University Of Minnesota

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Predicting Late Talkers in Infants who are at Elevated Familial Likelihood for Autism Autistic children are late to say their first word, and these early challenges persist with over 70% of autistic preschoolers having language impairment. Siblings of autistic children unaffected by autism themselves are at a 4-5-fold increased risk of developing language challenges. The field lacks screening tools with strong predictive power to identify late talking autistic children during the birth-to-three early intervention period. The proposed project makes significant steps towards early identification of language impairment and understanding the developmental sequelae of language in autistic toddlers by leveraging data from the Baby Sibling Research Consortium database, the largest collection of language data of infants who develop autism. Aim 1 of the proposed study is create a normative model sensitive to the heterogeneity of autistic language development using summary-level data from the MacArthur-Bates Communicative Development Inventories (CDI). This model will then be used to predict late talkers. The normative modeling framework quantifies individual differences in scores, flagging individuals for further follow up. This framework moves away from the limitations of simple “case- control” designs by allowing for the heterogeneity that is inherent to developmental disorders. We will make our normative model publicly available, to be used by investigators interested in samples that tend to be smaller in size, increasing the significance and

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Acute Lymphoblastic Leukemia (ALL) is the most common form of childhood cancer. Despite dramatic improvements in initial remission rates over the past 50 years, relapsed ALL remains a significant challenge and is the second leading cause of cancer-related death in children. Thus, ALL continues to pose a substantial health concern. The BCR-ABL fusion protein, resulting from a chromosomal translocation, is a key driver of leukemogenesis in ALL and serves as a promising target for immunotherapy due to its tumor-specific and essential role in oncogenesis. Studies have indicated that the presence of T cells specific for the BCR-ABL fusion epitope correlates with better leukemia control; however, vaccine-based strategies to elicit such protective T cell responses have not been successful. Our lab, together with our collaborator, has previously generated MHC class II tetramers linked to the BCR-ABL fusion peptide (BAp), which identified a small population of CD4+ T cells capable of recognizing this leukemia antigen. These BAp CD4+ T cells expanded in the presence of BCR-ABL+ leukemia but were often converted into immunosuppressive FOXP3⁺ regulatory T cells (Tregs) or dysfunctional type 1 regulatory (Tr1) like cells producing IL-10, limiting their effectiveness. Interestingly, combining tyrosine kinase inhibitors like nilotinib with anti-PD-L1 antibodies promoted Th1 CD4+ T cell responses and prevented relapse in murine models, suggesting that effective activation of BAp-specific T cells can enhance leukemia control. A key question that emerges is how to identify and activate T cells capable of sustaining durable anti-leukemia responses without succumbing to exhaustion or immunosuppressive conversion. We have collaborated with Dr. Dileepan to develop "affinity-enhanced" peptide-MHC class II tetramers (BAp) by augmenting the interaction between the MHCII β chain and the CD4 co-receptor, allowing us to identify and potentially activate these low-affinity T cells. I propose that activating low-affinity BAp-specific T cells using these affinity-enhanced reagents can generate potent and sustained anti-leukemia immunity. Thus, my central hypothesis is that a proper activation of low-affinity BAp CD4+ T cells is crucial for preventing relapse in ALL. This hypothesis will be tested in the following two specific aims: (1) Determine whether enhancing low-affinity BCR-ABL-specific T cell responses limits relapse, and (2) Determine the relationship between functional low-affinity BCR-ABL-specific T cells and clinical outcomes in ALL patients. Completion of these aims will further advance our understanding of how low-affinity T cells can be harnessed to generate durable anti-leukemia responses and how perturbation of these responses can lead to relapse in ALL. This research may lead to new immunotherapeutic strategies that improve survival outcomes for ALL patients and provide insights applicable to other cancers. Finally, this project will provide me with important training in the use of cutting-edge research tools and models, which are important skills for me to acquire as I embark on a career as a physician-scientist focused on cancer immunotherapy.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT CD4 T cells govern host control of M. tuberculosis (Mtb) infection. The diverse phagocytic myeloid cells that harbor intracellular Mtb in the lungs require CD4 T cell help to activate antibacterial effector mechanisms. Protection against tuberculosis (TB) depends on this cellular interaction, but it remains incompletely defined. In our preliminary studies, we discovered that myeloid cells in the lungs of Mtb infected mice critically depend on T cells to deploy a broad program of transcriptional activity that controls intracellular infection. These pathways, constituting the spectrum of T cell help, included some pathways known to be due to adaptive immune function, but also others that are canonically considered innate. Despite the heterogeneity of myeloid cells in the lungs, most T cell dependent pathways were shared by MHCII+ antigen presenting cells (APCs) and non-antigen presenting cells, like neutrophils. Using adoptive transfer, we found that CD4 T cells were both necessary and sufficient for induction of the acute inflammatory response by MHCII+ APCs, as marked by expression of SAA3. Concordantly, we found that expression of MHCII by monocyte derived APCs was required for control of Mtb in the lungs. To advance understanding of how cell-cell contact mediated by immune synapse formation between CD4 T cells and Mtb-infected APCs contributes to protection, we developed a technique to identify and isolate physically interacting CD4+, MHCII+ multiplets from the lungs. With these preliminary data and tools, we now aim to define mechanisms of CD4 T cell help in TB, characterized as either direct (local, requiring cell-cell contact with APCs) or indirect (global, impacting both APCs and non-APCs). Using adoptive transfer of CD4 T cells from wild type or knockout mice into T cell deficient recipients, we will define how distinct CD4 T cell-derived effector functions drive myeloid cell activation and control of intracellular Mtb among APCs and non-APCs. By advancing our studies of mice that lack MHCII among monocyte derived APCs in the lungs, we will newly define how of antigen presentation in the lungs drives different CD4 T cell functions and determine whether this differs depending on the antigen recognized. We will test the hypothesis that antigen presentation by monocyte derived APCs governs mechanisms of both direct and indirect help. With our innovative approach to isolating physically interacting multiplets composed of CD4 effector T cells and Mtb-infected MHCII+ cells, we will discover bidirectional gene expression programs that are highly associated with immune synapse formation in the lungs. We will use ChipCytometry on sorted multiplets to spatially characterize the protein expression characteristics of each cell contributing to the synapse. Using bone marrow chimeric mice, combined with bulk and multiplet-RNA-Seq, we will determine the role and requirement of essential CD4 T cell effector functions in direct CD4 T cell help delivered at the immune synapse. By providing new information about the ways that CD4 T cells protect the host against TB, the studies will inform and accelerate the development of more effective TB vaccines.

NIH Research Projects · FY 2025 · 2025-08

Project summary This proposal seeks the acquisition of a LUMICKS C-Trap instrument at the University of Minnesota. The C-Trap is the only commercially available laser optical trap that combines TIRF fluorescence microscopy with integrated microfluidics in a user-friendly, reliable, engineered instrument. Single molecule force and fluorescence spectroscopies have transformed the fields of cellular and molecular biology, biochemistry, biophysics, and biomedical engineering, providing answers to crucial biological and health-related questions. These tools are currently limited to laboratories with specialized expertise and custom-built single molecule instruments. The C-Trap is designed as a turnkey system, enabling laboratories without specific single molecule force or fluorescence spectroscopy expertise to conduct advanced single molecule force, position, and fluorescence localization, as well as FRET analysis, with unparalleled spatial and temporal resolution. Key features of the C-Trap include dual optical traps for manipulating biomolecules with sub-pN force resolution/detection; 3-color laser TIRF and widefield fluorescence detection for visualizing biological processes; precise temperature control; micro and nano-stage control for exact sample positioning; laminar flow microfluidics for high sample throughput and varying ambient media while maintaining molecular interactions; and user-friendly software for integrated, controlled, and automated instrument operation. The intuitive instrument and software interface, along with the automation package, will enable non-expert users to run experiments and collect high-quality data with minimal training. The proposal represents five major users (all with NIH R01/R35 funding) who will primarily use the instrument, and eight minor users with NIH or other funding. These users come from four colleges across University of Minnesota (College of Biological Sciences, College of Science and Engineering, School of Dentistry, and the Medical School) and six different departments. The C-Trap will significantly advance the NIH-funded projects of these researchers by enabling high-impact single molecule mechanobiological and fluorescence studies on a range of important biomedical questions in areas such as cell surface mechanosensing, muscular dystrophy pathogenesis, cytoskeletal structure and dynamics, viral assembly and cell entry and nucleic acid processing. The major users and technical advisors possess extensive relevant expertise with the technology, and will provide guidance to all users. The instrument will be housed in the University Imaging Center, a Nikon Center of Excellence, which provides support and training for all of their instruments as well as expert advice and consultation, from experiment planning to image acquisition and analysis. Establishing this resource will significantly enhance numerous research programs at the University of Minnesota and contribute to the discovery of impactful scientific, biomedical, and healthcare-related knowledge.

NIH Research Projects · FY 2025 · 2025-08

Summary With advances in modern technologies, an enormous amount of high-throughput neuroimaging and genomics data are available through large-scale public databases such as the Adolescent Brain Cognitive Development Study (ABCD) project. These expansive "big multi-modal data" repositories have spurred the rapid growth of brain imaging genomics studies to improve understanding of the pathophysiological processes and genetic architecture of mental disorders. However, these large-scale databases also present significant challenges in data management and analysis, given the common characteristics of large open data including high dimensionality, data inconsistency, and complex correlation structures. In addition, a wide gap exists between neuroimaging and statistical genomics researchers, causing extra barriers to reproducible research in brain imaging genomics. While many training programs provide neuroimaging or genetics data analysis training, very limited (if any) of them focus on studying the relationship between neuroimaging and genomics underlying mental health. This proposal outlines a three-week summer workshop designed to equip graduate-level students or junior researchers with knowledge and skills to conduct reproducible analysis of brain imaging genomics. The primary focus will be on learning in best practices how to estimate meaningful associations of genetic variants on brain structure and function, along with their implications for cognitive functions and broader mental health outcomes. Our proposed workshop will address the critical need for training on neuroimaging genomics to build a bridge between cognitive neuroscience and genomics and promote rigorous and robust analyses of large open brain imaging genomics data with statistical considerations that are specific to large open data . The workshop also aims to promote diversity with trainees having different social and academic backgrounds and prioritizing the attendance of underrepresented scholars. The workshop will feature in-person training with an interdisciplinary faculty team comprising experts in statistical and machine learning methodology, genomics, and brain imaging research. We expect that by end of the workshop the trainees will be able to (1) Learn to access and use large, open brain research databases and understand analytical and statistical considerations specific to large open data; (2) know the important scientific questions in brain research and learn to design and conduct neuroimaging-genotype association studies with clinically meaningful phenotypes; and (3) develop best practices of coding and documentation for reproducible research of brain imaging genomics. All lectures, codes, and lab exercises will be made available on GitHub and the course website, ensuring the widespread dissemination of our training program within the broader community.

NIH Research Projects · FY 2025 · 2025-08

Abstract Mammarenaviruses, or arenaviruses, can cause a range of diseases in humans, including congenital disorders, encephalitis, meningitis, and severe hemorrhagic fevers (HFs). Arenaviruses are divided into Old World (OW) and New World (NW) groups. Several NW arenaviruses, such as Junín (JUNV), Machupo (MACV), Guanarito (GTOV), Chapare (CHAPV), and Sabiá (SABV), are endemic in South America and can cause HF infections in humans with fatality rates reaching up to 30%. Other uncharacterized or unknown NW arenaviruses may include potential pathogens. To date, there are no FDA-approved vaccines or therapeutics for arenaviruses. Candid#1, a live attenuated JUNV vaccine, is licensed for use in Argentina against JUNV-caused Argentine HF, but is unlikely to be approved by the FDA due to safety concerns. There is an urgent need for vaccines to prevent current and future arenavirus outbreaks. Our long-term goal is to create safe and effective vaccines against pathogenic arenaviruses. The objective of this R01 proposal is to leverage the unique properties of a viral vaccine vector rP18tri based on a non-pathogenic NW arenavirus, Pichinde virus (PICV), to develop safe and broadly protective vaccines against NW arenaviral pathogens. PICV has no to low seroprevalence in humans, even in endemic areas in Colombia, South America. The rP18tri-based live-attenuated vaccines show limited replication in the vaccinated animals, with no evidence of viremia or virus shedding. They induce a balanced level of antigen-specific antibody and T-cell responses, which can be further enhanced by homologous boosting. Our compelling preliminary data demonstrate that the rP18tri-based JUNV vaccines are safe and can provide complete protection against virulent JUNV challenges in guinea pigs. Furthermore, the rP18tri vector alone can confer partial protection against JUNV, underscoring its potential to elicit broadly protective immunity against diverse NW arenaviruses. We hypothesize that the non-pathogenic arenavirus vector (rP18tri)-based vaccines expressing multivalent antigens from JUNV and MACV will elicit robust and broadly protective immunity against various NW arenaviruses. We have assembled a multi-institutional team of investigators with complementing expertise in molecular virology, viral vector and vaccine development, guinea pig T cell immunology, and animal modeling of arenavirus HFs in the BSL3/4 facilities. We will produce a panel of rP18tri-based NW arenavirus vaccines expressing one or two antigens (glycoprotein GPC and nucleoprotein NP) from JUNV and MACV, evaluate their immunogenicity and protective efficacy against homologous (Aim 1) and heterologous (Aim 2) virus challenge in established guinea pig models, and characterize vaccine-induced protective immunity (Aim 3). Impact: The study will generate safe and broadly protective NW arenaviral vaccine candidates for subsequent preclinical and clinical evaluation, generate new knowledge about the protective antigen(s) and immunity to guide the design of next-generation arenavirus vaccines, and advance the rP18tri platform for developing vaccines and immune therapies against communicable and noncommunicable diseases, such as cancers, in humans.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Ovarian cancer remains the deadliest gynecologic malignancy, with a five-year survival rate below 50% and a high likelihood of recurrence. Current treatment strategies, centered on surgical debulking and chemotherapy, have shown limited advancements over the past several decades, underscoring the urgent need for novel therapies. Immunotherapy holds immense promise in this context, as increased natural killer (NK) cell activity has been correlated with improved outcomes in ovarian cancer patients. Unlike T cells, NK cells are inherently non-antigen-specific, enabling them to respond to heterogeneous tumors without prior sensitization, while also avoiding complications like graft-versus-host disease. Like T cells, however, NK cells activity is hindered by the immunosuppressive tumor microenvironment. To overcome this, our lab has developed and rigorously tested the Tri-specific Killer Engager (TriKE) platform, which enhance NK cell activity by simultaneously targeting CD16 on NK cells, binding tumor-associated antigens, and delivering an interleukin-15 (IL-15) cytokine domain. Together, the TriKE promotes NK cell migration, activation, proliferation, and sustained cytotoxicity. However, as with any immunotherapy targeting only a single antigen, the therapeutic application of the TriKE molecules could be hindered by the risk of antigen escape, particularly in ovarian cancer’s highly heterogeneous tumor microenvironment (TME). To address this challenge, we have developed a dual-antigen targeting NK cell engager platform. A Poly-Antigen Cytokine Complex (PACC), which binds to the TriKE’S IL-15 domain, was made by linking IL-15 receptor subunit alpha (IL-15Rα) to a tumor-targeting antibody domain. This innovative system combines the well-established benefits of TriKEs with targeting of a second tumor-associated antigen. By simultaneously engaging B7H3 and CD133, the TriKE-PACC is designed to exert greater immunologic pressure, reducing the likelihood of antigen escape and improving therapeutic efficacy. Aim 1 will evaluate the ability of the TriKE-PACC to prevent antigen escape in vitro and in vivo using ovarian cancer cell lines with CRISPR-modulated antigen expression to model antigen escape. Aim 2 will assess the preclinical efficacy of the TriKE-PACC against patient-derived xenograft (PDX) models of ovarian cancer to capture the full heterogeneity of clinical tumors. Together, these studies will establish the therapeutic potential of the TriKE-PACC and provide a foundation for its clinical translation. By targeting a dual-antigen approach to enhance NK cell immunotherapy, this project seeks to provide a novel treatment avenue for ovarian cancer patients, particularly the unmet need of those with recurrent disease who currently lack effective therapeutic options.

NIH Research Projects · FY 2025 · 2025-07

The mission of the University of Minnesota Medical Scientist Training Program (MD/PhD) is to train and support an exceptional community of dedicated students to become physician scientists who are able to integrate their scientific, clinical, and leadership skills to promote human health. Our program objectives are to: 1) recruit, admit, and matriculate trainees with the potential and commitment to successfully pursue a sustained physician scientist research career; 2) provide rigorous training so that trainees gain the technical, operational, intellectual, and professional skills required for sustained success, and successfully graduate and confidently move forward to the next stage of their physician scientist career; 3) utilize a rigorous evaluation and assessment plan to make evidence-based program improvements that will help us meet our program goals and mission. Our training program emphasizes integration of research and medical training through all phases, flexibility to meet the needs of individual trainees, rigorous training in the highest standards of practice in biomedical research by outstanding faculty committed to mentoring, and a cohort-based training approach that builds professional identity as a physician scientist and promotes student retention, success and well-being. Trainees pursue PhD training in a wide range of scientific disciplines, including biomedical engineering, public health, medicinal chemistry, and bioinformatics and computational biology. A new revised MD curriculum provides additional protected time for research early in training. Structured clinical continuity experiences during the PhD phase provide opportunities for career discernment, continued development of clinical skills, and expansion of a mentor network. Program activities guide trainees through key transition points in training. The program provides ongoing academic and professional development support with an MSTP-specific Individual Development Plan (IDP), regular progress meetings, and a commitment to always being accessible to trainees and knowledgeable about their goals and aspirations. We successfully matriculate 10 trainees per year from a large applicant pool that has increased in the last 5 years. Our trainees have an outstanding publication record and a high rate of success in obtaining external fellowships. Our graduates match into outstanding PSTP and residency programs and are well positioned to advance their physician scientist research careers. We seek NIH support so that we can continue to strengthen the physician scientist workforce, contributing to important biomedical progress by successfully training and graduating the next generation of physician scientist leaders.

NIH Research Projects · FY 2025 · 2025-07

Summary Immunological tolerance prevents autoimmune disease by deleting self-reactive T cell clones or impairing their function. While there is much know about how CD4+ self-reactive T cells become anergic or acquire suppressive functions, there is less known about non-deletional forms of CD8+ tolerance. The proposed research builds on our previous findings regarding CD8+ T cell tolerance to the melanocyte antigen Trp2, where non-deletional tolerance is prominent. The paucity of CD8+ T cells that react to a given antigen like Trp2 has presented a challenge to acquiring deeper mechanistic understanding. Here we employ two new TCR single chain transgenic models where either deletion or non-deletional tolerance prevails, and the frequency of Trp2 reactive clones is sufficiently high to allow mechanistic studies. Our approach will employ single cell genomics analysis (scRNAseq and scATACseq) to define the gene-regulation that inhibits non- deleted self-reactive CD8+ T cells. Further experiments will use deep sequencing of the TCRα repertoire and generate TCRα retrogenic models to define how the repertoire is shaped by self-antigens and provide functional classification of different self- reactive clones into 4 types: imperfectly deleted high-avidity clones, low-avidity anergized clones, low avidity non-anergized clones and “dangerous” clones that evade tolerance mechanisms altogether. Finally, we will use an established model of vaccination-induced vitiligo to determine which types of tolerance put animals most at risk of autoimmune disease when provoked by inflammation. This work will test the central hypothesis that while self-reactive CD8+ T cell clones with the highest avidity for self are purged from the repertoire, other self- reactive cells survive and are epigenetically repressed to maintain tolerance.

NIH Research Projects · FY 2025 · 2025-07

ABSTRACT: Development of effective and robust treatments for diseases caused by apicomplexan parasites is an ongoing medical and veterinary challenge. Since natural products are a proven source of effective and robust therapeutics against parasitic infections, we mined marine sources for anti-Toxoplasma and anti-Cryptosporidium activity. These screens identified a marine natural product, tartrolon E (trtE), with broad anti-apicomplexan activity in vitro and in vivo. Repeated attempts to select trtE-resistant T. gondii or P. falciparum mutants were unsuccessful implying that conservation of the trtE target is essential for parasite viability. To identify the target of trtE, we conducted a drug affinity response target stability (DARTS) assay. Proteins that were protected from proteolysis by the presence of trtE were identified by quantitative mass spectrometry. Two rhoptry proteins were highly enriched in trtE treated samples, TgROPLMF, and TgROP14. Both proteins are integral membrane proteins with a lipase maturation factor (LMF) domain. LMF-domain containing proteins are present in most all apicomplexans (ortholog group OG6_106580) consistent with trtE’s broad spectrum activity. The only apicomplexan lacking an obvious ortholog for the LMF domain containing proteins is Cryptosporidium. Our preliminary data lead us to hypothesize that TgROPLMF and TgROP14 are targets of trtE’s activity, and that trtE’s inhibition of the LMF domain proteins has downstream effects on proteins that interact with TgROPLMF. We will test these hypotheses through the completion of the following aims. Aim 1: To determine if the LMF domain proteins mediate the anti-parasitic activity of trtE: Toxoplasma parasites lacking TgROPLMF1, TgROP14, both proteins, or parasites overexpressing TgROPLMF, will be generated, validated and characterized. The susceptibility of the recombinant parasites to trtE, as compared to wild-type parasites, will be quantified in assays that evaluate inhibition of invasion and intracellular replication. Aim 2: To identify the interactome of TgROPLMF and TgROP14 and evaluate trtE’s ability to disrupt the TgROPLMF interactome. The interactome of the LMF domain containing proteins will be identified by proximity labeling with TurboID. Interacting proteins will be validated by endogenous tagging and tracking in wild type and recombinant parasites, and the ability of trtE to alter the TgROPLMF interactome tested. Aim 3: Identify putative trtE targets in Cryptosporidium parvum. Cryptosporidium is the only apicomplexan susceptible to trtE that lacks LMF domain proteins. In this aim our DARTS strategy will be repeated with sporozoites to identify putative targets in C. parvum. If the LMF domain proteins are trtE targets, we will have identified eminently druggable targets common to apicomplexan pathogens, providing lead compounds for new therapeutic strategies for diseases caused by apicomplexan parasites.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY An estimated 3 million US adults suffer from inflammatory bowel disease (IBD), a group of conditions primarily including ulcerative colitis (UC) and Crohn's disease (CD) and are characterized by chronic or recurring inflammation of the gastrointestinal tract. While anti-inflammatory and immunosuppressant drugs are available to control symptoms (remission), the relapsing rate can be as high as 75% and challenges exist to effectively maintain remission and improve quality of life. Prebiotic fibers and short-chain fatty acids (SCFAs) generally have beneficial effects on intestinal physiology, and butyrate is the preferred oxidizable fuel for colonocytes. On the other hand, dietary fat exacerbates experimental colitis, and its overconsumption is associated with the global increase in the prevalence of UC. The proposed project aims to understand how excessive fat in diet perturbs colonic metabolism of butyrate and subsequently leads to elevated mucosal inflammation. One notable alternative fate of butyrate in colonocytes is to generate ketone bodies including acetoacetate (AcAc) and D-β- hydroxybutyrate (D-βHB). There is a significant knowledge gap regarding the pathophysiological function of mucosal ketone bodies. In Aim 1, we propose to test the hypothesis that terminal oxidation of butyrate in colonocytes suppresses while ketogenesis promotes intestinal inflammation. We will first perform stable isotope tracing experiments to determine butyrate’s fate during colitis. Using genetic mouse models, we will determine functional outcomes of terminally oxidative vs. ketogenic metabolism of butyrate in colitis. In Aim 2, we seek to understand how high-fat diet (including ketogenic diet) promotes the colonocyte-macrophage ketone shuttle to drive inflammation. We will test the hypothesis that the fat-sensitive PPARα signaling, by counteracting the tissue-reparative type 2 immune response, activates colonic beta-oxidation and ketogenesis to potentiate gut inflammation. Intestinal macrophages, predominantly derived from monocytes via a “waterfall” differentiation process, are the gatekeepers of mucosal homeostasis. In Aim 3, we hypothesize that the utilization of locally produced ketone body, specifically AcAc, induce the formation of Spp1+ macrophages to drive inflammation. Taken together, we expect to find that dietary fat induces the metabolic switch from butyrate terminal oxidation to ketogenesis in colonocytes, which reprograms intestinal macrophages to form a pro-inflammatory milieu. The proposed study will provide valuable insights into the metabolic underpinnings of UC etiopathogenesis and the development of future therapies and dietary advice for UC patients.

NIH Research Projects · FY 2025 · 2025-07

ABSTRACT CD8 T cells are potent killers of cells presenting foreign antigens, making them powerful to fight microbial infections. The fetal host immune defense against pathogenic microbes is underdeveloped and ill-equipped to defend against microbial infection and heavily relies on maternal host immune responses to prevent the dissemination of pathogenic microbes. However, the fetus also expresses immunologically foreign allo- antigens, which if recognized by CD8 T cells, could threaten its survival. The maternal-fetal interface (MFI), where fetal and maternal mucosal tissues intertwine, is a critical location for preventing the dissemination of pathogenic microbes and maintaining tolerance for allo-antigens. Major gaps in knowledge relating to CD8 T cell biology, tolerance, and pathology at the MFI have persisted due in large part to necessary restriction on using human cells and tissues for research. In addition, conventional specific pathogen free (SPF) mouse models fail to recapitulate human CD8 T cell biology and diversity, both systemically and in tissues, making them inadequate for studies on CD8 T cells at the MFI. In SPF mice, CD8 T cells make up 0.5-2% of MFI leukocytes and have a naïve phenotype. Conversely, we recently discovered that natural microbial exposure expanded CD8 T cells to 6% of leukocytes at the MFI and phenocopied human CD8 T cells with high fidelity. This proposal uses innovative natural microbial exposure as a more faithful model system of human CD8 T cell biology. Natural microbial exposure expanded CD8 T cell numbers and generated additional CD8 T cell diversity including effector, memory, tissue resident and dysfunctional CD8 T cell types that we also observed at the human MFI. We utilize this model as well as human tissues to gain mechanistic understanding of factors that modulate CD8 T cell tolerance and host immune defense against pathogenic microbes at the MFI. We will compare the immune regulation of CD8 T cells are a result of exposure to a natural microbiome or pathogens both systemically in blood and lymphoid tissues as well as locally in the mucosal tissues. These mechanistic investigations of adaptive CD8 T cell responses will identify tissue localized CD8 T cell types, their receptors and specificity, as well as the beneficial and pathogenic host factors providing immune protection for pathogenic microbes while maintaining immune tolerance. Further, these experiments will lay the groundwork for improved preclinical models investigating biomarkers, treatments, and preventions for immune pathologies at the MFI.

NIH Research Projects · FY 2025 · 2025-07

Temporomandibular disorders (TMDs) are the most common cause of orofacial pain not related to teeth, affecting 5-12% of the population, and 15% of patients seeking treatment will develop chronic TMD with a greater prevalence in females. Jaw muscle pain (myalgia) is the most common TMD diagnosis, usually mildly present at rest which gets aggravated with function, e.g., chewing, or parafunctional habits, e.g., teeth clenching. Such habits are frequently reported by patients, are strong predictors for TMD incidence and experimentally they can induce jaw pain/fatigue in controls and aggravate symptoms in patients. Putative factors for jaw pain onset and persistence include intrinsic muscle metabolic features related to function. Jaw function tests that evoke symptoms mimicking “real-life” situations are necessary to determine mechanisms of jaw pain and fatigue in chronic TMD. Example models include teeth clenching at maximum force, chewing wax/gum or clenching at pre-defined forces relative to the individual maximum voluntary bite force. Outcome measures include clenching endurance and magnitude of pain/fatigue intensities. Common to most models is a focus on fixed parameters, typically muscle contraction at pre-specified levels. However, little attention has been directed to models whose stimulus-induced outcome is a targeted magnitude of sensation, e.g. moderate pain/fatigue, and the jaw muscle effort required for induction. There is a critical need for new models of jaw function that closely emulate known risk factors for TMD pain. This proposal will address this unmet need by developing and validating a novel jaw metabolic stressor (JMS) task to determine the jaw muscle metabolic stress and brain functional characteristics related to jaw pain perception during function. The JMS task is designed for chairside and magnetic resonance imaging (MRI) uses at high (3T) and ultra-high (7T) field. The following aims are proposed: 1. Develop and validate a JMS task able to evoke targeted intensities of jaw pain and fatigue in pain-free controls; 2. Determine the jaw muscle effort required to evoke moderate jaw pain and fatigue in chronic TMD pain cases and matched controls using a JMS task; 3. Demonstrate feasibility of using a JMS task during functional MRI to evoke brain functional activations secondary to targeted jaw pain and fatigue levels in chronic TMD pain cases and matched controls. In Aim 1, a psychophysics-based JMS task will be validated by contrasting it to traditional models of jaw function by comparing the targeted jaw pain/fatigue intensities and jaw muscle blood oxygenation evoked by each model, and its reliability will be determined from test-retest assessment. Aim 2 will use similar methods as Aim 1 to compare the jaw muscle effort required to achieve a targeted intensity of jaw pain/fatigue for chronic TMD pain cases and matched pain-free controls. Finally, Aim 3 includes feasibility testing for JMS task performance during MRI in both high and ultra-high fields. It is anticipated that a valid and reliable JMS task designed for chairside and MRI uses will render a new method to investigate peripheral and central neural mechanisms of jaw pain/fatigue in chronic TMD.

NIH Research Projects · FY 2026 · 2025-07

Project Summary/Abstract Autoimmune hemolytic anemia (AIHA) is a severe hematologic disorder caused by the antibody-mediated destruction of red blood cells (RBCs). The disease causes significant morbidity and mortality for patients: With today's treatments, patients face a significant risk of death, high relapse rates, and only a rare chance at full remission. Better therapies are possible, but our limited understanding of the genetic and immunologic mechanisms that lead to AIHA are roadblocks to making new treatments a reality. Fortunately, dogs provide an exceptional naturally occurring model for studying genetic risk factors and T cell biology in AIHA. The disease is naturally occurring in both humans and dogs, and shares similar clinical, genetic, and immunologic underpinnings in both species – while occurring significantly more frequently in dogs compared to humans. The goal of this study is to lay the groundwork for a canine model system to study AIHA by identifying genetic risk factors for the disease and by characterizing the autoreactive T cell population in two highly predisposed dog breeds. Specifically, we will test the hypothesis that we will identify multiple genetic risk variants affecting humoral immunity, along with autoreactive CD4+ T cells that are activated by peptides derived from RBC surface antigens. Once we identify genetically predisposed dogs and the T cell epitopes that trigger AIHA, we can begin to develop and test approaches that manipulate or downregulate the aberrant immune response in highly predisposed animals. Ultimately, we expect to use this canine model system to drive advances in our understanding of AIHA and foster the development of novel immunotherapies for humans. The work proposed in this application will be completed by a highly collaborative team of scientists anchored at the renowned Center for Immunology at the University of Minnesota Medical School, the University Minnesota College of Veterinary Medicine, and the University of Georgia College of Veterinary Medicine.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY / ABSTRACT The safe and effective ways to care for patients after acute kidney injury (AKI) is a significant unknown in the medical field and one of growing attention. There is significant uncertainty about use of kidney-protecting medications in patients who have had AKI because many of those medications, paradoxically, reduce kidney function when initiated or resumed before their kidney-protecting qualities become apparent. This leads to deferral of therapy for many patients who have had recent AKI and other risk-factors, like chronic kidney disease (CKD) and diabetes mellitus. These patients are actually at heightened risk for multiple adverse outcomes that kidney-protecting medications prevent. Such deferrals may even become indefinite. The newest of these medication classes being impacted by these post-AKI practices are sodium-glucose cotransporter-2 (SGLT2) inhibitors, which are first-line therapy for type 2 diabetes and are known to prevent progression of CKD and cardiovascular events. Observational studies, including those published by the applicant Dr. Daniel Murphy, show favorable outcomes when SGLT2 inhibitors are provided after AKI to those with type 2 diabetes. Now Dr. Murphy seeks to translate these findings into a pilot, pragmatic, cluster-randomized clinical trial (RCT) based in primary care clinics to demonstrate the feasibility of an intervention to increase use of SGLT2 inhibitors after AKI (Aim 1). Nested within the pilot RCT, Dr. Murphy seeks to conduct a qualitative study focused on the experience of primary care providers and pharmacists and, separately, patients, within the pilot RCT. These interviews will identify barriers to prescribing, receiving, and taking a SGLT2 inhibitor and the perceived impact of our study’s intervention, which will help Dr. Murphy craft changes to the study intervention for designing a future RCT based on all his K23 results (Aim 2). In parallel with these efforts, Dr. Murphy will use electronic health record-data from patients with type 2 diabetes and AKI to determine predictors of SGLT2 inhibitor receipt, which could be used in identifying patients to recruit for a future RCT after this pilot study (Aim 3). Paired with applicable training, these research questions in this K23 Mentored Patient-Oriented Research Career Development Award will position Dr. Murphy as an independent clinical researcher equipped in both pragmatic RCT design and conduct alongside his skillset in using observational data to identify potential treatments and strategies that may improve clinical outcomes. His career will include investigating treatments to care for patients who have suffered AKI though observational and interventional means.

NIH Research Projects · FY 2025 · 2025-07

ABSTRACT Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States, with limited treatment options, especially for patients with microsatellite stable (MSS) tumors, which are largely resistant to immune checkpoint inhibitors (ICIs). Despite advances in chemotherapy and targeted therapies, CRC treatment remains suboptimal, particularly in overcoming immunotherapy resistance. Therefore, there is an urgent need to identify novel therapeutic strategies that can modulate the immune microenvironment and enhance the efficacy of ICIs to increase survival outcomes in advanced-stage CRC. Our high-throughput metabolomic and transcriptomic data analysis from CRC patients identified significant upregulation of the essential amino acid (eAA) transporter LAT1 in CRC tissues, particularly in MSS tumors. Elevated LAT1 expression correlates with poor prognosis and immunosuppressive M2-like macrophages, suggesting that LAT1 contributes to tumor immune evasion. Our preliminary studies showed that LAT1 inhibition decreases tumor growth and improves T cell infiltration in the tumor. Based on these observations, we hypothesize that LAT1 inhibition will reduce tumor growth and enhance antitumor immune responses by increasing M1-like macrophages and CD8+ T cells in the tumor environment. We propose targeting LAT1 with the JPH203, a LAT1-specific inhibitor, to synergize with ICI therapy to improve therapeutic outcomes in advanced-stage CRC models. Aim 1: Evaluate the impact of LAT1 inhibition on tumor growth and immune cell infiltration using an orthotopic CRC model. Aim 2: Evaluate the effect of the LAT1-specific inhibitor JPH203, alone and in combination with ICIs, in reducing tumor burden and enhancing immune responses in advanced-stage CRC models. Impact: This project will elucidate the role of LAT1 in CRC and explore the therapeutic potential of LAT1 inhibition in combination with ICIs. By targeting LAT1, we aim to overcome immunotherapy resistance and improve outcomes for CRC patients, particularly those with MSS tumors.

NIH Research Projects · FY 2025 · 2025-07

This is a new application seeking support for the Molecular Pharmacology and Therapeutics (MPaT) graduate program at the University of Minnesota (UMN). The mission of the MPaT program is to cultivate the next generation of pharmacologists who will lead or contribute to efforts yielding new therapeutic approaches for our most vexing health challenges. While recent program changes have fostered growth, enhanced its governance structure, and addressed succession planning concerns, there remain several opportunities for improvements that could enhance the training experience for students and faculty alike. The overarching objective of efforts described in this application is to transform the MPaT graduate program so that it better equips our students with the skills needed to conduct impactful research and become leaders in the evolving biomedical workforce. This goal will be achieved by implementation of a plan that includes critical updates to the MPaT curriculum, investments in recruitment and retention, augmented career development activities, mentorship training for preceptors, and continual improvement driven by comprehensive program evaluation. The plan is guided by student-facing objectives that align with desirable competencies related to scientific knowledge, technical and analytical skill development, communication, and the self-efficacy that develops over the course of an independent research project, as well as program-facing objectives including enhanced retention rate and research outcomes for all students. NIGMS support is requested for 5 students per year for the first 2 years in the program. This support, combined with institutional funding, will help the MPaT program meet its goal of matriculating 12 incoming students per year and training approximately 60 students across all cohorts. All MPaT students will complete 7 courses that give them a solid grounding in quantitative and systems pharmacology; underscore the importance of rigorous experimental design, responsible conduct of research, and team-based learning and problem-solving; and afford many opportunities to refine skills in scientific communication. This core curriculum will be supplemented by a computational course and data management workshop, and electives that permit program individualization. Student engagement with the Pharmacology Seminar Series and Retreat will foster awareness of research challenges and opportunities in contemporary pharmacology and provide networking opportunities. For their mentored research experience, students will select an advisor from among a talented and dedicated group of preceptors who share an interest in translating molecular, cellular, and organ systems-level insights into novel approaches to diagnose and/or treat human ailments and disease. Students can further tailor the training experience by engaging with experiential learning opportunities designed to give them exposure to careers-of-interest and facilitate their transition into the biomedical workforce. The training experience overall will be enhanced by the vast resources at UMN that foster safe, rigorous, and impactful research, and the strong institutional commitment to program success.

NIH Research Projects · FY 2025 · 2025-07

Primary Immune Deficiencies (PID) are a heterogeneous class of diseases ascribed to individuals exhibiting deficiency or severe impairment of cellular and/or humoral immunity. This proposal focuses on a form of PID associated with radiation sensitivity and defects in V(D)J rearrangement caused by mutations in the DCLRE1C (Artemis) gene, “ART-SCID”. Artemis is a hairpin-opening enzyme that plays an essential role in V(D)J recombination, immunoglobulin and T cell receptor gene rearrangement in immunodevelopment. The only effective therapy for ART-SCID is allogeneic hematopoietic stem cell transplant (HSCT), for which there is a significant risk of morbidity and mortality. Our team demonstrated that ex vivo lentiviral correction of Artemis deficiency in a mouse model of SCID-A was most effectively accomplished using the endogenous Artemis promoter to regulate expression, forming the basis for our teams’ clinical trial initiated in 2018 (NCT03538899). However, results from clinical trials using viral vectors have highlighted some of the limitations of genomically dispersed vector-mediated integration, such as genotoxicity associated with insertional oncogene activation, genomic instability, transgene silencing, and disrupted proliferative control. What we envision in this proposal is the next generation of genetic therapies for Art-SCID by chromosomal site-directed correction of the endogenous Artemis gene in hematopoietic stem cells. The Specific Aims of this proposal address key questions in the development of a targeted genetic approach for therapy of ART-SCID and PID in general: Aim 1: What is the feasibility of targeted modification of the Artemis locus at a single genetic lesion for restoration of immune function? To address this question, we have developed an Adenosine Base Editing (ABE) approach that functionally restores a TAA nonsense mutation in the Artemis gene that is prevalent in Navajo and Apache Native Americans. We have also established a new mouse strain transgenic for the human TAA nonsense mutation (SCID-A mice). Using electroporation conditions that we have shown to be effective for introduction of CRISPR gene editing reagents, in Aim 1 we will deliver ABE along with guide RNA targeting the Artemis TAA stop codon in mouse hematopoietic stem cells transplanted into recipient SCID-A mice, evaluating the animals for restored immunity. Aim 2: How can targeted genetic modification be extended from ex vivo to in vivo delivery of CRISPR reagents targeting the Artemis gene? In Aim 2 we will test adenovirus vectors and AAV vectors targeted to hematopoietic stem cells for in vivo delivery of ABE and restoration of immunity in SCID-A mice. Results from these studies will have immediate implications for genetic therapy of ART-SCID and by extension for therapy of PID in general.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT Ovarian cancer is the most lethal gynecologic malignancy. The majority of ovarian carcinomas are diagnosed at an advanced stage for which the treatment options are considerably limited and the 5-year survival rate is <30%. Despite their widespread use in the clinic, current immunoassay- and transvaginal ultrasound-based diagnostic tests for women with ovarian cancer have had a limited impact on reducing mortality rates due to their low sensitivity and specificity. Consequently, these methods have minimal utility to screen the general population for ovarian cancer. Targeted mass spectrometry (MS) assays that measure ovarian cancer protein biomarkers can fulfill a significantly unmet need in establishing sensitive and specific diagnostic biomarkers. Serum is an ideal biological fluid within which to measure these diagnostic biomarkers due to its non-invasive method of collection. We have identified a panel of candidate ovarian cancer protein biomarkers in patient serum, which may serve to differentiate healthy patients versus patients with serous ovarian cancer – the most common subtype of epithelial ovarian cancer. In the proposed work, we will develop and validate a sensitive and specific multiplexed targeted MS assay to quantify 9 of these diagnostic serous ovarian cancer protein biomarkers in serum. This will be accomplished using Parallel Reaction Monitoring (PRM) MS, which is a highly sensitive, selective and specific targeted MS method with a wide dynamic range and low limits of quantification, enabling streamlined method development and robust, multiplexed protein quantification. We will evaluate the analytical and clinical performance of our multiplexed PRM assay in measuring these prioritized biomarker candidates using a panel of >1,300 serum samples obtained from a geographically diverse cohort of women receiving gynecologic care at five cancer centers and academic health centers in the U.S. and one in Europe. Our multidisciplinary investigative team brings together researchers with diverse expertise in analytical chemistry, clinical chemistry, ovarian cancer biology, epidemiology, and biostatistics. The successful completion of the high impact pre-clinical studies described in this proposal will enable the differentiation of serous ovarian cancer patients from healthy patients based on the abundance of diagnostic serum protein biomarkers. This work will provide a foundation for the development and validation of novel multiplexed PRM assays that could readily be applied to other ovarian cancer patient cohorts to support diagnosis and early detection efforts.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited lethal neurodegenerative disease caused by 39- 82 CAG repeats in the ATAXIN-1 (ATXN1) gene. Patients with SCA1 suffer from progressive gait and balance deficits, and severe degeneration of Purkinje cells (PCs) in the cerebellum. There are no effective disease modifying therapies currently available for SCA1, indicating a critical need for better understanding of disease pathogenesis. Studies using limited postmortem brain tissue from patients are complicated by practical and ethical concerns of tissue availability, late disease stage with the loss of neurons, and manipulation. Mouse models of SCA1 have been extremely useful in increasing our understanding of SCA1, but have important limitations due to the inherent species differences as well as genetic modifications, such as overexpression and much longer CAG expansions than seen in patients, needed to model disease. Because of these limitations, we propose to complement studies using mouse models with the investigations of human cells, including models of the human cerebellum. Recent study developed a robust and reproducible protocol to generate human cerebellar organoids from iPSCs. We propose to use this protocol and our iPSCs lines from SCA1 patients and sibling controls to establish a human cerebellar organoid model of SCA1. This human cerebellar organoid SCA1 model will enable us to determine how ATXN1 with CAG expansions commonly seen in patients impacts human cerebellar cells and cerebellar activity, as well as provide a platform to help identify and test molecular targets for intervention.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY In 2020, approximately 12 million adolescents were diagnosed with a neurodevelopmental disorder (NDD) which arise from early life disturbances in key neurodevelopmental processes such as axonogenesis, synaptic pruning, and neuronal migration. These disruptions in brain development lead to debilitating neurobehavioral and cognitive disabilities such as sensory processing disorders, language delays, and social deficits, which can present as early as 6 months old. As such, the most effective therapies for NDD rely on early identification and intervention. This proposal aims to determine therapeutic targets during pregnancy with the goal of preventing neurodevelopmental and neurobehavioral consequences implicated in NDDs. Prenatal risk factors such as maternal nutrition, immune activation, and stress modify the maternal gut microbiota and disrupt offspring brain development and behaviors, suggesting the maternal gut microbiota and its associated metabolites are poised to be critical regulators of neurodevelopment and offers the potential for microbiota-based therapeutics for NDDs. The study of maternal microbiome-based therapies for NDDs align with the mission of the NICHD to “...improve reproductive health, enhance the lives of children and adolescents, and optimize abilities for all.” My preliminary data supports my central hypothesis that the maternal gut microbiota regulates key in utero microbial metabolites that promote thalamocortical development, allowing for typical adolescent somatosensory and behaviors. Further, my data provides evidence that maternal gut regulated metabolites act on embryonic microglia activity to shape thalamocortical circuit development and function that underlie adolescent somatosensory behavior. These findings support my rationale that the absence or disruption of the maternal gut microbiota decreases the in utero bioavailability of key microbially modulated metabolites that are necessary to shape microglia development and phagocytic activity at developing thalamocortical axons that then mediate somatosensory circuit dysfunction and behaviors in adolescence. I propose to test my central hypothesis with the following aims: Aim 1: Determine how 4MM regulates embryonic thalamocortical neuron and microglia morphology, growth, and function; Aim 2: Investigate the role of in utero 4MM on adolescent somatosensory circuitry and behaviors. The mechanistic approach that I have outlined in this proposal will elucidate a causal role of maternal gut microbiota associated metabolites on neurodevelopment and highlights the potential for microbiome-based interventions to improve childhood outcomes and livelihood. By completing the proposed research, I will identify a novel mechanism for how the maternal gut microbiota can regulate offspring brain development and behavior. My proposed research is essential to my predoctoral training as I will develop and master technical skills, experimental design and analysis, effective mentorship, and promote my contribution to the scientific field

NIH Research Projects · FY 2026 · 2025-07

Project Summary. The rodent-borne arenaviruses can cause severe hemorrhagic fevers; several of these potential pandemic pathogens have been noted to cause high rates of maternal mortality, congenital infection, and/or fetal demise when pregnant people are infected. The objective of this project is to determine how arenavirus infection causes adverse pregnancy outcomes. Clinical observations that Lassa virus (LASV) replicates to high titer in the placenta prompted us to conduct a preliminary study using in vitro models of the human placenta and experimental guinea pig infections with an arenavirus that is not pathogenic in humans, Pichinde virus (PICV), to ascertain the role of infection at the maternal fetal interface in severe disease. We found that PICV replicates to high titers in undifferentiated and differentiated first trimester human trophoblasts and explants derived from term placenta. Furthermore, infection during pregnancy led to fetal demise, high viral loads in the placenta and decidua, and low rates of congenital infection in guinea pigs. We hypothesize that the placenta and decidua serve as a reservoir for arenavirus replication and that the antiviral immune response to infection at the maternal-fetal interface leads to placental dysfunction. This project will use a combination of methods and models that include diverse New World (PICV and Junin virus [JUNV]) and Old World (LASV and lymphocytic choriomeningitis virus [LCMV]) viruses to reveal how arenaviruses circumvent host defense to infect the placenta and cause severe disease. The placenta is generally an effective barrier against the bloodborne transmission of pathogens and primary trophoblasts are broadly restrictive to viral infection. Using a genetically tractable model of the first trimester placenta, human trophoblast stem cells, and term placental explants, we will investigate the viral factors that allow arenaviruses to circumvent innate and intrinsic immunity and establish productive infections in trophoblasts (Aim 1). We will next use guinea pigs to assess how the immune response to arenavirus infection during pregnancy either causes placental dysfunction or may be harnessed to protect the mother and fetus (Aim 2). A detailed study of PICV pathogenesis at the maternal-fetal interface will be complemented with experiments using JUNV and LASV to determine whether these highly pathogenic viruses have a similar tropism for and effects on the placenta and decidua. The completion of this project will address a critical knowledge gap by revealing the mechanisms by which arenaviruses damage the placenta and cause pregnancy complications, informing the future development of much-needed therapies to minimize harm to pregnant people and their offspring.

NIH Research Projects · FY 2025 · 2025-07

Chronic pain is more prevalent in women, and the rapidly increasing list of mechanistic sex differences in pain signaling has underscored the importance of considering sexual dimorphism in the development of therapies for chronic pain management in women. Our long-term goal is to investigate sex differences in spinal dopaminergic mechanisms of persistent pain. The objective of this exploratory project is to define dorsal horn (DH) Drd1-expressing neurons and the function of Drd1 in a model of neuropathic pain. Our central hypothesis is that Drd1 participates in nerve injury-induced hypersensitivity in a sexually dimorphic manner due in part to differential expression in DH neuronal subpopulations in male and female mice. Preliminary electrophysiological recordings demonstrate a striking difference in the distribution of firing patterns of Drd1-expressing DH neurons in males and females. This observation suggests sex differences in the expression of Drd1 among subsets of DH neurons that could contribute to sex differences in spinal dopaminergic signaling under conditions of persistent pain. Drd1 is expressed in a heterogeneous population of excitatory and inhibitory DH neurons and partially overlaps with markers of excitatory subpopulations that participate in tactile hypersensitivity. Therefore, excitatory Drd1 neurons may participate in DH circuits, mediating nerve injury-induced hypersensitivity. On the other hand, increased activation of DH Drd1 inhibitory neurons may contribute to disinhibition-mediated hyperexcitability under conditions of persistent pain. We will address the potentially distinct functions of Drd1 in excitatory and inhibitory neurons using intersectional deletion of the receptor. The overall rationale for the proposed research is that it will identify specific mechanisms contributing to sex differences in spinal dopamine signaling, which could be harnessed for more effective chronic pain management in women. In Aim 1, we will identify the pattern of Drd1 distribution among subsets of DH neurons in males and females. We will use fluorescent in situ hybridization analysis to determine the expression of Drd1 in transcriptionally defined subtypes of DH neurons. We will also define the firing patterns and primary afferent inputs of excitatory and inhibitory Drd1 neurons using patch clamp electrophysiology and will label recorded neurons with neurobiotin for neuroanatomical analysis. In Aim 2, we will define the contribution of Drd1 in excitatory and inhibitory DH neurons to nerve injury-induced hypersensitivity in males and females, using an intersectional AAV-mediated CRISPR/Cas9 gene ablation approach; we will determine the functional impact of Drd1 ablation behaviorally. Impact: The proposed project will advance understanding of the mechanisms that underlie sex differences in chronic pain by elucidating the function of Drd1 in nerve injury-induced hypersensitivity in female and male mice. This outcome will provide proof-of concept for comprehensive investigation of sex differences in spinal dopamine signaling, extending to 1) other persistent pain models, 2) the other dopamine receptors, which are also expressed in spinal cord, and 3) the descending dopaminergic inputs, which originate in hypothalamus and have been implicated in pain chronification.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT Many types of resident leukocytes live within the tissues of the body, acting as frontline defenders against infection as well as fulfilling homeostatic tissue support roles. Tissue resident memory T cells (TRM) are poised to respond to antigen-specific and non-specific signals at sites of tissue injury. Extracellular adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+) are evolutionary conserved “danger signals” released by cell damage. In mice and humans, extracellular ATP (eATP) is recognized by purinergic receptors. Among those receptors, P2RX7 is a cell membrane channel preferentially expressed in immune cells that can support T cell activation along with the development and maintenance of memory T cell subsets at physiologic levels of eATP. However, P2RX7 activity is finely balanced: too little activity discourages memory cell homeostasis whereas persistent, strong activation culminates in cell death via formation of a non-specific pore structure. In mice, ARTC2.2 covalently modifies P2RX7 in response to extracellular NAD+ (eNAD+), amplifying the sensitivity of P2RX7 eATP and encouraging its pro-apoptotic capacities. Previous work has suggested ARTC2.2 and P2RX7 function to limit inappropriate T cell activation at sites of tissue injury, perhaps curtailing development of harmful immune responses that would produce tissue damage in turn. However, autonomous, cell-intrinsic effects of ARTC2.2 upon TRM are poorly defined. Utilizing a newly-generated, germline knockout mouse strain deficient in ARTC2.2, this proposal aims to (1) characterize the cell-intrinsic contribution of ARTC2.2 to CD8+ TRM homeostasis in primary immune responses and (2) assess ARTC2.2 effects upon TRM in the setting of tissue injury incurred by sterile inflammation and secondary infection. Using murine models of primary, secondary, and bystander immune responses, multi-parameter spectral flow cytometry and advanced, high-resolution quantitative microscopy will identify and enumerate memory CD8+ T cell subsets from lymphoid and non-lymphoid tissues. As release of eNAD+ and eATP can occur during processing of experimental tissue, these studies purport to surmount technical challenges inherent to investigation of tissue-resident T cells. Ultimately, these studies will help elucidate the role of ARTC2.2 in memory CD8+ T cell homeostasis and will provide key insights on how “danger signals” and the sensing of cellular damage help build long-term immunity against pathogens and limit deleterious immune activation following tissue injury.

NIH Research Projects · FY 2025 · 2025-07

Project Summary Transplantation of islets has restored tight glycemic control in patients with type 1 diabetes, but the toxicity of chronic immunosuppression used to prevent rejection has precluded the application of cell replacement in diabetes care. By inducing immune tolerance to transplanted islets, we achieved long-term, drug-free islet transplant survival in nonhuman primates (NHPs). To justify the clinical translation of the protocol, a deep understanding of the immunobiology that determines maintenance and loss of immune tolerance is required. Using highly multiplexed immunophenotyping and imaging methods customized for NHPs, our preliminary studies comparing tissues from tolerant vs. rejecting NHPs led us to hypothesize that the spleen plays a crucial role in maintaining tolerance to islet transplants in macaques. The retention of allospecific CD4+ T cells, the abundance of exhausted T cells, their spatial arrangement within the spleen, and the ongoing Treg vs. Teff cell crosstalk via the Areg-EGFR axis are key contributors to sustained tolerance. We propose two Specific Aims to address this hypothesis. AIM 1: To comprehensively profile allospecific Areg+ ST2+ Tregs and immune cell clusters in the spleen testing the hypothesis that sustained tolerance to islet allografts in NHPs is linked to an abundance of unique Tregs and Tex cell clusters in the spleen. Splenic Tregs in tolerant NHPs are enriched with elevated Areg and ST2 expression and exhibit an elevated interaction with Teff and Tex cells via Areg-EGFR signaling. AIM 2: To analyze the spatial interaction of allospecific Areg+ ST2+ Tregs with Teff, Tex, and myeloid cells clusters in the spleen testing the hypothesis that the retention of allospecific effector cells in special microdomains of the spleen and their subsequent exhaustion, caused by their proximity to Areg+ ST2+ Tregs in the spleen, plays a role in the consolidation of tolerance to islet tx The studies proposed herein will be the first to harness the capabilities of highly multiplexed immuno-phenotyping of single cells and tissues to investigate the role of Areg-EGFR signaling in splenic Teff cell retention and their subsequent exhaustion in maintaining nonchimeric transplant tolerance in NHPs. The generated dataset will represent a valuable resource for the transplant tolerance community, trigger additional studies on the anatomy of tolerance in the spleen, and promote the clinical translation of tolerance induction to donor- and stem cell- derived islet cell transplants.