immunity

Regulation of neutrophil endoplasmic reticulum stress response by IRE1a

Project Summary/Abstract: The lungs are exposed to pathogens and environmental toxins that trigger stress and cause numerous respiratory diseases. Effective host defenses against lung infection by bacterial pathogens, including methicillin- resistant Staphylococcus aureus (MRSA), rely on innate immune cells including neutrophils, prominent early responders to sites of infection. If host defenses are ineffective, MRSA causes serious lung infection, resulting in severe morbidity and a significant economic burden on healthcare facilities, where it is endemic. MRSA infections have a mortality rate of up to 14% and an estimated $500 million in healthcare costs in the US alone. Increasing resistance to vancomycin, the last resort antibiotic for MRSA infections, underscore the urgent need for innovative treatment approaches. Although directly targeting pathogens with antibiotics has been a successful approach for treating infections, many pathogens, including MRSA, eventually will become resistant to these drugs. As an alternative, immunomodulatory strategies to enhance host defenses, such as those shown to be effective against cancer cells, have the potential for treating drug-resistant pathogen infections. Recently, we showed that the inositol-requiring enzyme 1-α (IRE1α), an endoplasmic reticulum (ER) stress sensor, is required for clearance of MRSA in a murine skin abscess model, where neutrophils are robustly recruited to the site of infection. Further, IRE1α coordinates signaling events upstream of calcium (Ca2+) mobilization, histone citrullination, and production of mitochondrial reactive oxygen species (mitoROS), all of which are important for neutrophil inflammatory responses including the formation of antimicrobial neutrophil extracellular traps (NETs). Because excessive neutrophil activation and NET release can be detrimental to vital organs, it is not clear whether neutrophil IRE1α-mediated stress responses aid or impede the resolution of infection in the lungs. While IRE1α activation has been linked to the development of lung fibrosis through the regulation of alveolar epithelial- to-mesenchymal transition in the context of chronic inflammatory diseases, its role in pulmonary neutrophil defenses is unknown. Thus, there is a gap in our knowledge of how cellular stress responses modulate pulmonary neutrophil defenses and infection outcomes in the lungs. The overarching goal of this proposal is to elucidate the mechanisms by which neutrophil IRE1α signaling influences production of mitoROS and Ca2+ mobilization to drive NET release, injure lungs, and regulate pulmonary host defense against MRSA. We will accomplish the following Aims: (1) Define the molecular mechanisms underlying IRE1α-mediated mitoROS hyperactivation of human and mouse primary neutrophils and excessive NET release, and (2) Elucidate the role of neutrophil IRE1α signaling in excessive NET release, lung injury, and immunity in vivo using a MRSA pneumonia infection mouse model. These studies will yield mechanistic insight into how IRE1α-driven ER stress responses impact pulmonary neutrophil defenses and lung injury revealing potential targets for anti-microbial immunotherapies.

Antibody-guided design of a human astrovirus vaccine

PROJECT SUMMARY Viral diarrheal diseases cause substantial global morbidity and mortality. Diarrheal disease is the second leading cause of childhood mortality in the world, accounting for over 10% of all deaths of children under 5 years old. Gobally, over 1 billion cases of diarrheal diseases occur every year, making prevention of these diseases a public health concern of the highest priority. Human astrovirus (HAstV) infection is a leading cause of viral diarrhea in children and has been shown to cause chronic gastrointestinal disease and fatal neurological disease in immunocompromised patients. There are nearly 4 million cases of HAstV infection each year in the United States alone, and there are no clinically approved HAstV-specific vaccines or therapeutics. Antibody-guided vaccine development leverages a deep understanding of productive antiviral antibody responses in order to design vaccine immunogens that deliberately focus the induced response toward highly conserved epitopes with the goal of reliably inducing broad, durable immunity. Using a cutting-edge monoclonal antibody (mAb) discovery approach based on next-generation antigen barcoding, single cell multi-omics, and sophisticated bioinformatics, we will exhaustively screen the HAstV- specific antibody repertoires of geographically distinct donor cohorts to uncover the structural and immunogenetic features that differentiate broad and potently neutralizing HAstV mAbs. A more complete understanding of these exceptional – and potentially very rare – mAbs will accelerate the development of HAstV vaccines and therapeutics. We have assembled a collaborative, multidisciplinary group of investigators with a long history of productive collaboration and with highly complementary areas of expertise. We expect our work will result in the discovery of thousands of novel anti-HAstV mAbs from cohorts of healthy adult and pediatric participants. Detailed genetic, functional, and structural characterization of these mAbs will reveal conserved sites of viral vulnerability, uncover the precise molecular mechanisms of viral neutralization, and inform our development of a broadly protective HAstV vaccine.

The role of endogenous chimeric mRNA encoded GasderminD fusion proteins in immunity

Project Summary: Programmed inflammatory cell death, or pyroptosis, is a crucial innate defense mechanism that protects hosts against infection and orchestrates subsequent immune responses. Central to this process is Gasdermin D (GSDMD), a protein that forms plasma membrane pores upon activation, enabling the release of pro- inflammatory cytokines such as IL-1β and driving cell lysis. Although GSDMD-mediated pyroptosis has been conventionally understood to be controlled mainly at the post-translational level, through proteolytic cleavage by inflammatory caspases, we have discovered compelling evidence that alternative RNA processing may introduce additional, previously unappreciated complexity in GSDMD regulation. Our laboratories have developed and optimized a highly innovative long-read direct RNA sequencing pipeline, which bypasses conventional cDNA synthesis to avoid artifacts and enables unbiased discovery of native chimeric mRNA (chRNA) in mammalian cells. Using this approach, we have uncovered a remarkably diverse repertoire of chRNA species, including over a thousand unique fusions in murine macrophages and more than two thousand in human inflamed tissues. Among the chRNA found in mice, we identified a chRNA joining the effector domain of GSDMD with a novel C-terminal region encoded by Tmem106a, giving rise to the GSDMD:TMEM106A fusion protein. Functional studies demonstrate that GSDMD:TMEM106A is not only produced in response to inflammatory signals in macrophages but is critical for GSDMD-dependent cytokine release and optimal pyroptosis. Genetic loss of GSDMD:TMEM106A in mice results in reduced cytokine secretion and increased susceptibility to bacterial infection, while in vivo delivery of Gsdmd:Tmem106a mRNA is sufficient for protective immunity. Intriguingly, we have also identified a putative human counterpart, GSDMD:S100A6, which is highly inducible in colon biopsies from patients with inflammatory bowel disease. In this application, we propose a comprehensive exploration of this newly defined class of naturally occurring GSDMD fusion proteins. The specific aims are: (1) to elucidate the subcellular localization, protein-protein interactions, and pore-forming function of GSDMD:TMEM106A during canonical and non-canonical inflammasome activation; (2) to determine the transcriptomic, proteomic, and physiological consequences of GSDMD chRNA expression in vivo during infection, sepsis, and inflammatory disease, and to validate and functionally characterize GSDMD:S100A6 in relevant immune and barrier cell populations. Collectively, this work will establish chimeric splicing as a fundamental source of immunoregulatory protein diversity, redefining the landscape of cell death control in the immune system. By revealing new layers of gasdermin regulation and function, our studies have the potential to identify novel therapeutic strategies for infectious, auto-inflammatory, and immune-mediated diseases.

Defining Microbial and Host Pathways Driving Asymptomatic C. difficile Colonization Associated with Aging and High-Sugar Diets

SUMMARY Clostridioides difficile infection (CDI) is a leading cause of healthcare-associated diarrhea, with rising incidence in community settings and a growing burden of asymptomatic colonization. Asymptomatic car- riers, particularly among the elderly and individuals consuming high-sugar diets, represent a critical but underexplored reservoir for transmission and disease progression. This proposal introduces novel, anti- biotic-independent mouse models demonstrating that both dietary sugar and aging independently pro- mote asymptomatic C. difficile colonization. We hypothesize that these factors disrupt colonization re- sistance (CR) through distinct but overlapping microbial, metabolic, and immune pathways. In Aim 1, we will define how traditional and emerging dietary sugars alter the gut environment to permit C. difficile colonization using in vitro bioreactors and in vivo models. Aim 2 will identify age-associated changes in microbiota and mucosal immunity that impair CR, using longitudinal studies and fecal micro- biota transfer. Aim 3 will functionally validate C. difficile genes upregulated during asymptomatic carriage using CRISPR-Cas9 mutants in both sugar- and age-induced models. This integrative, multi-omics approach will uncover the mechanisms enabling asymptomatic colonization and identify microbial and host targets for intervention. The findings will inform microbiome-based strat- egies to prevent CDI in vulnerable populations and shift current paradigms in CDI risk assessment and prevention.

From B-cell decisions to antibody repertoires

PROJECT SUMMARY/ABSTRACT Vaccine responses are highly variable across the population and not without risk for debilitating side-effects. Antibody-mediated immunity is generated by a Darwinian process to generate B-cells that contain B-cell receptors (BCR) that have high affinity for the pathogen-derived antigen, while also eliminating B-cells that happen to react to self-antigens. This process depends on cell fate decisions such as (i) death vs survival, (ii) entry into a proliferative program, (iii) differentiation into antibody-secreting plasma cells. According to clonal selection theory, B-cell fate decisions are made based on the genetically encoded affinity of the the BCR to the antigen (Signal 1) and the cognate T-cells’ TCR to the antigen peptide (Signal 2). However, single-cell resolution studies have revealed that fate decisions of genetically identical B-cells are remarkably heterogeneous. Our studies of the previous funding period revealed that B-cell epigenetic heterogeneity is in fact dynamically controlled: it is generated during the selection process but remains largely stable during the proliferative burst. This leads to our newly proposed Aim 1 to examine how the dynamic control of epigenetic state variability affects antibody responses. An innovative multi-scale model of Darwinian evolution directs and interprets experimental studies by life cell video microscopy in vitro and in immunization studies in vivo. Our previous studies also found that B-cells are capable of sensing the time gap between signal 1 and 2, suggesting a temporal proofreading mechanism for negative selection. This leads to newly proposed Aim 2 which seeks to identify the regulatory circuits that control the stringency of negative selection, as well as contextual germinal center (GC) cytokines that could be manipulable in vivo. These in silico and in vitro studies are followed by in vivo immunization to extend their physiological relevance. Finally, in Aim 3, we will ask what determines the time-gap of signal1 and signal 2, which occur in the immune- induced structure of the GC. We will develop a new model that simulates B-cell fate decisions as a function of their interactions with antigen-presenting stromal cells and T-cells that may be cognate or non-cognate. Model simulations will be used to interpret spatial transcriptomic data to test different adjuvants and predictions will be tested in in vivo immunization studies. With mouse models of inflammation and aging we will examine how adjuvants alter vaccine efficacy and risk.

Perturbation of mammary immunoglobulins during maternal antibiotic administration

Project Summary Prescribed in up to 40% of pregnancies, antibiotics represent the most commonly used class of medication during pregnancy. Although this practice is often necessary for maternal health, accumulating evidence suggests that antibiotic exposure may have unintended consequences for the mother-infant dyad. Epidemiologic studies associate maternal antibiotic exposure, especially in the absence of infection, with increased risk of neonatal complications including late-onset sepsis (LOS) and necrotizing enterocolitis (NEC), yet the mechanisms driving these associations remain poorly understood. Secretory IgA (sIgA) in milk is an essential component of neonatal mucosal immunity, shaping early gut microbial colonization and providing protection against enteric pathogens. The mechanisms by which maternal physiology regulates the abundance and microbial specificity of these antibodies in milk remain poorly understood. In animal models, the maternal gut–mammary axis governs the generation of milk IgA: IgA-committed lymphocytes from the maternal intestine migrate to the mammary gland during advancing pregnancy via CCL- 28/CCR10 signaling. Our preliminary data suggest that maternal antibiotic exposure disrupts this process leading to a decrease in milk IgA. However, the timing and extent of antibody dysbiosis are undefined; the downstream effects on neonatal intestinal health are unknown; and the underlying mechanisms—whether due to altered microbial stimulation, impaired recruitment of IgA⁺ cells to the mammary gland, or both—remain to be elucidated. Our central hypothesis is that maternal antibiotic exposure reduces pathogen-reactive IgA in milk by impairing gut-to-mammary immune cell trafficking thereby compromising neonatal mucosal immunity and increasing infection susceptibility. We will address this hypothesis through three integrated aims: (1) Determine the magnitude and duration of antibiotic-mediated mammary antibody dysbiosis in women who deliver preterm and at term; (2) Identify microbial targets of mammary antibodies diminished by maternal antibiotic exposure and (3 Determine the role of maternal antibiotics in the disruption of mammary resident IgA+ plasma cells in animal models. This integrative human and animal study will uncover critical mechanisms by which maternal antibiotic use alters the maternal-infant immune axis. The results will provide mechanistic insight into the risks associated with perinatal antibiotic exposure and inform clinical strategies to mitigate risk to neonatal health.

The role of GPR132 in regulating T cell responses in infection and cancer

PROJECT SUMMARY. CD8 T cells play a critical role in protection from a variety of infectious microorganisms, and pathogen-specific CD8 T cells undergo robust expansion, with an individual T cell clones expanding up to 10,000-fold in a matter of days. After infection is resolved, the majority of these T cells die, leaving a small population of memory cells to provide protective immunity from secondary challenge. T cell expansion and contraction are tightly orchestrated processes that involve a delicate balance between stimulatory and inhibitory signals to ensure proper immune function. Dysregulation of the T cell response can have detrimental effects; too little proliferation and the host fails to mount a successful immune response, while excessive proliferation and persistence of effector T cell populations can lead to tissue damage. This proposal aims to determine the role of the G protein coupled receptor GPR132 in the regulation of CD8 T cell responses during infection and tumorigenesis. GPR132 detects oxidized endogenous and microbial lipids, and this can lead to cell cycle arrest; however, the role of GPR132 in CD8 T cells remains unexplored. Here we identify GPR132 as a critical regulator of CD8 T cell expansion and memory differentiation. Completion of the proposed aims will: 1) uncover the temporal role of GPR132 in regulating T cell accumulation and function during infection and tumorigenesis, 2) examine the abundance of GPR132-activating ligands within the tissue during health and disease, and 3) determine how altering GPR132 ligand availability could be used to enhance/inhibit T cell responses. Overall, these studies will provide fundamental insights into the regulatory mechanisms that dictate the magnitude of T cell responses and how they can be modulated therapeutically, which would allow us to boost responses to pathogens/tumors or inhibit pathogenic responses in the context of autoimmune disease.

The Role of the Intestinal Microbiota in Sepsis Mortality

Project Summary/Abstract Sepsis is a life-threatening condition characterized by a dysregulated host response to infection that can cause multi-organ damage and death. As the leading cause of in-hospital mortality, sepsis mortality rates reach up to 50%, and account for approximately 270,000 deaths and $38 billion annually in health care costs in the United States. Notably, patients with similar medical backgrounds can have vastly different sepsis outcomes— some survive with medical treatment while others die. The reasons for this dichotomy are unknown but is seen across all forms of bacterial bloodstream infections, is not specific to any strain-level differences in the infecting pathogen and cannot be explained by human genetic differences. Human microbiota studies suggest that gut microbial dysbiosis is associated with sepsis mortality and that these alterations influence gut barrier breakdown, leading to gram-negative bacteremia—one of the most common causes of sepsis and mortality. However, there are a lack of studies that investigate the causal role of the intestinal microbiota in sepsis mortality. This K08 proposal will elucidate the role of the intestinal microbiota in sepsis mortality. Utilizing the well- established murine model of sepsis by intraperitoneal injection of lipopolysaccharide (LPS), we combine microbiota taxonomic sequencing and metagenomics, advanced bioinformatic techniques and prediction modeling, with knowledge of mucosal immunity and germ-free mouse systems to characterize the microbiota features and members that correlate with, predict, and cause sepsis mortality. This proposal is organized into two specific aims: (1) identify baseline stool microbial features associated with and predictive of sepsis outcomes and (2) determine how colonization with immunostimulatory microbes heightens sepsis mortality. In this work, I will holistically characterize the host immunologic and microbiota features that are associated with and predictive of mortality and experimentally identify microbes and microbial pathways that cause death in our model. These findings will reveal new microbial and host biomarkers of sepsis mortality and identify novel targets for sepsis prevention and treatment to reduce the overall mortality rate of this deadly disease. My long-term goal is to become an independent physician-scientist who integrates cutting-edge computational methods with experimental biology to identify predictive biomarkers of disease onset and outcomes, investigate how they influence disease processes, and develop novel therapeutic and preventive strategies to improve patient care. This proposal details specific research aims and a structured career development and training plan that will allow me to acquire focused, in-depth and multidisciplinary training under the guidance of an internationally recognized team of experts in clinical infectious diseases, host-microbiota interactions, immunology, immunometabolism, and computational biology. The knowledge generated will address the fundamental role of the microbiota in sepsis outcomes and inform future preventative and therapeutic strategies that will lower the sepsis mortality rate worldwide.

Targeting VIP–VPAC Signaling to Reverse Immune Exclusion and Enhance Immunotherapy Response in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer that is largely unresponsive to chemotherapy and current immune checkpoint blockade drugs, highlighting a critical need for the development of innovative therapeutic strategies. This R01 proposal targets vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide overexpressed in PDAC, which signals through VIP receptors (VPAC) on cancer cells, T cells, and myeloid cells within the tumor microenvironment. Based on our recent success in developing selective and potent VPAC receptor antagonists, we hypothesize that blocking VPAC signaling will reverse immunosuppression in the PDAC TME by reducing immune checkpoint expression, enhancing chemokine-driven infiltration of cytotoxic T cells, and disrupting immunosuppressive interactions between T cells and myeloid cells, ultimately leading to durable anti-cancer immunity. We propose three specific aims to explore the immunosuppressive roles of VPAC signaling in PDAC. Aim 1 will identify the primary sources of VIP in PDAC tumors and characterize the effects of VPAC signaling on immune cell function and phenotype within the tumor microenvironment. Aim 2 will investigate how VPAC signaling influences immune cell migration into tumors by modulating chemokine receptors and directional signaling. Aim 3 will determine how VPAC signaling regulates interactions between T cells and immunosuppressive myeloid cells, particularly tumor-associated macrophages, and the resulting impact on anti-cancer immune responses and immunological memory. Our preliminary findings indicate that combined inhibition of VPAC signaling and PD-1 significantly enhances the regression of PDAC tumors in multiple mouse models, generating lasting protective immunity in cured mice without triggering autoimmune responses. We will use novel methods to pursue our aims, including inducible genetically engineered mouse models (GEMM) of PDAC, long-acting VPAC antagonists engineered with immunoglobulin Fc domains to improve their plasma half-life, and advanced microfluidics technologies to analyze immune cell movement within tumors. Animal experiments will be used to validate the translational potential of observations from in vitro organoids and microfluidic experiments. The GEMM and orthotopic mouse models of PDAC are necessary to provide critical insights into the 3-D structure of the TME and tumor regression in response to our novel immunotherapy. This research will be conducted by a multidisciplinary team with complementary expertise that will clarify the therapeutic potential of VPAC signaling inhibition in PDAC using sophisticated experimental tools and single-cell RNA sequencing. Ultimately, these findings could significantly improve the development of immunotherapeutic strategies for PDAC, potentially enhancing patient outcomes in pancreatic cancer and other malignancies expressing high VIP levels.

Exploring in vivo Treg function in T1D through the lens of expanded Tregs

PROJECT SUMMARY/ABSTRACT A critical barrier to optimally treating Type 1 Diabetes (T1D), an autoimmune disease in which the islet beta cells are destroyed by immune cells, is understanding how autoimmunity is regulated in vivo. Several lines of evidence suggest that defective CD4+FOXP3+ regulatory T cells (Treg) likely contribute to the loss of tolerance in T1D. Yet, less is known about how human Treg function in vivo. In the Sanford T-rex study in which adolescents diagnosed with T1D were treated with a single dose of polyclonal autologous in vitro expanded Treg (expTreg), we found that a lower degree of in vitro Treg expansion significantly correlated with better preservation of C- peptide (a biomarker of insulin secretion and beta cell function) a year after treatment. This correlation could not be explained by age, expTreg phenotype or in vitro expTreg suppressive function. However, we did identify an expTreg gene signature that correlated with better C-peptide preservation and this expTreg signature was consistently expressed over time within individuals. Further, lower- and higher- expTreg differed phenotypically and transcriptionally by signatures implicating metabolic, homing and suppressive functions. Together, these data suggest that intrinsic features of an individual’s Treg may contribute to the extent of in vitro Treg expansion. They also suggest that strong activation and expansion can differentially amplify or alter the state of Tregs, leading to changes in homing and function that may impact clinical response. Based on these findings, we hypothesize that Treg proliferative capacity is driven by the activation and metabolic state of Treg resulting in differential in vitro fold expansion, homing potential and in vivo suppressive function that impacts clinical outcome. We will test this hypothesis by leveraging existing primary human samples from both the T-rex clinical trial and the Benaroya Research Institute Registry and Repository that includes individuals with known degree of in vitro Treg expansion and known C-peptide decline. In Aim1, we will identify how activation states of pre- and post- expansion Treg and longitudinal Treg in T-rex participants contribute to proliferative capacity and outcome using cellular, transcriptomic and epigenetic assays. In Aim 2 we will determine how metabolic shifts during Treg in vitro fold expansion alter Treg suppressive function, thereby impacting clinical outcome. In Aim 3, we will compare the in vivo suppressive function of lower- versus higher-expTreg from clinical samples using a xenogeneic graft versus host disease (GvHD) mouse model in addition to assessing in vivo expTreg homing and function using the assays from Aims 1 and 2 and a novel in vitro assay of cell trafficking to pancreatic islets. Successful completion of these aims will reveal mechanisms regulating Treg proliferative capacity and in vivo function that impact clinical outcome. Understanding these mechanisms will guide development of next generation Treg activation and expansion protocols for Treg therapies and help tailor the Treg expansion process to an individual’s baseline Treg signature.

Structural and functional characterization of autoimmune antibodies against NMDAR

Project Summary. The goal of this project is to understand the origins and molecular mechanisms underlying the anti-cancer autoimmune response against the N-methyl-D-aspartate receptor (NMDAR) and its correlation with anti-N-methyl-D-aspartate receptor autoimmune encephalitis (NMDARAE). While anti-cancer immune responses can promote tumor elimination, they may also lead to the production of self-reactive antibodies that trigger autoimmune diseases. NMDARAE is the most common form of immune-mediated encephalitis, which results in prominent neuropsychiatric symptoms, including seizures, psychosis, and memory deficits. NMDARs belong to a family of ligand-gated ion channels expressed exclusively in the central nervous system. They are involved in various aspects of brain development and function, including learning and memory. They respond to the neurotransmitter glutamate and a co-agonist, glycine or D-serine, to mediate excitatory neurotransmission, which plays a central role in synaptic plasticity. NMDARAE is associated with ovarian teratomas, where aberrant NMDAR expression is believed to trigger an autoimmune response. In NMDARAE, anti-NMDAR antibodies, as well as B cells and antibody-secreting cells, cross the blood-brain barrier via unknown mechanisms, resulting in the presence of anti-NMDAR antibodies at high titers within the brain and cerebrospinal fluid (CSF). These antibodies target NMDARs, modulating their function and contributing to disease pathology. Emerging evidence, supported by our preliminary data, suggests that NMDARs are also expressed in triple-negative breast cancer (TNBC), extending the relevance of anti-NMDAR autoimmunity beyond ovarian teratomas. In our TNBC mouse model, which ectopically expresses NMDARs (TNBC-NMDAR), we observed the onset of anti-NMDAR autoimmunity, where the produced antibodies cause both anti-tumor activity and symptoms such as lowered seizure threshold, mirroring key features of NMDARAE. Here, we will establish this TNBC mouse model as we develop molecular methods to characterize it. Aim 1 will focus on establishing and characterizing the TNBC- NMDAR mouse model. We will develop a detection method utilizing the intact tetrameric NMDAR channel proteins and a method to isolate B cells expressing B cell receptors against NMDAR from biological samples by using fluorescently labeled intact NMDAR proteins, followed by single-cell RNA sequencing. Aim 2 will utilize single-particle cryo-electron microscopy (cryo-EM) to investigate the interactions between NMDAR and the cloned antibodies, providing insights into epitope recognition, NMDAR subtype specificity, and conformational changes induced by antibody binding. Aim 3 will assess the impact of the cloned antibodies on NMDAR channel activity using electrophysiology. We will also assess anti-tumor activity and NMDARAE onset by each antibody clone. Together, the proposed research will gain insights into the link between anti-cancer anti-NMDAR autoimmunity and NMDARAE. It will also elucidate which functional properties of the cloned antibodies promote anti-tumor activity while contributing to NMDARAE, thereby informing potential therapeutic strategies.

Mechanisms of Commensal- Specific CD8+ T Cell Differentiation, Restraint and Dysregulation in Intestinal Inflammation

PROJECT SUMMARY Our understanding of immunity largely stems from models of infection with pathogenic microbes. However, the vast majority of microbial-immune encounters occur as a symbiotic relationship with the commensal microbiota. Recently, the contribution of commensal-specific T cells to host physiology has received significant attention. These commensal-specific responses not only control microbiota containment but also promote immune tolerance within the gastrointestinal tract. While commensal-specific CD4+ T cell responses in the lamina propria have dominated models of mucosal immune regulation, these are vastly outnumbered by CD8+ intraepithelial lymphocytes within the epithelium. How CD8+ T cell responses to gut microbiota are primed, differentiate and function under homeostasis has not been addressed. Conversely, aberrant immunity to commensal microbes has been proposed to underlie pathologies of barrier tissues, including inflammatory bowel disease (IBD), where commensal-specific T cells accumulate in blood and intestinal tissues of afflicted patients. A better understanding of the properties and functions of commensal-specific T cell responses is therefore fundamental to studies of tissue immunity in health and disease. Our long term goal is to better understand how commensal-specific T cell responses contribute to barrier tissue homeostasis, and the objective in this application is to investigate the mechanisms regulating induction of commensal-specific CD8+ T cells in homeostasis and how they become dysregulated in IBD. Our rationale for the proposed work is that uncovering these mechanisms has the potential to translate into new therapeutic approaches. Our central hypothesis is that commensal-specific CD8+ T cells develop as functionally restrained intraepithelial lymphocytes (IEL) under homeostasis, but that perturbation of local immune regulation within the intestinal epithelium, in the case of patients with ulcerative colitis, by autoantibody-mediated blockade of integrin avb6 results in aberrant CD8+ effector T cell responses in IBD. Based on strong preliminary data, we will test three specific aims: (1) Determine key antigen-presenting cells (APC) priming SFB-specific CD8⍺β+ IEL. (2) Identify how cell-intrinsic pathways drive differentiation, maintenance and restraint of SFB-specific CD8⍺β+ pIEL. (3) Determine how pathogenic KLRG1+Eomes+ CD8+ T cells arise and contribute to inflammation in murine models of ulcerative colitis Our approach is innovative as it investigates new mechanisms of immunity unique to commensal-specific CD8+ T cell responses. The proposed work is significant because it will establish new insights into the interaction and communication between commensal microbes and immune cells in the gut environment and identify potential targets for therapeutic intervention in conditions of chronic intestinal inflammation.

Transcriptional control of activation induced deaminase (AID) function

SUMMARY Somatic hypermutation (SHM) and class switch recombination (CSR) are vital for the generation of high affinity antibodies with appropriate effector function, protection against infection, and vaccine efficiency. They are initiated when the activation induced deaminase (AID) deaminates cytidines in single-stranded DNA in the context of transcription by RNA polymerase 2 (Pol2). Aberrant DNA deamination by AID is an important driver of genetic instability and the development of B cell malignancies. Understanding the factors and mechanisms that coordinate AID-mediated deamination with Pol2 transcription is an important objective in the study of humoral immunity and the central goal of research under this grant. Our preliminary data demonstrate that Pol2 pause factor NELF, Super Elongation Complex (SEC) components MLLT1/3, and the phosphatase module of the Integrator-protein phosphatase complex (INT-PP2A) are required for SHM, with MLLT1/3 but not NELF being required for AID binding to its chromatin targets. Our findings yield a new conceptual framework and model for AID-Pol2 collaboration in which NELF and a balance between kinase and phosphatase activities of SEC and INT-PP2A regulate Pol2 pausing/elongation to generate the critical stalled Pol2 complex on which AID acts. Further, our work has yielded major methodological advances that allow us to overcome obstacles that have stymied progress in the field. In this proposal, we take advantage of these conceptual and technical advances to pursue our central goal through the following two aims: Aim 1: Determine the molecular mechanisms by which NELF and other Pol2 regulatory factors enable AID-Pol2 collaboration and SHM/CSR. It has previously been very difficult to assess the role of cell-essential factors in SHM. By combining our new Rapid Assay for SHM (RASH) cells with degron technology, we will determine the mechanism of action of our newly discovered regulators of SHM using genomic, transcriptomic, and interaction assays that assess Pol2 distribution, phosphorylation, and activity, and the chromatin binding profiles of and interactions between AID and components of NELF, SEC, and INT-PP2A. AID and MLLT1 appear to co-associate in a complex and we will test for a direct interaction between AID and MLLT1/3. Factors will be tested for roles in CSR and validated in human cell line and germinal center B cell models and in mice. Aim 2: Hypothesis testing and deep mechanistic analysis through perturbation of the balance between Pol2 pause/arrest and elongation. We will rigorously test our new model for AID-Pol2 collaboration using degron, reconstitution, mutagenesis, and small molecular inhibitor approaches to perturb the balance between Pol2 pausing and elongation, revealing how altering NELF-Pol2 interactions and the balance between SEC kinase and INT-PP2A phosphatase activities influences SHM efficiencies and AID binding. Together, our proposed studies are significant for the development of new technologies and for understanding mechanisms of antibody gene diversification and causes of genome instability and cancer.

Airway Epithelial Defense Mechanisms in Combating STAT3-Deficiency-Related Lung Infections

Airway Epithelial Defense Mechanisms in Combating STAT3-Deficiency-Related Lung Infections Signal transducer and activator of transcription 3 (STAT3) regulates the expression of genes essential for various cellular processes, including survival, proliferation, differentiation, self-renewal, angiogenesis, and immune response. Abnormal and persistent STAT3 activation is detected in diverse human cancers, driving multiple pro- oncogenic functions. Multiple antitumor drug development targets the inhibition of STAT3 to treat various types of cancer. Unfortunately, downregulated STAT3 significantly increases host susceptibility to recurrent infections, especially pneumonia. Additionally, individuals with genetic polymorphisms associated with lower STAT3 expression are more susceptible to severe tuberculosis. Furthermore, patients with autosomal dominant hyper- IgE syndrome (AD-HIES), also known as Job Syndrome, which is caused by de novo STAT3 mutations and substantially decreased STAT3 expression, have a significantly increased susceptibility to bacterial and fungal infections, with high mortality rates and a shortened life span often associated with Pseudomonas aeruginosa infections. Gram-negative bacteria, particularly P. aeruginosa, are opportunistic pathogens that frequently cause hospital-acquired infections. The problems are worsened by the emerging P. aeruginosa with multidrug resistance (MDR), especially in patients with repeated antibiotic treatments, such as Job Syndrome sufferers. Notably, airway epithelial cell-derived proteins play a significant role in the antimicrobial milieu, promoting effective host defense against invading pathogens. One of the most critical STAT3-regulated antimicrobial molecules is bactericidal permeability-increasing protein fold A1 (BPIFA1, also known as SPLUNC1), a multifunctional innate immunity molecule and indispensable host defense protein that is abundantly secreted in the lungs. This application aims to elucidate how STAT3 deficiency impairs host epithelial defense against microbial infections and whether BPIFA1-mediated innate immune responses can sufficiently restore effective antimicrobial protection to prevent pneumonia. The long-term objective is to advance our understanding of the respiratory innate immune response, particularly in relation to epithelial cell-specific antimicrobial defense. We characterized BPIFA1 as an airway lining fluid protein secreted apically in the airway lumen and in primary human airway epithelial cultures. In this study, we hypothesize that mucosal BPIFA1 is an essential antimicrobial protein that plays a critical role in host defense against microbial infections in STAT3-deficiency- associated pneumonia. Our proposed studies will assess innate immunity mechanisms regulating the antimicrobial activity of the airway epithelium in STAT3 deficiency-associated lung infections. By focusing on the crucial epithelial-derived protein product, BPIFA1, our study will provide an alternative treatment for respiratory infections by augmenting native host defense mechanisms in high-risk individuals, including AD-HIES, cancer, and immunocompromised patients.

Bridging Local and System-Wide Autoreactive, Extrafollicular B Cell Signatures in a TLR7-Driven Model

Project Summary A substantial body of literature has described the development of autoreactive humoral responses in the context of autoimmune disease and recently discerned an exciting new avenue for investigation. While early work focused on canonical mechanisms of activation through the germinal center (GC) response, recent studies have found GC infrastructure to be dispensable for the onset of chronic autoimmunity. It has become clear that an alternative pathway of B cell activation, the extrafollicular (EF) pathway, can drive the onset of new autoreactivity in multiple human disorders including rheumatoid arthritis and systemic lupus erythematosus (SLE). In comparison to the GC pathway, the EF pathway represents a less stringent method for B cell activation, leads to accelerated antibody-secreting cell (ASC) formation, and thus has a higher propensity for the production of autoreactive B cell effectors and ASCs. Recently, our group has identified a similar skew toward the EF response in the context of severe viral infection, tied to acute tolerance loss, increased disease severity, and complicated recovery from infection. These findings highlight how further study of the EF response is crucial to our understanding of autoimmune induction across multiple areas of disease. Toll-like receptor 7 (TLR7) stimulation has been identified as a key contributor to EF B cell development in SLE, and several studies have now linked TLR7 overstimulation to chronic autoimmune disease. While EF effector B cell populations have now been identified in both murine models and humans, substantial gaps in our knowledge remain to be answered concerning i) the origins of these cells and ii) the system-wide and microenvironmental signaling and organization that drive this differentiation pathway. We propose to address these gaps, here, by utilizing a TLR7 agonist (R848) in a murine model to characterize the autoreactive response within the blood and draining lymph node through innovative high-throughput analytical techniques. Systemic shifts in proteomic signatures and immune cell phenotype will be monitored in the blood throughout the induction of autoreactivity, using novel applications of machine-learning based classification. These signatures will then be connected to developing inflammatory microenvironments identified within the draining lymph node by applying a customized set of software tools to spatial transcriptomic data. This work will deepen our understanding of the immunologic mechanisms by which the EF pathway can lead to “run-away” autoreactive B cell development, with the added potential for identification of early blood-based biomarkers for this developing autoreactivity. The above proposed work will provide an ideal training opportunity for the candidate to develop experience with advanced immunologic laboratory techniques, rigorous bioinformatic analysis, a systems-level view of immunology, and scientific communication. The Woodruff and Sanz Labs are highly experienced within the autoimmune disease space with extensive experience with the required techniques and established routes for clinical collaboration to act on these findings.

Transposable element silencing as a regulator of salivary gland immune homeostasis

PROJECT SUMMARY/ABSTRACT Sjogren’s syndrome (SjS) is a chronic autoimmune disorder marked by salivary and lacrimal gland dysfunction, lymphocytic infiltration, and progressive secretory decline. While traditionally viewed as immune cell–driven, emerging evidence suggests that epithelial cells may initiate local inflammation. However, the molecular triggers originating from epithelial cells remain poorly defined. Transposable elements (TEs), including endogenous retroviruses (ERVs) and LINEs, are normally repressed through DNA methylation, histone modifications, and heterochromatin organization. Failure of TE silencing mechanisms due to aging, hormonal changes, or stress results in cytoplasmic dsRNA accumulation, nucleic acid sensor activation, and type I interferon signaling. These TE-derived nucleic acids are increasingly recognized as endogenous triggers of immunological stress that disrupt cellular homeostasis. Our preliminary data show widespread TE derepression and upregulation of interferon-stimulated genes in salivary glands from patients with SjS. To mimic this phenomenon, we will inducibly delete Setdb1, a key histone H3K9 methyltransferase, in defined epithelial compartments of the salivary gland. This will allow us to model compartment-specific TE derepression and assess its impact on both innate immune activation and adaptive immune responses. We will also test how aging and estrogen deficiency disrupt TE repression in basal/ductal versus acinar cells using lineage tracing and epigenomic profiling. Finally, we will evaluate the therapeutic potential of reverse transcriptase inhibitors and chromatin-modifying drugs in attenuating TE-driven inflammation. This exploratory study will uncover how failure of TE silencing contributes to epithelial-driven autoimmunity in SjS and will provide a foundation for future targeted epigenetic manipulations in human tissues and patients.

Targeted Prodrug Cytokines for Metastatic Breast Cancer Immunotherapy

Project Summary. Our approach directly addresses key limitations in targeting and treating metastatic breast cancer, where we propose the selective activation of modular immune-modulating cytokines within the hypoxic and ROS-active TME for delivery across the BBB, providing the necessary pre-clinical data for future clinical translation. The in vitro and in vivo investigations of this novel immunotherapeutic in immunocompetent models will allow our team to study the interplay between tumor-driven immune activation, cytokine signaling, and anti-tumor immunity in both primary and metastatic sites, and establish a robust groundwork for subsequent clinical validation within the OSUCCC. This proposal addresses two key challenges in developing a novel immunotherapy strategy for breast cancer by answering two hypotheses: (1) can a modular immunotherapy platform with tumor-selective activation of prodrug recombinant cytokines overcome these limitations in drug delivery, and (2) can the development of nanobody-cytokine fusions that can selectively target primary breast cancer tumors and cross the BBB to reach metastatic tumor sites? The first hypothesis focuses on achieving tumor environment-specific activation of prodrug-based recombinant cytokines. Protein cytokines are highly potent, and while others have tried to block their activity using a fused genetic linker to ‘mask’ functionality, no one has yet attempted to use a non-canonical-based chemical strategy to achieve this inhibition. Immune-modulating cytokines will be recombinantly expressed with integrated ncAAs that block cytokine activity until the function is regenerated in the breast cancer TME. Once the cytokine activity is controlled, our second hypothesis will be to achieve selective delivery of the cytokine via fusion to nanobodies. While success has been found in targeting primary tumors in drug and protein delivery, a key challenge remains in reaching secondary metastatic tumors in hard-to-reach sites (i.e., brain). Engineered nanobodies, with affinity for breast cancer tumors and the ability to bind to BBB transcytosis receptors, will enable selective delivery to metastatic breast-to-brain tumors, resulting in tumor- specific activation, immune responses, and improved therapeutic outcomes. This system can significantly improve therapeutic outcomes for patients with mBC by integrating selective activation and delivery mechanisms to reduce off-target effects and enhance tumor-specific immune responses in both primary and secondary metastatic tumor sites. Optimizing drug delivery systems to tune immune responses could offer more effective and less invasive treatment options when compared to traditional and engineered cell-based approaches. Our momentum towards precision medicine and targeted therapies holds significant promise for improving outcomes for mBC patients, and has the potential to serve as a pan-cancer treatment for aggressive metastatic cancers from the following aims: (1) generating a modular platform for tumor-specific activation of prodrug cytokines, (2) evaluating cytokine delivery and anti-cancer immune phenotypes in mBC.

Optimizing gamma-delta T cell receptor-mediated signaling to improve cancer immunotherapy

PROJECT SUMMARY The recent development of T cell-based cancer immunotherapies, including checkpoint blockade (anti-PD-1, anti-CTLA-4 and others) or adoptive cell therapy (ACT) using modified patient T cells, has led to improved patient outcomes for a variety of cancers. However, durable responses are observed in only a fraction of patients. Further progress can be made by studying and targeting different T cell subpopulations, such as the gd T cells which are known to possess antitumor activities. Further, gd T cells are mostly independent of MHC-restriction, unconstrained by neoantigen burden, preferential homing to peripheral tissues and possess unique properties of T cells as well as natural killer cells making them an extremely attractive cancer immunotherapy target. One way of gd T cell activation involves the gd T cell receptor (gdTCR)-CD3 signaling pathway. gd T cell recognition of antigen by the gdTCR and the resulting proximal signaling through surrounding CD3 subunits are key steps of gd T cell activation. Even though the individual components of the gdTCR-CD3 and abTCR-CD3 complexes remain the same except for the TCRs, the complete gdTCR-CD3 complex extracellular structure is unknown. Identification of the specific extracellular interactions between the gdTCR and CD3 subunits could offer precise guidance for the development of immunotherapeutic strategies that modulate gdT cell immunity by targeting signaling through the gdTCR-CD3 complex. Our previous data showed that mutating residues in the constant domain of the abTCR resulted in altered ab T cell cytokine responses. Based on this data, our hypothesis is that gdTCR-CD3 signaling can also be modulated by targeting specific regions of the gdTCR by mutagenesis to improve gd T cell antitumor activities. To test our hypothesis, in Aim 1, we will use a novel photo-crosslinking and computational docking methodology to solve the complete extracellular structure of a gdTCR-CD3 complex. Further, we will use an in silico structure-based TCR design approach to identify gdTCR mutants that enhance signaling. In Aim 2, we will use an in vitro retroviral TCR display method using degenerate primers to create gdTCR mutant libraries at specific gdTCR sites such as Cg helix 3 and connecting peptide (CP) regions. In both instances, identified mutants will be tested for improved functionalities in an MHC-independent gd TCR (G115 Vg9Vd2 TCR) using in vitro cytokine and tumor-killing assays. Overall, the newly identified enhanced gd T cell clones could potentially lead to a new wave of effective cancer immunotherapy strategy by leaning into the largely untapped potential of gd T cells.

Structure-Based Development of Nucleotide-Competing Inhibitors Against HIV-1 and LINE-1 Reverse Transcriptases

PROJECT SUMMARY Reverse transcriptases (RTs) from retroviruses and endogenous retroelements are essential polymerases that catalyze RNA- and DNA-dependent DNA synthesis. Nucleoside inhibitors (NIs) remain central to HIV-1 therapy and are also used against other viral infections and in cancer, but toxicity, limited selectivity, pharmacokinetic (PK) liabilities, and the emergence of drug resistance highlight the need for alternative RT inhibitor mechanisms. In contrast to NIs, nucleotide-competing inhibitors (NCIs) block the polymerase active site without requiring incorporation into nucleic acids. Structural studies by PI Ruiz have defined the NCI mechanism of action for HIV- 1 RT and revealed conserved binding modules shared across multiple polymerase families. These advances now enable rational discovery of improved NCIs. LINE-1 (L1) ORF2 RT is an emerging therapeutic target in cancer, autoimmunity, and aging, yet NIs are the only inhibitors known to act against L1 RT. Notably, the NCI-binding region is structurally similar between HIV-1 RT and L1 RT, suggesting that NCI recognition principles may extend across these two biologically distinct polymerases. This R21 seeks to establish proof-of-concept for NCI development against both enzymes. Aim 1 will discover and structurally optimize NCIs targeting HIV-1 RT by combining binding modules from known NCI chemotypes and determining their biochemical activity and co-crystal structures. Aim 2 will determine whether HIV-1 RT NCI principles translate to L1 RT by solving L1 RT/nucleic acid/NCI structures, evaluating enzymatic inhibition, and applying AI-based structure prediction and generative design to propose L1-specific NCI candidates. Cellular retrotransposition assays will test mechanism of action. Aim 3 will develop a fragment library tailored to protein–nucleic acid interfaces and perform fragment screening of HIV-1 and L1 RT/nucleic acid complexes to identify additional chemotypes that engage the NCI binding region. Successful completion will yield NCI scaffolds and mechanistic insights applicable to HIV-1 RT and L1 RT, define structural principles governing NCI recognition across two evolutionarily related polymerases, and establish new avenues for RT inhibitor development. The PI is highly qualified to lead this work, with extensive expertise in RT structural biology, drug design, and fragment-based discovery.

A Novel Mitochondrial-Targeted Inhibitor of NLRP3 Inflammasome Activation

PROJECT ABSTRACT Inflammasomes are multiprotein complexes of the innate immune system that assemble upon detecting specific molecular patterns associated with pathogens and cellular damage. Once assembled, activated inflammasomes trigger a cascade of downstream events that culminate in cell death and inflammation. Aberrant activation of the NLRP3 inflammasome contributes to the pathogenesis of numerous inflammatory and degenerative diseases, including gout, atherosclerosis, type 2 diabetes, and Alzheimer’s disease. Despite its central role in innate immunity and inflammation, there are no FDA-approved therapies that directly target the NLRP3 inflammasome. Current strategies rely on biologics that inhibit downstream pro-inflammatory cytokines produced from inflammasome activation, such as interleukin-1β (IL-1β), but do not block upstream inflammasome assembly or pyroptotic cell death, highlighting a critical unmet need for selective small-molecule inhibitors with novel mechanisms of action. To address this gap, we identified a covalent small molecule, Compound-2 (C-2), that robustly inhibits NLRP3 inflammasome activation in murine and human immune cells. C-2 suppresses multiple downstream events triggered by inflammasome activation, including IL-1β secretion and pyroptosis, with no apparent toxicity. Chemoproteomic profiling revealed that C-2 interacts with SLC25A3, a mitochondrial phosphate and copper transporter, suggesting a previously unrecognized regulatory node in inflammasome signaling. This R21 project aims to (1) elucidate the mechanism by which C-2 suppresses NLRP3 activation and (2) define the molecular interaction between C-2 and SLC25A3 and its functional consequences. Our studies will integrate biochemical, cellular, and in vivo approaches to uncover a novel mitochondrial mechanism of inflammasome regulation and validate C-2 as a first-in-class inflammasome inhibitor. Successful completion of this project will lay the foundation for future therapeutic development targeting mitochondrial- inflammasome crosstalk in inflammatory disease.

Breaking Tolerance: Trichloroethylene Provides Survival Signals to Autoreactive CD4s in the Liver

PROJECT SUMMARY The industrial solvent and widespread environmental contaminant, trichloroethylene (TCE) has been linked to autoimmune disease in humans. How TCE impairs tolerance (i.e., unresponsiveness) to self-antigens leading to autoimmunity has not been explored. Autoimmune diseases (ADs) are a class of disorders that affect many different organs and tissues. However, all autoimmune diseases share a feature in common which is the ability of potentially pathogenic autoreactive cells to evade deletion. During early life, peripheral CD4+ cells are primarily comprised of recent thymic emigrants (RTE) which home to the liver. The liver is known to efficiently retain and tolerize self-reactive CD4s to where they are functionally unresponsive to their antigen. Thus, the liver is the first checkpoint in the periphery to filter, retain, and enforce tolerance to autoreactive CD4+ RTEs. The liver is also the site of TCE metabolism. Our Aims are designed to test the hypothesis that TCE, through its metabolite TCAH, delivers costimulatory signals to liver CD4 RTEs via CD28, thereby overriding inhibitory CTLA-4 signaling. This disruption promotes the survival of self-reactive CD4 RTEs by impairing CTLA-4-dependent tolerance mechanisms contributing to the development of ADs. This research will significantly advance the fields of toxicology and autoimmunity, where the origins of environmentally induced AD remain poorly understood. Aim 1 will assess TCE’s effects on RTE migration patterns in real-time in transgenic mice. Aim 2 will investigate TCAH-mediated costimulatory signaling in CD4 RTEs in vitro. Successful completion of these studies will determine how TCE alters key tolerance pathways in the liver resulting in a greater proportion of self-reactive effector memory (EM) peripheral CD4s capable of promoting AD.

Glycoengineering core a(1,3)-fucose motifs to enhance HIV-1 envelope vaccine immunogenicity

Project Summary The HIV-1 envelope glycoprotein (Env) is the sole target of neutralizing antibodies (NAbs). We previously developed a vaccine platform integrating three innovations: (1) the uncleaved prefusion-optimized (UFO) trimer design to stabilize Env; (2) multilayered single-component self-assembling protein nanoparticles (1c-SApNPs) for multivalent trimer display; and (3) enzymatic trimming of oligomannose glycans on CHO cell-produced Env immunogens. Glycan trimming substantially improved Env immunogenicity by enhancing tier 2 NAb elicitation, reducing off-target responses to immunodominant glycan sites, and increasing responder rates. These vaccine candidates are now in phase 1 clinical trials (NCT06541093; NCT06905275). Building on this foundation, we propose a novel strategy to enhance immunogenicity by incorporating core α(1,3)-fucose into HIV-1 Env. Core α(1,3)-fucose, a key allergenic epitope in many plant and insect glycoproteins, is highly immunogenic in humans and other mammals. Our central hypothesis is that the targeted introduction of core α(1,3)-fucose will convert the glycan shield from an immune-evasive barrier into an immunogenic trigger that promotes NAb induction. Glycoengineered cell lines expressing α(1,3)-fucose will enable production of highly immunogenic Env vaccines suitable for preclinical and clinical testing. Importantly, particulate display of these Env trimers on 1c-SApNPs can suppress IgE-mediated allergic pathways by inducing high-affinity protective IgGs, ensuring vaccine safety. Aim 1 will focus on producing core α(1,3)-fucosylated HIV-1 Env immunogens. We will begin by developing a transient insect cell expression system using BTI-TN-5B1-4 (“High Five” or Hi5) cells to produce Env with short paucimannose glycans bearing native α(1,3)-fucose. To further enhance α(1,3)-fucosylation, we will co-express exogenous core α(1,3)-fucosyltransferases in insect and CHO cells. We will validate glycan profiles and characterize the biochemical, biophysical, structural, and antigenic properties of the resulting immunogens. Aim 2 will assess the immunogenicity of these glycoengineered HIV-1 Env immunogens. Using our previously established glycan-trimmed Env immunogens as benchmarks, we will immunize mice, rabbits, and nonhuman primates (NHPs). Mice will be used for early-stage immunogen and adjuvant screening; rabbits to evaluate glycan hole-targeting NAb responses; and key vaccine formulations will advance to NHP studies. We will assess autologous and heterologous tier 2 NAb responses and vaccine responder rates. Aim 3 will elucidate the functional, structural, repertoire, and mechanistic basis of vaccine-induced immunity. We will isolate NAbs via Env-specific single-cell sorting and antibody cloning, map epitopes by electron microscopy (EM) and X-ray crystallography, perform next-generation sequencing (NGS) of B-cell repertoires, and trace NAb lineages. Finally, we will investigate antigen trafficking, retention, presentation, and germinal center (GC) reactions in lymph nodes. Together, these studies will define a new class of glycoengineered HIV-1 vaccines and establish core α(1,3)-fucose as a novel immunomodulatory tool to overcome glycan shield-mediated immune evasion.

The Role of GPCR Family Mrgprs in Itch, Pain, and Innate Immunity

From the guts to the brain through adaptive immunity in the prevention of Alzheimer’ disease

Dr. Pasinetti is the Saunders Family Chair and Professor of Neurology at Icahn School of medicine at Mount Sinai, New York. His studies allowed him to develop novel therapeutic approaches through investigation of preventable risk factors including mood disorders in the promotion of resilience against neurodegenerative disorder. In his presentation Dr. Pasinetti will discuss novel concepts about the gut-brain axis in mechanisms associated to peripheral adaptive immunity as therapeutic targets to mitigate the onset and the progression of Alzheimer’s disease and other form of dementia.

Hallucinating mice, dopamine and immunity; towards mechanistic treatment targets for psychosis

Hallucinations are a core symptom of psychotic disorders and have traditionally been difficult to study biologically. We developed a new behavioral computational approach to measure hallucinations-like perception in humans and mice alike. Using targeted neural circuit manipulations, we identified a causal role for striatal dopamine in mediating hallucination-like perception. Building on this, we currently investigate the neural and immunological upstream regulators of these dopaminergic circuits with the goal to identify new biological treatment targets for psychosis

Engineering an inhibitor-resistant human CSF1R variant for microglia replacement

Humoral immunity at the brain borders in homeostasis and a scRNA-seq atlas of immune cells at the CNS borders

https://www.cnsbordercellatlas.org/

Meningeal macrophages protect against viral neuroinfection

https://doi.org/10.1016/j.immuni.2022.10.005

Social immunity in ants: disease defense of the colony

Social insects fight disease as a collective. Their colonies are protected against disease by the combination of the individual immune defenses of all colony members and their jointly performed nest- and colony-hygiene. This social immunity is achieved by cooperative behaviors to reduce pathogen load of the colony and to prevent transmission along the social interaction networks of colony members. Individual and social immunity interact: performance of sanitary care can affect future disease susceptibility, yet also vice versa, individuals differing in susceptibility adjust their sanitary care performance to their individual risk of infection. I present the integrated approach we use to understand how colony protection arises from the individual and collective actions of colony members and how it affects pathogen communities and hence disease ecology.

Remembering Immunity, Central regulation of peripheral immune processes

Thoughts and emotions can impact physiology. This connection is evident by the emergence of disease following stress, psychosomatic disorders, or recovery in response to placebo treatment. Nevertheless, this fundamental aspect of physiology remains largely unexplored. In this talk, I will focus on the brain’s involvement in regulating the peripheral immune response and explore the question of how the brain evaluates and represents the state of the immune system it regulates.

Remembering immunity: Neuronal representation of immune responses

Accumulating data indicate that the brain can affect immunity, as evidenced, for example, by the effects of stress, stroke, and reward system activity on the peripheral immune system. However, our understanding of this neuroimmune interaction is still limited. Importantly, we do not know how the brain evaluates and represents the state of the immune system. In this talk, I will present our latest study from our lab, designed to test the existence of immune-related information in the brain and determine its relevance to immune regulation. We hypothesized that the InsCtx, specifically the posterior InsCtx (as a primary cortical site of interoception in the brain), is especially suited to contain such a representation of the immune system. Using activity-dependent cell labeling in mice (FosTRAP), we captured neuronal ensembles in the InsCtx that were active under two different inflammatory conditions (dextran sulfate sodium [DSS]-induced colitis and zymosan-induced peritonitis). Chemogenetic reactivation of these neuronal ensembles was sufficient to broadly retrieve the inflammatory state under which these neurons were captured. Moreover, using retrograde neuronal tracing, we found an anatomical efferent pathway linking these InsCtx neurons to the inflamed peripheral sites. Taken together, we show that the brain can store and retrieve specific immune responses, extending the classical concept of immunological memory to neuronal representations of inflammatory information.

JAK/STAT regulation of the transcriptomic response during epileptogenesis

Temporal lobe epilepsy (TLE) is a progressive disorder mediated by pathological changes in molecular cascades and neural circuit remodeling in the hippocampus resulting in increased susceptibility to spontaneous seizures and cognitive dysfunction. Targeting these cascades could prevent or reverse symptom progression and has the potential to provide viable disease-modifying treatments that could reduce the portion of TLE patients (>30%) not responsive to current medical therapies. Changes in GABA(A) receptor subunit expression have been implicated in the pathogenesis of TLE, and the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway has been shown to be a key regulator of these changes. The JAK/STAT pathway is known to be involved in inflammation and immunity, and to be critical for neuronal functions such as synaptic plasticity and synaptogenesis. Our laboratories have shown that a STAT3 inhibitor, WP1066, could greatly reduce the number of spontaneous recurrent seizures (SRS) in an animal model of pilocarpine-induced status epilepticus (SE). This suggests promise for JAK/STAT inhibitors as disease-modifying therapies, however, the potential adverse effects of systemic or global CNS pathway inhibition limits their use. Development of more targeted therapeutics will require a detailed understanding of JAK/STAT-induced epileptogenic responses in different cell types. To this end, we have developed a new transgenic line where dimer-dependent STAT3 signaling is functionally knocked out (fKO) by tamoxifen-induced Cre expression specifically in forebrain excitatory neurons (eNs) via the Calcium/Calmodulin Dependent Protein Kinase II alpha (CamK2a) promoter. Most recently, we have demonstrated that STAT3 KO in excitatory neurons (eNSTAT3fKO) markedly reduces the progression of epilepsy (SRS frequency) in the intrahippocampal kainate (IHKA) TLE model and protects mice from kainic acid (KA)-induced memory deficits as assessed by Contextual Fear Conditioning. Using data from bulk hippocampal tissue RNA-sequencing, we further discovered a transcriptomic signature for the IHKA model that contains a substantial number of genes, particularly in synaptic plasticity and inflammatory gene networks, that are down-regulated after KA-induced SE in wild-type but not eNSTAT3fKO mice. Finally, we will review data from other models of brain injury that lead to epilepsy, such as TBI, that implicate activation of the JAK/STAT pathway that may contribute to epilepsy development.

Role of the gut microbiota in the development of alcohol use disorder

The gut microbiota is composed of a very large number of bacteria, viruses, fungi and yeasts that play an important role in the body, through the production of a series of metabolites (including neurotransmitters), and through an essential role in the barrier function of the gut and the regulation of immunity and stress response. In this lecture I will present, based mainly on human studies but also on preclinical studies, the evidence for a role of the gut microbiota in the development of alcohol use disorder. I will show the first results of trials to test the effects of nutritional approaches to address these deficits.

Reflex Regulation of Innate Immunity

Reflex circuits in the nervous system integrate changes in the environment with physiology. Compact clusters of brain neuron cell bodies, termed nuclei, are essential for receiving sensory input and for transmitting motor outputs to the body. These nucelii are critical relay stations which process incoming information and convert these signals to outgoing action potentials which regulate immune system functions. Thus, reflex neural circuits maintain parameters of immunological physiology within a narrow range optimal for health. Advances in neuroscience and immunology using optogenetics, pharmacogenetics, and functional mapping offer a new understanding of the importance of neural circuitry underlying immunity, and offer direct paths to new therapies.

Innate immune response in brain pathologies: Lost in translation?

Inflammation is a key component of the innate immune response. Primarily designed to remove noxious agents and limit their detrimental effects, the prolonged and/or inappropriately scaled innate immune response may be detrimental to the host and lead to a chronic disease. Indeed, there is increasing evidence suggesting that a chronic deregulation of immunity may represent one of the key elements in the pathobiology of many brain disorders. Microglia are the principal immune cells of the brain. The consensus today is that once activated microglia/macrophages can acquire a wide repertoire of profiles ranging from the classical pro-inflammatory to alternative and protective phenotypes. Recently, we described a novel ribosome-based regulatory mechanism/checkpoint that controls innate immune gene translation and microglial activation involving RNA binding protein SRSF3. Here we will discuss the implications of SRSF3 and other endogenous immune regulators in deregulation of immunity observed in different models of brain pathologies. Furthermore, we will discuss whether targeting SRSF3 and mRNA translation may open novel avenues for therapeutic modulation of immune response in the brain.

Neuroimmune interactions in Cardiovascular Diseases

The nervous system and the immune system share the common ability to exert gatekeeper roles at the interfaces between internal and external environment. Although interaction between these two evolutionarily highly conserved systems is long recognized, the pathophysiological mechanisms regulating their reciprocal crosstalk in cardiovascular diseases became object of investigation only more recently. In the last years, our group elucidated how the autonomic nervous system controls the splenic immunity recruited by hypertensive challenges. In my talk, I will focus on the molecular mechanisms that regulate the neuro-immune crosstalk in hypertension. I will elaborate on the mechanistic insights into this brain-spleen axis led us uncover a new molecular pathway mediating the neuroimmune interaction established by noradrenergic-mediated release in the spleen of placental growth factor (PlGF), an angiogenic growth factor potentially targetable with pharmacological approaches.

Interactions between the microbiome and nervous system during early development

The gut microbiota is emerging as an important modulator of brain function and behavior, as several recent discoveries reveal substantial effects of the microbiome on neurophysiology, neuroimmunity and animal behavior. Despite these findings supporting a “microbiome-gut-brain axis”, the molecular and cellular mechanisms that underlie interactions between the gut microbiota and brain remain poorly understood. To uncover these, the Hsiao laboratory is mining the human microbiota for microbial modulators of host neuroactive molecules, investigating the impact of microbiota-immune system interactions on neurodevelopment and examining the microbiome as an interface between gene-environment interactions in neurological diseases. In particular, our research on effects of the maternal microbiome on offspring development in utero are revealing novel interactions between microbiome-dependent metabolites and fetal thalamocortical axonogenesis. Overall, we aim to dissect biological pathways for communication between the gut microbiota and nervous system, toward understanding fundamental interactions between physiological systems that impact brain and behavior.

Meningeal lymphatics and peripheral immunity in brain function and dysfunction

Immune cells and their derived molecules have major impact on brain function. Mice deficient in adaptive immunity have impaired cognitive and social function compared to that of wild-type mice. Importantly, replenishment of the T cell compartment in immune deficient mice restored proper brain function. Despite the robust influence on brain function, T cells are not found within the brain parenchyma, a fact that only adds more mystery into these enigmatic interactions between T cells and the brain. Our results suggest that meningeal space, surrounding the brain, is the site where CNS-associated immune activity takes place. We have recently discovered a presence of meningeal lymphatic vessels that drain CNS molecules and immune cells to the deep cervical lymph nodes. This communication between the CNS and the peripheral immunity is playing a key role in neurophysiology and in several CNS disorders. Interestingly, meningeal lymphatics are impaired in aging and their dysfunction may be related to age-related cognitive decline as well as to Alzheimer’s pathology. In addition to providing new insights into age-related disorders, meningeal lymphatics may also serve as a novel therapeutic target for these diseases and are worth of in-depth mechanistic exploration.

A mechanosensory system in the spinal cord for posture, morphogenesis & innate immunity

Influence of perinatal inflammation on meningeal immunity and neurodevelopment

Сellular immunity in patients with concussion and possible association with subsequent cognitive impairment withdrawn by author

Dissecting the effects of distinct VTA projections on peripheral immunity

FENS Forum 2024

Modelling neurodevelopmental disorder risk in inborn errors of immunity

FENS Forum 2024

N-aglycosylated extracellular loop of the potassium channel KCNJ10 as contributor to autoimmunity in multiple sclerosis

FENS Forum 2024