a natural immunoregulatory mechanism in response to viral infection. Conclusions These results demonstrate that SARS-CoV-2 infection can increase autoantibody levels to ACE2 and other immune factors. The levels of these autoantibodies are associated with COVID-19 disease severity.

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Abstract Pre-cancerous cells are normally recognized and eliminated by immune cells. Cancer progresses only when this immunosurveillance system fails. Although immunotherapy has driven the most significant and exciting advances in cancer treatment in modern times, current approaches are running into barriers. An emerging area of interest includes evidence for rare but highly potent stem cell-like T cells. Stem-like T cells combine long-term persistence and high potency with immunological memory. Our recent findings reveal insights into the nature and regulation of stem-like T cells and their potential anticancer activity. Minimal residual disease (MRD) underlies therapeutic resistance, dictates treatment escalation, and predicts patient outcomes. We have shown that the Wnt and PI3K pathways promote the transformation of hematopoietic stem cells (HSCs) into chemoresistant leukemia stem cells (LSCs), which form the root of leukemia initiation and post-treatment recurrence. High-throughput screening revealed that LSCs could be targeted by low dose anthracycline treatment. Mechanistically, low dose, but not high dose anthracyclines indirectly target LSCs by inhibiting their unique properties of immune escape, allowing for their elimination by CD8+ T cells. We employed single-cell genomic and proteomic analysis to investigate immunological changes during development of HSCs, LSCs, and their progeny in response to low dose anthracycline treatment. While leukemia progression results in exhaustion of differentiated CD8+ T cells, stem-like T cells accumulate. However, low dose anthracycline treatment reverses this imbalance. Comparing MRD+ and MRD- patient samples, we found that differential proportions of stem-like T cells persist in the bone marrow of leukemia patients. While T cells of MRD- patients recover following chemotherapy induction, T cell recovery is severely attenuated in MRD+ patients. Furthermore, failure of immunological recovery is driven at least partially by a lack of stem-like T cells in MRD+ leukemia patients. Overall, our studies have revealed that low dose anthracyclines, in contrast to high dose, induce opposing, dichotomous effects on LSCs vs. HSCs and stem-like vs. differentiated T cells. Citation Format: Fang Tao, Sara McElroy, Jacqelyn Nemechek, Irina Pushel, John Szarejko, Santosh Khanal, Todd Bradley, Doug Myers, John M. Perry. Stem-like T cells maintain latent anticancer activity and underly therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5557.

Severe COVID-19 often leads to secondary infections and sepsis that contribute to long hospital stays and mortality. However, our understanding of the precise immune mechanisms driving severe complications after SARS-CoV-2 infection remains incompletely understood. Here, we provide evidence that the SARS-CoV-2 envelope (E) protein initiates innate immune inflammation, via toll-like receptor 2 signaling, and establishes a sustained state of innate immune tolerance following initial activation. Monocytes in this tolerant state exhibit reduced responsiveness to secondary stimuli, releasing lower levels of cytokines and chemokines. Mice exposed to E protein before secondary lipopolysaccharide challenge show diminished pro-inflammatory cytokine expression in the lung, indicating that E protein drives this tolerant state in vivo. These findings highlight the potential of the SARS-CoV-2 E protein to induce innate immune tolerance, contributing to long-term immune dysfunction that could lead to susceptibility to subsequent infections, and uncovers therapeutic targets aimed at restoring immune function following SARS-CoV-2 infection.

Abstract Enterovirus A (EV-A) has been responsible for recent outbreaks of hand-foot-mouth disease (HFMD); however, there is no vaccine or effective antiviral treatment. The goal of our project is to develop broad vaccines targeting the diverse EV-A serotype viruses that cause HFMD. We used the EV-A viral capsid protein 1 (VP1) as the vaccine antigenic target, because of its genetic diversity across EV-A serotypes, and prior work that showed VP1 has linear neutralizing antibody epitopes. We computationally designed VP1 proteins to create mosaic antigens that optimize B and T cell epitopes among diverse EV-A viral sequences. We identified that a hexavalent mosaic vaccine cocktail had the highest EV-A sequence coverage. We then immunized mice in three groups with recombinant VP1 proteins in adjuvant; Group 1 was given the EV-A Consensus VP1 (Consensus), Group 2 was given the EV-A strain EVA-71 VP1 protein (EVA-71, strain specific) and Group 3 was given our newly designed hexavalent vaccine (EV-A Consensus + 5 mosaic VP1). We measured the antibody binding levels to 9 EV-A VP1 antigens after immunization and found that the EV-A hexavalent vaccine resulted in improved antibody binding breadth during in vivo vaccination. Our results demonstrate the feasibility of using polyvalent VP1 proteins to increase the breath of antibody epitopes among EV-A serotypes. Our approach has the potential to treat and block EV-A infection and serve as a model for responding to other emerging viral diseases.