La Jolla Institute For Immunology

NIH Research Projects · FY 2026 · 2026-06

PROJECT SUMMARY Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by self-reactive antibodies (autoantibodies), and this chronic disease results in significant loss of quality of life and irreversible tissue damage that can result in organ failure and death. With SLE being a disease that affects many organs with a range of severity, newer targeted therapies are needed. Despite advances in understanding SLE, most human studies have relied on peripheral blood, limiting insights into the lymphoid tissue compartments which are likely to be the primary sites where autoreactive T and B cells are activate. As a result, major gaps remain in our understanding of tissue-specific immune mechanisms in SLE pathogenesis and treatment response. To address these knowledge gaps, we propose a novel approach using minimally invasive nasal swab lymphoid tissue sampling combined with high-dimensional immunologic analysis in SLE. Unlike blood-based testing, this method enables direct study of immune cells in a lymphoid tissue. Our team has developed and validated a safe and reproducible nasal swab protocol, paired with flow cytometry and transcriptomic profiling, to measure lymphoid tissue immune cell populations with high resolution. This method allows direct interrogation of immune responses in lymphoid tissue over time, which will be a key advance for SLE research. Importantly, such measurements can be done repeatedly, longitudinally over time, allowing for novel kinetic insights into SLE pathophysiology. This approach, combined with our human immunology expertise, can potentially provide unprecedented insights into SLE immune events. We will (1) characterize immune cell populations in lymphoid tissue from SLE patients vs. healthy controls; (2) identify features that correlate with disease activity over time; and (3) assess pre- and post-treatment immune dynamics in patients receiving common and new SLE therapies. Our central hypothesis is that longitudinal analysis of lymphoid tissue immune signatures will uncover critical drivers of SLE pathogenesis and treatment response. Additionally, we aim to identify key features of these T and B cells that could serve as targets for novel therapeutic interventions and/or biomarkers of disease activity and responses to treatment, thus potentially identify novel SLE therapeutic interventions and biomarkers. The project is entirely focused on human immunology in SLE patients, directly examining lymphoid tissue. Furthermore, we will, for the first time, collect detailed longitudinal data from lymphoid tissues from SLE patients, including patients receiving therapies. By advancing understanding of human adaptive immunity in lupus, this work could significantly improve patient stratification and treatment outcomes in SLE.

NIH Research Projects · FY 2026 · 2026-05

Abstract Mumps virus is one component of the MMR vaccine, which has been successful in the global effort against measles, mumps and rubella. However, of the three viruses, protection against mumps is the least effective. Cycles of mumps virus outbreaks continue to occur, especially among young adults and even in the vaccinated. 10% of patients in a recent San Diego County outbreak were hospitalized. Breakthrough infections could be linked to sequence differences between the vaccine strain and the newly emerged mumps genotype G. Surprisingly, although we have had this vaccine since 1971, medical science still does not know what the human antibody response is against mumps virus, from either vaccination or natural infection. No human antibodies against mumps have been yet described, and no structures of any antibodies against mumps have yet been determined. Thus, we are lacking understanding of which antibodies and which epitope target sites on the surface proteins HN and F are most effective, and we are lacking the antibodies themselves which might be incorporated into post-exposure treatments. In this program, we will develop panels of human monoclonal antibodies against mumps HN and F from both vaccinated and convalescent individuals. Results obtained from this program will illuminate what humans target in their immune responses, and which target sites, or epitopes, lead to broadest viral genotype recognition, especially between vaccine-derived genotype A and currently circulating genotype G of the virus. The results of this program will also provide the essential mAbs themselves from which we may develop post-exposure treatments and guide development of boosters.

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY: From the parent award: We propose that the features of germinal center biology important for developing high affinity B cells to a very difficult epitope, such as a tier 2 nAb epitope on HIV Env trimer, are very different than the well characterized features of GC biology for conventional antigens, such as haptens. We have demonstrated that slow delivery immunization changes fundamental aspects of the immune response, which can result in dramatic improvements in nAb responses (Cell 2019). Conventional immunization strategies will likely be insufficient for the development of a bnAb vaccine to HIV or other difficult pathogens due to the immunological hurdles posed, including B cell immunodominance and GC quantity and quality. We found that two independent methods of slow delivery immunization of RMs resulted in more robust Tfh cells and more GC B cells with Env-binding, tracked by longitudinal lymph node (LN) fine needle aspirates (FNA) 1. Improved GCs correlated with the development of> 20-fold higher titers of autologous tier 2 neutralizing Abs (nAbs). BCR sequencing and Ab mapping demonstrated targeting of immunodominant non-neutralizing (nnAb) epitopes by conventional bolus immunized animals, while slow delivery immunized animals targeted a more diverse set of epitopes, including multiple tier 2 nAb epitopes. We will continue these groundbreaking studies to use novel slow release technologies to probe the biology of germinal centers relevant to affinity maturation against a difficult HIV trimer immunogen in non-human primates (NHP, rhesus macaques).

NIH Research Projects · FY 2026 · 2026-04

Project Summary Peripheral blood is the primary sample type collected to evaluate a vast range of medical conditions. However, peripheral blood testing may not reflect processes occurring in tissue, like the upper airway. The SARS- CoV-2 (SARS2) pandemic highlighted the importance of being able to sample the upper airway for purposes including evaluation of viral infection and clearance. Although the upper airway represents the primary site of infection for many human pathogens, critical questions remain regarding upper airway immunity and protection. Nasal cavity swab sampling can be used to bridge knowledge gaps regarding upper airway immunity to important respiratory pathogens like SARS2 and to address important immunologic questions where peripheral blood sampling is inherently insufficient. I established novel methods for reproducible, longitudinal sampling of upper airway immune cell populations using swabs and demonstrated that sufficient numbers of viable immune cells could be collected to allow for high resolution downstream analyses, including multiparametric flow cytometry and single cell RNA sequencing. Upper airway resident memory B and T cell populations, including SARS2- specific memory B and T cells were characterized using these methods. There is great interest in developing next generation vaccines, including vaccines that can elicit robust mucosal immune responses. This requires knowledge of the immunologic contexts in which mucosal immunity is generated and the requirements for maintenance of upper airway immune memory. I hypothesize that local antigen exposure is required to develop durable upper airway immune memory, and intramuscular immunization alone may fail to elicit upper airway immunity despite generating circulating immunity. I will test this hypothesis by studying upper airway memory B and T cell frequencies, diversity, kinetics and durability in distinct immunologic contexts including SARS2 breakthrough infection (BTI) and COVID-19 booster vaccination using this award. Other respiratory pathogens of public health importance will also be examined. If funded, this K08 will assist with my transition to becoming a successful independent physician scientist investigator in human immunology and infectious diseases. The La Jolla Institute for Immunology (LJI) is a superb training environment and located on the campus of another excellent academic research and medical institution, the University of California, San Diego (UCSD). I will receive ongoing mentorship from an established investigator with a track record for producing successful mentees, Professor Shane Crotty, PhD, a world- renowned expert in the field of immunology. LJI and UCSD are closely affiliated, and Dr. Crotty has an adjunct appointment at UCSD. I will have access to resources at both LJI and UCSD for this career development award. As an Associate Physician in UCSD’s Division of Infectious Diseases I will receive additional mentorship in academic career development, and maintain my clinical acumen by providing part-time medical care and consultation services for medically complex patients.

NIH Research Projects · FY 2026 · 2026-04

Project Summary/Abstract: Filoviruses encompass a broad group of highly lethal pathogens, including orthoebolaviruses (Ebola (EBOV), Bundibugyo, Sudan, and Taï Forest viruses) and orthomarburgviruses (Marburg (MARV) and Ravn viruses). These viruses cause sporadic disease outbreaks, but which virus will emerge and where cannot be predicted. As such, residents of affected areas, as well as military, aid workers and travelers in Central and West Africa continue to be at risk of infection. Further, currently approved monoclonal antibody (mAb) treatments and vaccines are available only for EBOV. A few known mAbs can cross-react among orthoebolaviruses, but none are yet known to offer broad filovirus neutralization. It is possible that our ability to uncover human mAbs of broad neutralization was limited by the nature of the human samples used for discovery. Prior antibody discovery campaigns to date isolated antibodies from individuals who had been infected once with just one of these viruses (usually EBOV, one with MARV, etc.), and many studies were performed on returning Americans who would not have had more than one filovirus exposure. This proposal, however, utilizes a different cohort. Serology studies in the Democratic Republic of the Congo, where multiple filoviruses are endemic, revealed 23 individuals with extraordinarily broad and strong antibody responses against a broad range of filovirus surface glycoproteins (GP). Some of these individuals have strong reactivity to all of them: all four pathogenic orthoebolaviruses and both orthomarburgviruses. These individuals have lived all or most of their lives in remote villages that have had known outbreaks and have occupations that likely led to multiple filovirus exposures over time. In this project, we will analyze, in detail, the antibody repertoires of these multiply exposed individuals. We will analyze reactivity at the polyclonal and monoclonal levels alike. We expect that mAbs identified in these individuals will have greater breadth and broader filovirus activity than have been discovered before. The results from this study could lead to the discovery of a novel, broad-spectrum filovirus mAb therapeutic, guide the development of more broadly applicable vaccines, and will deepen our understanding of how a lifetime of exposure shapes the humoral immune response to these viruses.

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY/ABSTRACT The Mammarenavirus genus of the Arenaviridae family, encompasses several severe human pathogens, including Old World arenaviruses (OWAs) like Lassa virus and New World arenaviruses (NWAs) such as Machupo and Junín viruses, each of which cause hemorrhagic fever. Currently, the only available vaccine, the live-attenuated Candid #1 for JUNV, is not FDA-approved and provides limited neutralization breadth, leaving populations vulnerable to diverse arenaviruses. Monoclonal antibody (mAb) therapies represent one of the most important treatment opportunities in modern medicine. They are being used to treat and cure numerous diseases, including different types of cancer and autoimmune diseases. Since the 1980s, mAbs like Synagis against RSV, and Ebanga™ and Inmazeb™ against Ebola virus disease have been used to treat viral health threats. However, there remains no licensed therapies for treatment of any arenavirus infection. As such, development, especially of broadly applicable therapeutics, is an urgent need. Pioneering discoveries from our group illuminated the “Achilles Heels” on the Lassa virus surface glycoprotein and led to a first-in-class pre- clinical therapeutic for Lassa virus infection. Here, we extend this cutting-edge work to New World arenaviruses. Employing a novel panel of prefusion-stabilized NWA GPCs, we will elucidate the structural and functional properties of these proteins, providing essential templates to interpret antibody responses. Our approach includes advanced methods such as high-resolution cryo-electron microscopy to visualize nAb interactions at a molecular level. We will isolate a diverse array of broadly protective nAbs from immunized animal models and humans vaccinated with Candid #1, assessing their breadth and potency to identify candidates for therapeutic development. Furthermore, we will conduct comprehensive structural and biochemical analyses to delineate the antigenic landscape of NWA GPCs and establish mechanistic rules underlying their neutralization. This multifaceted investigation promises to yield groundbreaking insights into the immune response to NWAs, paving the way for the development of effective vaccines and immunotherapeutics to combat these unpredictable and often fatal infections.

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY One third of the population suffer from allergic diseases including asthma, rhinitis, atopic dermatitis and food allergy. House-dust mite (HDM) is the most common allergen with nearly ubiquitous presence in US homes and ~30% sensitization. It is an enigma why some people develop allergy to a ubiquitous allergen such as HDM while others don’t. HDM is also the most important allergen driving sensitization and risk of allergic diseases like asthma. But, again, only some people with HDM sensitization develop asthma and/or rhinitis, for unknown reasons. In this proposal, we will explore the mechanisms and cell types that drive either natural tolerance or allergen sensitization and the development of asthma. We focus on CD4+ helper T cells (TH) because of their importance in allergy and asthma. To determine the diversity of these TH cell subsets in allergy and asthma, we performed one of the first single-cell transcriptomic studies of HDM allergen-specific TH cells. We identified of a novel subset of HDM-specific TH cells characterized by an interferon response (IFNR) gene signature (THIFNR cells), which was negatively associated with HDM sensitization. In addition, HDM- specific TH2 cells with increased expression of IL9 were increased in HDM-sensitized subjects with asthma compared to those without asthma. Based on these findings, we hypothesize that HDM-specific THIFNR cells protect against HDM-sensitization, and IL9-expressing TH2 cells in the airways, specifically tissue-resident memory T cells (TRM cells) promote the development of HDM-allergic asthma. In Aim 1, we will determine the association between HDM-specific THIFNR cells and protection against HDM sensitization. We will assess subjects enrolled in the Isle of Wight Whole Population Birth Cohort (IOWBC; n=1456). We will identify subgroups of participants (n=140) with: (i) Persistent HDM-sensitization since childhood (ii) Never HDM- sensitization since childhood, (iii) Adult-onset HDM-sensitization, and (iv) Adult-onset HDM tolerance. Using longitudinally-collected peripheral blood mononuclear cells, we will isolate HDM-specific T cells and perform single-cell transcriptome and TCR-seq analysis to enumerate the frequency, properties, persistence and clonality of HDM-specific T cell subsets including the novel THIFNR cell subpopulation, and determine their association with protection against development of HDM-sensitization at different ages and with adult-onset tolerance. In Aim 2, we will identify HDM-specific T cell subsets in the blood and airways associated with the development of asthma in subjects with HDM-sensitization. We will enroll 125 subjects with HDM-allergy from the IOWBC (n=55 with asthma, n=70 without asthma), obtain blood and airway samples longitudinally collected, and in a subgroup following HDM-allergen bronchial challenge. We will perform single-cell transcriptome and TCR-seq analysis to determine the frequency, properties, persistence and clonality of HDM- specific TH cell and TRM subsets in the blood and airways, and their association with asthma development.

NIH Research Projects · FY 2026 · 2026-02

PROJECT SUMMARY Lung infections remain a significant source of morbidity and mortality across all age groups. The recent SARS- CoV-2 pandemic underscores the critical need for the scientific community to urgently develop strategies that enhance mucosal immunity against a wide range of pathogens. While vaccines offer protection against severe disease, their effectiveness are not often durable and for many infections, such as tuberculosis, they are not effective. Therefore, gaining a deeper understanding of the mechanisms supporting long-term protective immunity at barrier sites, like the respiratory tract, is vital for developing therapies that boost mucosal immune responses. Tissue-resident memory T cells (TRM cells) have emerged as the guardians of barrier immunity. TRM cells are a distinct population of memory T cells that reside within tissues and respond immediately against pathogens invading barrier tissues, thus representing the first-line of defense. Dr. Vijayanand’s team (LJI PI) has long-standing expertise in human lung TRM cells having shown their importance in driving natural and anti-PD1 therapy-induced T cell responses in lung cancer. Given the profound importance of TRM cells in protective immunity, the generation and maintenance of robust TRM responses is considered important for the success of vaccines aimed at preventing severe lung infections. However, currently, there are no therapeutic options for boosting or maintaining natural and vaccine-induced TRM responses in the lungs. Our transcriptomic studies have identified a novel orphan G-protein coupled receptor (GPCR) that is expressed at high levels in lung TRM cells. Using genetic knock-out models, we found that this GPCR plays a key role in the development of lung TRM cells. In this proposal, our objective is to de-orphanize this GPCR. We have already detected ligand activity in murine thymic extracts using the b-arrestin recruitment assay in HEK293 cells. In Aim 1, Dr. Changlu Liu’s team (PI) at SBP, will identify and purify the ligand from thymic extracts and thymic-derived cell types, as described in their previous work to deorphanize other GPCR like GPR183 and GPCR135. We will purify the ligand from thymic tissue through organic extractions followed by High-performance liquid chromatography (HPLC). Fractions with ligand activity will be analyzed by mass spectrometry to identify the nature and sequence of the ligand. The putative ligand will then be tested in a dose-dependent manner to determine affinity and specificity of the ligand to the orphan GPCR. In Aim 2, we will test functional activity of the putative GPCR ligand in primary T cells. We initially test in vitro the functional activities of thymic tissue/cellular extracts in T cells. Then, we will perform similar studies using the purified ligand and determine its activity on T cell signaling, activation, proliferation, cytokine production and TRM cell generation in vivo. Studies in this aim will identify the physiological ligand that can enhance the activity of an important target (GPCR) in TRM cells. This ligand or its analogues have the potential to enhance the generation of lung TRM cells, thereby boosting protective mucosal immune responses in humans.

NIH Research Projects · FY 2026 · 2026-02

PROJECT SUMMARY/ABSTRACT Systemic lupus erythematous (SLE) causes substantial mortality and morbidity. Current treatments are not highly effective in modifying disease progression, especially in severely affected patients with renal involvement. Hence, there is a large unmet need to develop novel therapeutic targets. Genome-wide association studies (GWAS) offer an unbiased approach to identify genes that play an important role in driving disease pathogenesis. To identify risk genes in immune cell types relevant to SLE pathogenesis, we performed the first large-scale single-cell eQTL study on activated CD4+ T cells and found that SLE-risk variants were strongly associated with increased expression of the poorly understood gene ILRUN. Our study provides the first direct evidence that activated CD4+ T cells are the key cell types in which SLE-risk variants increase the expression of ILRUN. In Aim 1, we will identify the functional SLE-risk variants associated with ILRUN expression in activated CD4+ T cells. We will employ CRISPRi assays to determine functional enhancers that overlap ILRUN eQTLs, perform luciferase reporter assays to determine functional variants in ILRUN promoter and enhancers, and perform ChIP assays to identify the functional ILRUN eQTLs that directly perturb the binding of key transcription factors and modulate ILRUN expression. In Aim 2, we will assess the functional role of ILRUN in CD4+ T cells in the context of SLE and determine whether ILRUN influences the activation, apoptosis, proliferation, differentiation and cytokine production of CD4+ T cells. Overall, the studies examining the expression, regulation and function of ILRUN in CD4+ T cells will provide important mechanistic insights into the genetic basis of SLE.

NIH Research Projects · FY 2026 · 2026-02

PROJECT SUMMARY As a key immune response, inflammation unites innate and adaptive immunity to fight against infections, injuries and other danger signals. However, chronic inflammation can cause and accelerate autoimmune diseases, organ disorders and cancers. At a molecular level, immune balance is orchestrated by pro- and anti- inflammatory signals that are initiated and mediated by cytokine/receptor interactions. Interleukin-1 receptor 8 (IL-1R8) is a potent immunoregulatory factor that not only blocks inflammatory signals by acting as a decoy receptor for numerous pro-inflammatory Toll-like receptors (TLRs) or IL-1Rs, but can also transduce IL-37- induced anti-inflammatory signaling as a cytokine co-receptor. IL-1R8 is widely expressed and dampens pro- inflammatory signals in many tissues, and the downregulation or dysfunction of IL-1R8 is associated with many inflammation-promoted diseases. On the other hand, silencing of IL-1R8 can unleash NK or CD8+ T cell- mediated cytotoxicity against infections and cancers. These multi-faceted features make IL-1R8 a promising target with superior druggability. However, the structural bases of IL-1R8-mediated anti-inflammatory mechanisms are still unknown, which hinders the development of therapeutics that target IL-1R8. In this project, we will fully interpret the anti-inflammatory mechanisms of IL-1R8 at an atomic level by solving the high-resolution structures of IL-1R8 in complex with pro-inflammatory TLR4 or IL-1R1, as well as the tripartite complex of IL- 1R8/IL-18Rα/IL-37, which mediates novel anti-inflammatory signaling. We will also discover, characterize and optimize IL-1R8-targeting monoclonal antibodies (mAbs) from mice immunized with an engineered IL-1R8 immunogen. Taking the structural information as a blueprint and mAbs as critical building blocks, we will design and evaluate a series of novel biological drug candidates with a high potential for precisely harnessing the anti- inflammatory and anti-tumor activities of IL-1R8 in various pathological conditions.

NIH Research Projects · FY 2025 · 2025-06

Project Summary – Overall The La Jolla HIPC team will focus on pathogens causing infectious diseases of the upper and lower respiratory tract that lead to substantial mortality and morbidity. Our approach is unique and innovative, as it focuses on defining immune signatures (IMS) of antigen-specific CD4 and CD8 T cells generated in response to natural infection with important respiratory pathogens such as SARS-CoV-2, Common Cold Coronaviruses (CCC), influenza, Respiratory Syncytial Virus (RSV) and Mycobacterium tuberculosis (Mtb). Likewise, our Program will investigate IMS of antigen-specific T cells generated following vaccination against a diverse array of pathogens in different platforms like attenuated pathogens (BCG, yellow fever (YF)), purified proteins (acellular Bordetella pertussis (PT) vaccines), viral vectors (J&J, SARS-CoV-2) and mRNA (Moderna and Pfizer). In Project 1, we will perform longitudinal analysis to determine persistence and plasticity of antigen-specific T cell responses following natural SARS-CoV-2 infection and vaccination. We will study T cell responses specific to SARS-CoV-2 following vaccination with different vaccine platforms in previously-unvaccinated donors, and in a longitudinal cohort of vaccinated individuals previously naturally-infected with SARS-CoV-2. In parallel studies, we will analyze T cell responses to SARS-CoV-2 in naturally-infected unvaccinated donors. We will also analyze T cell responses in two previously-enrolled cohorts who received YF and PT vaccinations; in both cohorts the natural evolution and persistence of T cell responses to CCC viruses will be investigated. In Project 2, we will perform longitudinal analysis of the IMS of Mtb-specific T cells. Here, we will build on our progress made during the previous HIPC funding period to characterize the IMS associated with latent and active TB disease as well as BCG vaccination. Specifically, we will characterize the longitudinal IMS of both active and latent TB during treatment. In parallel, we will characterize the longitudinal IMS of adults (re)vaccinated with BCG, and characterize the IMS of Mtb-specific T cells in the lung. In Project 3, we will determine the molecular properties of pathogen-specific lung tissue-resident memory T cells (TRM). Our goal is to establish a single-cell atlas of the transcriptome, epigenome, and T cell receptor (TCR) of antigen-specific lung TRM targeting common pathogens that infects the lungs such as: viral (influenza, RSV, para influenza, meta pneumovirus, SARS-CoV-2, CCC), bacterial (pneumococcus, PT, Mtb) and fungal pathogens. The longitudinal study design will enable assessment of plasticity and persistence of lung TRM cells following natural infection and vaccination. The synergy between Projects will allow the generation of cross- comparable large-scale single-cell T cell signatures for respiratory pathogens/vaccines.

NIH Research Projects · FY 2025 · 2025-05

PROJECT SUMMARY ALTernaria ALTernata (ALT) is the most common outdoor airborne fungus found worldwide. The prevalence of ALT sensitization ranges from 2% to 23.8% in the general population and much higher among asthmatics. ALT sensitization has been associated with airway hyper-responsiveness, asthma and adverse asthma outcomes. It is an enigma why some people develop sensitization to a common fungal allergen (ALT) while others don’t. Moreover, the reason why ALT sensitization is associated with increased risk of developing asthma is not known. In this proposal, we will explore the mechanisms and cell types that drive ALT sensitization and the development of asthma. We recently identified a novel subset of house dust mite (HDM)-specific T cells characterized by an interferon response (IFNR) gene signature (THIFNR cells), which was negatively associated with HDM sensitization. In a pilot study, focussed in ALT allergen, we found that the proportion of ALT-specific THIFNR cells was increased in healthy subjects without ALT sensitization when compared to asthmatic subjects suggesting that it may also play a protective role in ALT-sensitization and asthma. We also found that ALT-specific TH2 cells from asthmatics displayed pathogenic features that were distinct from HDM- specific TH2 cells. Based on these findings, we hypothesize that ALT-specific THIFNR cells protect against ALT-sensitization in healthy subjects, and a novel pathogenic TH2 subset in the airways, specifically tissue- resident memory T cells (TRM cells) promote the development of asthma in ALT sensitized subjects. In Aim 1, we will determine the association between ALT-specific THIFNR cells and protection against ALT sensitization. We will assess subjects enrolled in the Isle of Wight Whole Population Birth Cohort. We will identify subgroups of participants (n=100) with: (i) Persistent ALT-sensitization since childhood (n=25), (ii) Adult-onset ALT- sensitization (n=25), (iii) HDM-sensitized without ALT-sensitization since childhood (n=25), and (iv) Non- atopic since childhood (n=25). We will isolate ALT-specific T cells from blood and perform single-cell RNA-seq and TCR-seq to enumerate the frequency, properties, persistence and clonality of ALT-specific T cell subsets, including the novel THIFNR cell subpopulation, and determine their association with risk and protection against development of ALT-sensitization at different ages. In Aim 2, we will identify ALT-specific T cell subsets in the blood and airways associated with the development of asthma in subjects with ALT-sensitization. We will enroll 50 subjects with ALT-sensitization from the IOWBC (n=25 with asthma, n=25 without asthma), and as controls, 50 asthmatic subjects without ALT-sensitization (n=25 with HDM- sensitization, and n=25 with no sensitization to common allergens), obtain blood and airway samples (sputum). We will perform single-cell transcriptome and TCR-seq analysis to determine the frequency, properties, persistence and clonality of ALT-specific TH cell and TRM subsets in the blood and airways, and their association with asthma.

NIH Research Projects · FY 2026 · 2025-05

ABSTRACT T cell responses are critical in the pathogenesis, prevention, and treatment of a broad range of diseases from infection to cancer, autoimmune disease, and transplant rejection. HLA transgenic mice have been proven to predict human T cell responses; yet currently available HLA transgenic mice are specific to individual HLA alleles and based on genetic engineering technologies which date to before the turn of the century. Further, available HLA transgenic mice are incompatible for breeding to produce combined HLA class I and class II models. This project will create and validate a modular Cas9-expressing HLA transgenic mouse platform strain from which any HLA class I and or class II transgenic mouse can be generated with now broadly available CRISPR methods. Our published data demonstrate our track record of working with HLA transgenic mice and of designing, evaluating, and validating novel mouse models. Further, our established relationship with TransViragen Inc., established genetic engineering experts, assures that the mice will be engineered as precisely and efficiently as possible. As preliminary data we include information from our collaboration with Synbal Inc. which in less than 2 years’ time generated triple-humanized mice on both C57BL/6 and BALB/c backgrounds which are distributed by the The Jackson Laboratory. We will produce a Cas9-expressing platform mouse strain with humanized β2M, CD8 and CD4, and absence of mouse MHC II. The three key advantages of this strain will be: (a) investigators studying a broad range of diseases will be one CRISPR step or transgene injection away from a mouse model with HLA class I or class II allele of their choice, (b) crosses of allele-specific strains will allow investigators to model virtually any combination of HLA class I and II alleles beginning at F1, and (c) any platform-derived mouse for somatic mutagenesis experiments in the context of humanized CD4, CD8 and β2M. The specific aims are (1) Produce a CRISPR-ready platform strain and transgenic mouse strains that are HLA allele-specific for autoimmune diseases and the top HLA class I and II allele frequencies of US ethnic groups, (2) Test and validate T cell reactivity in HLA class I and HLA class II mice derived from platform strain. Mice will be deposited in the JAX Repository, and validation data will be compiled and published in a publicly viewable online database. This HLA transgenic platform strain will provide a broad range of investigators with a modular small animal in vivo model of human relevant T cell responses for the next decade and beyond.

NIH Research Projects · FY 2026 · 2024-12

Project Summary/Abstract: Our central hypothesis is that computational models can accurately predict influenza vaccine breadth and durability based on underlying immunological factors, including an assessment of innate responses triggered by the vaccine, antigen-specific B cells and T cells, the immune exposure history of the host, and genetic factors intrinsic to the host. We propose to capture these variables and their relationship to vaccine outcomes from public datasets and newly generated experimental data. We will build computational models with complementary approaches that use these data to predict vaccine outcomes, and we will host an annual prediction contest that is open to the public to evaluate the predictive performance of these models on unseen data. Our Aims are to: 1) Capture existing influenza vaccine data and generate new experimental data that connect immunological variables with vaccine breadth and durability. We will: 1.1) Collect and standardize data from existing studies that profile the breadth and/or durability of influenza vaccine responses using any of the key immune variables we will assess (genetic factors or innate, B cell, or T cell immunity). This will result in a comprehensive training set for our computational models. 1.2) Generate new experimental datasets that measure all of these variables, along with vaccine breadth and durability, to create an independent testing set for our annual competition. 2) Generate computational models predicting the breadth and durability of vaccine responses and the cascade of immune events leading up to it. We will: 2.1) Adapt and refine computational models developed by our center investigators to predict vaccine breadth, durability, and key immune events connected to these vaccine outcomes. 2.2) Implement models published in the literature to serve as a comparison. 2.3) Combine the best- performing models to develop an integrated understanding of influenza vaccine responses. 3) Rigorously evaluate model prediction performance on unseen data in an open competition. We will 3.1) Perform an annual competition using data from Aim 1.2) that was held back from the public to evaluate model performance. 3.2) Engage the broader scientific community to participate in the predictive modeling competition and thereby maximize the diversity of independently assessed computational modeling approaches. Overall, this open, transparent, and quantitative process to build and evaluate computational models of influenza vaccination-induced immunity will test our central hypothesis and quantify how well computational models predict vaccine breadth and durability.

NIH Research Projects · FY 2026 · 2024-06

Project Summary The long-term goal of this project is to develop a vaccine that confers robust and durable protection against transplacental transmission of Zika virus (ZIKV). Accomplishing this goal may be challenging in that the vaccine may need to induce both antibody and T cell responses to confer highly effective protection against ZIKV at the maternal-fetal interface (MFI). Studies with pregnant women in Brazil have suggested that antibody responses may contribute to pathogenesis of congenital Zika syndrome (CZS) and neutralizing antibody responses may not correlate with protection against CZS. Recent human studies also suggest that the anti-ZIKV antibody response may be less durable than T cell response. However, ongoing ZIKV vaccine development efforts are focused on eliciting mainly antibody responses. Based on our published data demonstrating a critical role for CD8 T cells in protecting against ZIKV infection in multiple mouse models, we will test our central hypothesis that a robust ZIKV vaccine-induced CD8 T cell response in mothers is required to provide strong and durable protection against transplacental transmission of ZIKV. Our replicon RNA vaccine expressing ZIKV premembrane (prM) and envelope (E) or nonstructural protein 3 (NS3) induces robust protection against ZIKV infection in pregnant mice but only partial protection in their fetuses. Therefore, we will use these replicon RNA vaccines and mouse models to achieve the following Specific Aims: 1) Improve vaccine-induced protection against ZIKV infection during pregnancy, and identify the maternal immune responses associated with the most protective and durable vaccines. 2) Test the role of CD8 T cells in vaccine-induced protection, and determine precise features of MFI CD8 T cells elicited by the most protective and durable vaccine. We have expertise in examining flaviviral pathogenesis and immunity using mouse models. We also have a longstanding collaboration with colleagues at our institute and UC San Diego to investigate virus-host interactions using genomics and histopathology-informed approaches.

NIH Research Projects · FY 2025 · 2024-06

Filoviruses emerge nearly annually and cause hemorrhagic fever with high mortality, and frequent unpredictable outbreaks. Development of broad-spectrum antiviral agents is needed and will be guided by a clear understanding of the structures and assemblies of essential viral machinery. The filovirus nucleocapsid forms the core of the virion and is essential for replication and transcription of the viral genome and host immune suppression. The viral replication and nucleocapsid assembly occur in viral replication factories in host cytoplasm. Assembly of these nucleocapsids in replication factories and their subsequent transport to the cell surface, and packaging into virions are all critical steps in the filovirus life cycle. Much about these essential processes, however, remains unknown, because the assembly processes happen inside cells, and traditional structural biology approaches involved use of purified, often recombinant, samples outside cells. Thus, we do not yet have structural understandings of what filovirus assembly, transport, matrix-interaction and virion-recruitment processes look like. For example, structures are available for the recombinant N-terminal half of the EBOV nucleoprotein (NP) coiled with cellular RNA, but we don’t yet understand the structure of the C-terminal half of NP that recruits the other NC proteins, nor do we understand how other viral proteins assemble to form complete nucleocapsid structure or how the nucleocapsid interacts with the virus matrix. A new technology, in situ cryo-electron tomography, now allows us to carry out structural biology analyses inside cells and allows us to collect previously inaccessible information about the structure of the assembling nucleocapsid inside infected cells. The goal of this project is to apply cryo-focused ion beam (FIB) milling-enabled in situ cryo-electron tomography (cryo-FIB-ET) to understand how the Ebola virus nucleocapsid is assembled inside cells and how the structures change before and after virion incorporation. Study of these complexes inside cells, from both transfected and infected cells, will provide us our first information about the assembly process of virus replication factories inside cells and complete structural model of filovirus nucleocapsid structure. The fundamental structural organization and critical protein interfaces revealed in this study are likely conserved among all filoviruses. Hence, the proposed work will provide insight into the fundamental structural principles of filovirus assembly. The knowledge and expertise gained here may also be relevant for understanding the structures of other negative-strand RNA viruses that have a similar protein structural organization.

NIH Research Projects · FY 2025 · 2024-05

The Old-World Arenavirus Lassa (LASV) can cause severe hemorrhagic Lassa fever (LF), which is associated with significant morbidity and mortality in West Africa every year. As the most exported hemorrhagic fever, LASV also poses a significant global health risk. LF is recognized as a “Priority disease” by the World Health Organization (WHO) and the Coalition for Epidemic Preparedness Innovations (CEPI). The trimeric glycoprotein complex (GPC) is the only viral protein expressed on the virion surface and responsible for LASV cell entry. Each monomer within the trimer is composed of the non-covalently associated stable signal peptide, the receptor-binding subunit, GP1, and the fusion machinery, GP2. LASV sequentially utilizes two host receptors for cell entry, the matriglycosylated alpha-dystroglycan (matri-α-DG) at the cell surface and the lysosomal-associated membrane protein 1 (LAMP1) in the endosome during endocytosis of the virion. The receptor switch requires an acidic pH-induced conformational rearrangement of the GPC trimer, called GPC priming. Structural biology has only captured atomic snapshots: prefusion GPC in complex with matriglycan, and then individual subunits: post-fusion GP2, and a low-pH incubated GP1 truncation presumed to be in the primed state. The inherent metastability of GPC has thwarted efforts to obtain high-resolution structures of trimeric GPC in priming states and the GPC:LAMP1 complex. In this project, we will fill in these important missing structures and illuminate pH-driven changes in the GPC trimer and mechanisms for receptor switching. The proposed structural work for this program is made feasible by a novel LASV GPC construct engineered by the PI that maintains native conformation at neutral pH and is capable of priming and LAMP1 binding at acidic pH, without dissociation of the GP1 and GP2 subunits. Importantly, insights from these studies can be applied to other medically relevant, pH-dependent receptor-switching arenaviruses, such as the ubiquitous Lymphocytic choriomeningitis virus and the rare, but potentially more fatal, Lujo virus.

NIH Research Projects · FY 2026 · 2024-05

Abstract We often depict signaling pathways by a dozen or so landmark phosphorylation events that culminate in the transcription of a gene or induction of a phenotype. In reality, a cascade of hundreds to thousands of phosphorylation sites create dynamic biochemical networks that orchestrate essentially every cellular process from expression of transcriptional programs and cell proliferation to migration and cytotoxic effector functions. T cell signaling is an important example. Signal transduction through the T cell receptor and co-stimulatory molecules is incredibly complex and leads to important but distinct downstream effects necessary for proper immune responses to pathogens, cancer, and vaccines. As humans age, T cell signaling becomes detrimentally altered, leading to “immunosenescence” and less efficient protection from malignancies. Mass spectrometry has enabled the profiling of tens of thousands of phosphorylation sites from a diverse range of cells and tissues. Unfortunately, functionally characterizing the tens of thousands of post-translational modifications cells use to coordinate essentially all cellular and organismal processes remains a fundamental challenge in biology. Here, we propose an innovative technology that will enable the functional assessment of thousands of phosphorylation sites in high throughput. We will employ CRISPR-mediated base editors, which mutate codons in genomic DNA, coupled to phenotypic screens to probe the signaling events that lead to specific T cell stimulation-specific gene expression programs or proliferation responses. Preliminary data from our laboratory strongly suggests that we can functionally screen tens of thousands of phosphorylation sites for their contribution to NFAT or NFKB signaling in stimulated T cells. In this proposal, we will further optimize this technology to create a robust, reliable screening platform. To validate our screening technology, we will characterize a subset of phosphorylation site mutants using conventional genomic engineering methods to understand how our screening platform compares to classic approaches to study phosphorylation site function, i.e. one mutation at a time. We will also extend this approach to primary human CD8+ T cells, to show that we can uncouple the signaling events that lead to cell proliferation or expression of the activation/exhaustion marker PD1. High throughput functional screening of phosphorylation sites in primary immune cells will revolutionize the way we study signaling pathways and cellular decision making, and the way we approach drug development or adoptive cell therapies to treat cancer and a variety of human ailments associated with aging.

NIH Research Projects · FY 2026 · 2024-04

SUMMARY Conventional vaccine strategies have not induced successful protection to diverse HIV strains. mRNA vaccine platforms enable novel modalities of antigen delivery for innovative vaccine strategies. The use of mRNA technology for HIV vaccines holds promise to conquer major barriers to induce protective responses via broadly neutralizing antibodies (bnAbs). However, mRNA vaccines to express HIV antigens capable of effectively conferring protective immunity have not been fully explored. Germinal center (GC) responses are paramount for prophylactic vaccines as they provide a highly specialized environment to affinity mature antibodies to difficult epitopes on HIV. We have demonstrated that efficient priming and longer duration of GC responses are likely favorable for broader neutralizing responses against HIV (Nature 2022). mRNA vaccines against SARS-CoV-2 have been shown to induce long-lasting GC responses after 2 doses, but the biology and effective manipulation of this process remains elusive. Particularly, a big knowledge gap is how mRNA vaccine-induced GCs differ from protein vaccines. A second knowledge gap is what mechanisms drive long- lasting GCs? A third knowledge gap is what immunological signals regulate the output of durable immune memory? HIV envelope (Env) trimer is the sole antigenic target for neutralizing antibodies. Many experimental HIV vaccines use soluble forms of Env that expose the immunodominant base of the trimer eliciting non- neutralizing antibodies. mRNA vaccines allow the realization of membrane-bound Env expression, which can present antigen in a more native form avoiding exposure of the base. Membrane-bound Env trimers can also be expressed on nanoparticles such as virus-like particles (VLPs) via mRNA delivery resembling higher density expression on native virions. Given the potential of long-lasting GC responses and the new modes of antigen delivery by membrane-bound Env or multimeric Env nanoparticles, we seek to investigate: Are HIV mRNA vaccines capable of inducing long-lasting GC responses? How can HIV mRNA vaccines optimally prime long- lasting GC responses? What sequential immunization strategies work best with HIV mRNA vaccines to induce diverse and durable memory responses? Non-human primates (NHPs) are an invaluable model for studying this biology and immunology, because of their relatedness to humans.

NIH Research Projects · FY 2026 · 2024-03

Abstract It is well established that aging is accompanied by chronic low-grade inflammation that correlates well with mortality and morbidity, but the association between aging and inflammation is not well understood. Similarly, changes in DNA cytosine methylation occur reproducibly with age, suggesting that DNA methylation contributes to the aging process, but the biology remains unclear. Certain families of transposable elements (TEs) are repressed in somatic cells via DNA methylation; moreover, TEs are activated in aging mice and in cultured human cells undergoing replicative senescence, and increased TE expression leads to DNA damage, mutations and inflammation. These consequences have all been observed in cells from aged individuals, but the underlying mechanisms are obscure. Understanding the mechanistic basis for the association of aging with inflammation and changes in DNA methylation will be essential to design rational interventions for age-associated disorders. Here we tackle the question of how changes in DNA methylation relate to inflammation during aging. DNA methylation is regulated by DNA methyltransferases (DNMTs) and TET methylcytosine oxidases, which control DNA methylation and demethylation respectively. DNMT deficiency predictably results in decreased DNA methylation, but TET deficiency is paradoxically also associated with a striking loss of DNA methylation in hetero- chromatin. Moreover, TET and/or DNMT-deficient cells show increased TE expression and cell-intrinsic inflammation, similar to that observed in cells from aged humans and senescent cells in culture. We will test the hypothesis that aging involves a progressive decrease in TET and/or DNMT activity with age, leading to selective losses of DNA methylation in heterochromatin where repressed TEs reside, and hence in increased TE expression and cell-intrinsic (“sterile”) inflammation. In Aim 1, we will define the DNA modification (5mC, 5hmC) status of transposable elements in immune cells (CD4+ T lymphocytes and monocytes) of young, middle-aged and old healthy subjects from the BLSA and GESTALT cohorts at NIA, and relate them to the observed increase in expression of TEs as a function of age. These studies probe the hypothesis that decline of TET and/or DNMT activity with age dysregulates TE expression. In Aim 2, we will correlate increased TE expression in immune cells from the BLSA and GESTALT cohorts with signatures of inflammation and serum levels of pro-inflammatory cytokines during aging. In Aim 3, we will perform complementary studies in genetically tractable mouse models of clonal hematopoiesis, a malady of both aging and inflammation. Using mice deficient in Tet2 and/or Dnmt3a, we will define the development of heterochromatic DNA hypomethylation and inflammation in immune cells of these mice with age, and determine the relation to increased TE expression. Our studies will test the novel hypothesis of a link between TET/DNMT deficiency, loss of heterochromatic DNA methylation and increased TE expression with age, and deepen our understanding of the elusive mechanisms that connect heterochromatin dysfunction with inflammation, aging and oncogenesis.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY/ABSTRACT Vaccines are the greatest bang for our public health buck, yet vaccine development is still sometimes hit or miss. Recent efforts with SARS-CoV-2 demonstrated that acceleration of vaccine programs is possible, especially when supported by prior studies characterizing successful immune responses against related prototype pathogens. The measles virus vaccination campaign is a global success story; the live-attenuated vaccine developed in the 1960’s still elicits highly protective antibodies to strains circulating today. Yet Measles outbreaks persist due to obstacles in vaccination programs such as global health inequities, vaccine hesitancy, extreme weather events related to climate change, and the SARS-CoV-2 pandemic. Furthermore, a growing population with compromised immune systems cannot receive live-attenuated viral vaccines. Thus, the threat of measles remains (there are still approximately 9-10 million cases annually), and innovative solutions will be needed to achieve global measles eradication. However, very little is known about the human antibody response to measles infection or vaccination beyond neutralizing antibody titers. Importantly, there are no structures of any antibodies in complex with any measles virus antigens. As a result, we do not know which sites on measles virus are immunodominant and protective. Much of this fundamental knowledge is lacking largely because measles was effectively eradicated from developed nations before needed scientific tools had been developed. In this work, we will use state-of-the-art tools to directly visualize the structure of human antibodies from vaccinee polyclonal sera bound to measles surface glycoproteins to understand which antigenic sites dominate in the human vaccinee response. We will also use microfluidics and rapid microscale multiplexed competition analyses to rapidly discover and analyze individual human monoclonal antibodies against measles virus for the first time. This work will reveal what components of the human immune response to this prototype paramyxovirus lead to vaccine-mediated protection, and will help guide development of modern vaccines for the immunocompromised and other vaccines against paramyxoviruses yet to emerge.

NIH Research Projects · FY 2025 · 2023-11

Project Summary The Mammarenavirus genus of the Arenavirus family contains multiple zoonotic pathogens with the potential to cause hemorrhagic fever. These include the South American viruses Junin virus (JUNV; Argentinian hemorrhagic fever) and Machupo virus (MACV; Bolivian hemorrhagic fever), and Lassa virus (LASV), which causes thousands of cases of Lassa Fever in West Africa each year. The case fatality rate for these viruses is 20-70%. Lujo virus (LUJV) is the most recently identified African arenavirus. This virus was responsible for five infections, of which four were fatal. Notably, this outbreak was characterized by human-to-human transmission rather than transmission between rodent and human, as is most common for other arenaviruses. Arenaviruses are genetically and geographically divided into New World (e.g. JUNV and MACV) and Old World (e.g. LASV) groups. Its African location placed LUJV into the OW group. However, LUJV is genetically divergent from other African arenaviruses and is phylogenetically equidistant between the NW and OW groups. Further, its glycoprotein GPC recognizes a different receptor and is antigenically distinct from the other arenaviruses. As the only protein expressed on the viral surface, GPC is responsible for receptor engagement, cell tropism and entry, and is the primary target of antibodies. Understanding the unique structure and surface chemistry of LUJV GPC in its native conformation is key to understanding receptor recognition in the native trimer context, what antibody targets might be on this divergent virus, and how we might design vaccines and therapeutics should the virus re-emerge. The premise of this proposal is that structures of the medically relevant LUJV GP alone and in complex with its cell surface receptor will reveal reasons for its unique cell entry requirements and any differences in its epitope landscape. We will use state-of-the-art biophysical techniques such as cryo electron microscopy, surface plasmon resonance and composition-gradient multiangle light scattering, to characterize the interaction of the prefusion-stabilized LUJV GP trimer (pfGP-TD) with its receptor NRP2. In Aim 2, we will identify antibodies from mice immunized with LUJV pfGP-TD using the Berkeley Lights Beacon platform. Results from the innovative research proposed here will launch multiple lines of inquiry for future studies and will help guide development of vaccines against a diverse range of arenaviruses.

NIH Research Projects · FY 2024 · 2023-07

SARS-CoV-2, the causative agent of an unprecedented global pandemic, has just four structural proteins. Of these, the nucleocapsid N is the most abundant protein in the virion, and plays essential roles in genome encapsidation and viral assembly. However, N has thus far defied structure determination of its full-length molecule. Indeed, there are currently no high-resolution structures of full-length N for any coronavirus, although multiple structures exist for individual domains within N. The lack of structural information on N, its assembly and its interactions and encapsidation of the genome stem from the inherent flexibility contributed by three intrinsically disordered regions. In previous work, N, in the absence of RNA or in the presence of random bacterial RNA derived from the expression system, was too flexible to allow determination of a high-resolution structure. The assembled capsid in the virion is also too heterogeneous in its flexibility, positions and conformations to afford high-resolution information. Through careful analysis using electromobility shift assays, size-exclusion and screening by electron microscopy, we have now identified portions of the SARS-CoV-2 genome that yield structurally homogeneous, purified N dimers, octamers, and 16-mers that are amenable to high-resolution structural analysis, and which represent the basic building block and likely assembly intermediates of the full capsid. We have further produced a polymerized full-length capsid in vitro that is also amenable to structural study. Here we propose cryoEM of the dimer, assembly intermediates and full length in vitro capsid, complemented by innovative native mass spectrometry and straightforward specific antibody-mediated domain identification. This work will illuminate (i) the structure and assembly of the coronavirus capsid; (ii) how the RNA genome interacts with multiple domains of the full- length N and connects along polymerized copies of N; (iii) conformational adjustments that occur in assembly, protein-protein and protein-RNA interaction sites; and (iv) sites that may be amenable targets for antiviral development.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ABSTRACT Inflammatory bowel diseases (IBD) cause substantial mortality and morbidity. Current treatments that block pathological inflammatory responses have improved clinical outcomes in some patients but they are not highly effective in modifying disease progression or preventing relapses. Hence, there is a large unmet need to develop novel therapeutic targets. Genome-wide association studies (GWAS) offer an unbiased approach to identify therapeutic targets in relevant immune cell types such as CD4+ T cells that play key roles in IBD pathogenesis. Because T cells are quiescent in the absence of extrinsic stimulation, it is not possible to fully examine the effects of disease-risk variants on functionally relevant effector genes under resting conditions. To identify IBD-risk genes in activated CD4+ T cells, we performed the first large-scale single-cell eQTL study on activated CD4+ T cells. We found that reduced expression of Leukocyte-specific protein 1 (LSP1), specifically in activated CD4+ T-cell subsets such as TH1 and TH17 cells, was associated with the risk of IBD. In this R01 proposal, we will investigate how reduced levels of LSP1 influences the differentiation and function of CD4+ T cells to drive disease pathogenesis, and will test the hypothesis that LSP1 plays a key role in restraining the re-programming of CD4+ T cells into a more pathogenic cell state in IBD patients. In Aim 1, we will determine the functional IBD-risk variants that reduce LSP1 expression in CD4+ T cells. We will employ luciferase reporter assays to determine functional variants in the enhancers and promoter of LSP1, perform CRISPR-mediated editing of prioritized IBD-risk eQTLs to define causal variants, and perform CRISPRi and ChIP assays to determine functional enhancers, relevant up-stream regulators and whether the functional LSP1 eQTLs directly perturb the binding of key transcription factors that modulate LSP1 expression. In Aim 2, we will determine the role of LSP1 in reprogramming of CD4+ T cells into the pathogenic state observed in IBD. To determine whether LSP1 influences the differentiation and pathogenic function of CD4+ T cells from healthy and IBD donors, we will reduce and increase LSP1 levels and assess the effects on CD4+ T-cell activation, apoptosis, proliferation and proinflammatory cytokine production. In an adoptive T cell transfer model of colitis, we will compare the ability of Lsp1- sufficient (wild-type) and Lsp1-deficient CD4+ T cells in driving colonic inflammation and pathogenic TH1 and TH17 differentiation. We will determine whether reducing Lsp1 expression in CD4+ T cells enhances their pathogenicity in mouse models of colitis, thus implicating an important T cell-intrinsic role for LSP1 in IBD pathogenesis. Overall, this study, examining the function, expression, activation and regulation of LSP1 in CD4+ T cells, will provide important mechanistic insights into the genetic basis of risk for inflammatory bowel disease.

NIH Research Projects · FY 2026 · 2022-08

Project Summary/Abstract O-GlcNAc is a single N-acetylglucosamine coupled to serine and threonine residues of nuclear and cytoplasmic proteins. Analogous to phosphorylation, O-GlcNAc signaling is dynamic, rapidly added and removed from proteins in a site-specific manner in response to cellular perturbations and extracellular cues. Because both modifications occur on the same residues it is hypothesized that there is a functional crosstalk between O- GlcNAc and phosphorylation, where one may affect deposition or removal the other. Unlike phosphorylation, however, which is catalyzed by over 500 kinases and roughly 300 phosphatases, the mammalian genome only encodes a single O-GlcNAc transferase (OGT) and a single hydrolase (OGA). While many kinases recognize specific amino acid sequences in their substrates, the determinants guiding OGT are unclear and likely manifold. This intracellular glycosylation is implicated in nearly every cellular process from gene expression and signal transduction to cell division and differentiation. Despite the ubiquitous nature of this post-translational modification in health and disease, the specific functions of OGT and the basic principles of O-GlcNAc signaling remain almost entirely elusive. This gap in our knowledge has been largely due to the major lack of tools and technologies available to study O-GlcNAc signaling or perturb the essential OGT. Here, we aim to uncover the basic principles of OGT and O-GlcNAc signaling, and their role in transcriptional regulation of cellular differentiation. We have recently developed a highly sensitive and specific enrichment reagent to analyze O-GlcNAc-modified peptides from cells and tissues by mass spectrometry. Using these new anti-O-GlcNAc antibodies we will elucidate the global, site-specific temporal dynamics of O-GlcNAc signaling during the transition from totipotency to naïve and primed pluripotency. Combined with phosphoprotemic profiling of the same samples, we will monitor for crosstalk between these two post-translational modifications. To gain insight into how OGT targets its diverse array of substrates, we will deconvolute the extensive OGT interactome employing chemical crosslinking and biochemical fractionation, followed by mass spectrometric analysis. To explore how OGT uses adaptor proteins to targets substrates, we will use an innovative approach, degrading specific OGT interacting proteins and assessing changes in downstream O-GlcNAc signaling using our new quantitative glycoproteomic approach. Integrating these two research programs, we will create a holistic, high- resolution understanding of the principles of O-GlcNAc signaling. The MIRA mechanism will not only enable the investigation of basic O-GlcNAc biology, but will provide the flexibility to conduct data-driven follow-up, functional analyses of dynamic O-GlcNAc/crosstalk sites and distinct OGT complexes, and their role in transcriptional regulation of some of the earliest development decisions.